m mpmno‘u AND STUDY
0; TH: CELLULASES AND HEMICELLULASES
or MYROTHECIUM vmumm}

Thai. for tho Dam of Ph. D.
MICHIGAN STATE UNIVERSITY
R. Merwin Grimes
1955

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TE“ PFI Phi-@310}! AID STUDY OF T11? CTLLUIACES

M33 i—E‘EICELLUL‘SFS 1: uncrsrmm vrzanuwzu

 

8?

E. HEREIN (BELTS

A?! AFSTFAC '1‘
Submitted to the School for Advancnd Graduate Studies
of menigan State University of Agriculture
and Applied Scionoo in partial fulfillment
of the "claimant.- for the demo of

DOCTOR OF I-‘dILCSOPifY

papa-aunt. o: Chum-try
In: 1955

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The present investig‘ation was. tmdr-rtalwn to study (1) the. possibln
Imltiplicity of cellulolytic pnzymca and (2) tho rolationships among the
collulane, hrnicr-llulase, and fi-glucoaida so activities of culture filtrates
of the fungus Exgothecium vcrrucaria. Previously; Whitaker (1953) obtained
widcnco indicating tnot opparontly a single cellulaao onzyno in elaborated .
by the fungus, but noose and Gilligan (1953) reported that that. or. tub
oopnroto enzyme. involved in the hydrolyail of ouch aolublo oubotitutod
onlluloco derivntivoa on carboxymothylcelluloco. Furthormnro, Jormyn (1952)
obtlinod evidence suggesting that several of tho protein component. in .
proporotiono from Asgcrgillua‘252322.pocoooo tho obility to catalyso tho
hydrolnio offl-glucooidoo on all u celluloao darintim.

Cultm film-eta: of II. vomcarin group in aubmrgad culturo gm
initially’concontratod by claw partial frvozing. Attampts to froctionoto tho
cancentrnte by salt and solvent precipitation and by chronntography'on colluloso
calm were gone-rally unauccoaaful, but aftcr further concentration 0!; the ‘
pfotcinl by'prccipitation with oovontyhfivo percent acetone or saturatod
ammonium oulfato, electr0phorooio-convection yieldnd propanationl containing
ditforont ration of the protein components. Froctionationo by thin mothod
IOf. controlled by estimation of onsymatic activity, using collulooo sodium
culthto no the Inbotrato, dotrrmination of protein nitrogen content, and
oloctrophorotic analyuio or non. of tho fractionl.

Six electrOphorotic components in pH 6.9, ionic otrongth 0.1h6 phosphat-
buffor Into oblorvcd in tho unfroctionotod enzyme concentrutoo. Th... component.

mun-bored.ltorting with tho Ila-ootnnigrating protein. Tho firot and moot
octivo of tho throo final onsyno propnrotiono ulod to study the onsymltic

proportion of tho comtituonto, contained primarily component! 1 and 2, along

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with a anal]. amount of component 3. Tho eooond preparation contained no
component 1, but had all the othoro, and tho third preparation woo devoid
of couponmto l and 2, but had all the remaining componento.

Colluloeo oodiuo sulfato, wheat otraw cellulooo, two henicolluloeo
preparationo from wheat otraw, and oalicin were uoed ao eubetratoo to otuch
the mymatic propertieo of tho threo enzyno preparationo. The opocific
activitioo (defined ao the milligrams of reducing ougare, calculated ao
glucooo, eplit from 100 milligram of the substrate under opooified conditions,
per Iilligram of protein nitrogen) of the three enzyme preparation acting
a: the five ouhotrateo were determined and cmpared. Specific activity-pf!
curveo for each oneymo preparation with each oubotrate were conotructcd. The
and producto obtained from celluloao aodium sulfate and the hemicelluloeo
preparation with each of tho onayno oolutiono were otudiod by papor chromato-
graphic techniquoo.

The preparation containing components 1, 2, and 3 wao tho moot active,
and tho one containing only conponmto 3, h, S, and 6 the loaot active. How-
ever, the roonlto indicated that more than one component woo enzymaticany
activo with all of tho polyoaccharido ouhotratoa. Compcnont 2 appeared to to
tho hoot active enzyme, but ito eharo of the total activity varied with tho
ouhotrato. Thie comment apparently contributed ooot of the hydrolytic
action ohoervod with ccllulooo, rout-what loco with collulooo Iodine oulfato,
and otill loco with tho henioollulooo preparationo. Although the dietribution
of the mining activity among the other componente no not clearly indicated
by the rooulto, conponent l, ao well ao one of the other proteino, probably
had oooo action on collulooe oodiuo eulfato and tho henicelluloeoo. The
multo indicated clearly that nearly all of tho fi-glucooidaoo ('ealicinaae")
activity no aoeociated with couponent l.

 

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FL. Herein Grimes
LITFMTUILF CITED

Jot-m, l. A. (1952) Auotralian J. Sci. Research, Ser. B, i, 103.
Boooo, F. 1.. and I. Gilligan (1953) Arch. Biochem. and Biophyl., 1.52, 71;.
mm, D. a. (1953) Aroh. Biochon. and BiOphye., 5;, 253.

THE PREPARATION AND STUDY OF THE CELLULASES

AND HEMICELLULASES OF MYRUTHECIUM VERRUCARIA

By

R. MERWIN CRIMES

A THESIS

Submitted to the School for Advanced Graduate Studies
of Michigan State University of Agriculture
and Applied Science in partial fulfillment
of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Department of Chemistry

1955

ii

A CK NOEfllED TEEN T S

The author wishes to express his sincere thanks to Dr. C. a. HOppert
and Prof. C. I. Duncan for their unfailing interest, valuable suggestions,
and considerable patience throughout the course of this investigation, and
for their help in the preparation of the manuscript.

Grateful acknowledgment is also due to Dr. E. J. Miller for his
interest in, and support of the work.

The writer is also greatly indebted to Mrs. Audrey M. Anderson and

Mrs. Jean Brehmer for their assistance in the preparation of the manuscript.

TABLE OF CCNTENTS

mTRODUCTIONQIOOOOIOOOOOOO.0O.0.OOOOOOCOOOOOCOOOOOOOO0......00....

The Importance of the Cellulases and Hemioellulases.........
Ravi“ Of manta-11.8.00000000.0.000000000000000000000000000.
Statement Of the Problem............................o.......

EXPERDVUMALoOooeeooeeeeeoeeeeecceoeeeeoeeooeoooee0000000000000...

Materials and MPthOdSooooooeoeoeeoeeeeeeeeeeeeesoeoeoeoon...
Production of Culture Filtrates........................
Concentration of Culture Filtrates.....................
Fractionation by Salt or Solvent Precipitation.........
Fractionation by Chromatography on Cellulose
Columns................................................
Fractionation by Flectrophoresis Convection............
Fstimation of Pnzymatic Activity.......................

Substrates........................................
Assay Procedure...................................
Electrophoretic Analysis...............................
Identification of End Products.........................

Results....... ..... .........................................
Production and Concentration of Culture Filtrates......
Fractionation by Salt and Solvent Precipitation........
Concentration by Precipitation with.acetone and
Ammonium.SUIfate.......................................
Fractionation by Chromatography on Cellulose
COlumn8eeeeeeeeeeeeoeoeeeooeeeeeeeeeeeoeeoeeeoeoeeoeso.
Fractionation by Electrophoresis-Convection............
‘ElectrOphoretic Analysis of Fractions Obtained by
ElectrOphoresis-Convection.............................
Action of M. verrucaria Enzymes onfl-Glycosides,
Extracted Wheat Straw;fiand'Wheat Straw Holocellulose
Preparations...........................................
Comparison of Cellulase, Hemicellulase, and
fi-Glucosidase Activities of Three Enzyme
Preparations...........................................
End Products of Fnzvmatic Hydrolysis..... ..... .........

 

DISCUSSION OF RESULTS............................................
SUMMARY..........................................................
LITERATURE CITED.................................................
APPENDIX A... ..... ...............................................

APPENDIX B.0OOOOOOOIIOOOOOOOOOOOOOOOOOO000......OOOOOOOOCCOOOOOOO

iii

PAGE

138
112

116
119

129
138
1ho
15h
170
176
179

185
191

INTRODUCTION

INTRODUCTION

The Importance 21; 2h: Cellulases and Hemicellulases

 

 

 

The enzymes concerned with the degradation of celluloses and the
hemicelluloses are of great importance to all life. Their action on
the polysaccharides of plant residues is an indispensable link in the
carbon cycle. By the process of photosynthesis, the green plants of
the earth fix carbcn dioxide at the rate of about three percent of the
atmospheric supply annually (Rabinowitoh, 191.5). At this rate, atmos-
pheric and reserve carbon dioxide available for photosynthesis would
be depleted in a relatively short time if it were not constantly re-
plenished by degradative processes.

A major portion of the biologically fixed carbon is found as
cellulose and related polysaccharides. Although other reactions, such
as the burning of coal, gas, petroleun, and wood, contribute measurably
to the replenishnent of atmospheric carbon dioxide, the greatest single
factor is the respiration of living organisms, especially the soil
bacteria and fungi (Meyer and Anderson, 1939). Since cellulosic
materials make up so large a portion of the biological carbon, and
the first step in their decomposition is caused by cellulases, the
great importance of these enzymes in the carbon cycle is obvious.

Incidental to the contribution of the cellulose-destroying micro-
organisms toward the perpetuation of the carbon cycle, but of nearly
as great significance is their role in soil fertility. Their activity

on plant residues, along with that of many other soil microorganisms,

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initiates a long and complex series of reactions leading to the forma-
tion of humus (Waksman, 1936). During this process, all the cellulose
and most of the hemicelluloses are destroyed, serving as a source of
energy and microbial cell substance. Certain nutrients from the plant
residues are released or changed into forms more accessible to succeed-
ing generations of plants. The lignin remaining after most of the other
plant constituents have been removed is relatively resistant to micro-
bial attack and serves as the nucleus for humus formation. The chemical
and colloidal properties of humus account for much of the difference
between ground and weathered rock and fertile soil.

The symbiotic relationship between their hosts and many species of
microorganism inhabiting the intestinal tracts of the higher animals
is well known. In the case of ruminants, this relationship has been
evolved to a high degree, and much of the credit for this successful
symbiosis is due to the cellulolytic organisms present in the rumen.
Their action not only converts part of the ingested cellulose and hemi-
celluloses into forms of carbohydrate, acids and proteins available to
the host, but also makes other nutrients more accessible (Huffman,
1953). Although feeds other than the cellulose-containing roughages
are necessary for an efficient ration, it is perhaps not an exaggeration
to state that our entire dairy and beef industries depend to a great
extent on the cellulases and hemicellulases.

The possibility of using celluloly'tic organisms for the industrial
Procluction of acids and alcohols has been caplored. Although this

a1591.:ication shows some promise, it is apparently not economically

feasible at present because of the presence in wood and plant wastes of
lignin and other encrusting materials which prevent a rapid utilization
of the cellulose and an adequate accmnulation of useful products (Sin,
1951). Among sane of the other commercial uses of cellulolytic organ-
ism may be mentioned the production of edible mushrooms, rubber pro-
duction, and sewage and garbage disposal.

Ihile it is difficult to overemphasize the importance of the
cellulases and hemicellulases to the material and energy balance of
the earth, man has perhaps concerned himself more with the harmful
aspects of their action. Given the proper conditions, cellulolytic
organisms will attack any suitable substrate. Textiles, lumber, paper,
almost every kind of cellulosic item manufactured, even living plants,
sustain a staggering amount of microbial damage. Such enormous losses
have stimulated much research on the subject, but most of this work
has been directed toward practical application, the mildew-proofing
of textiles in particular (Sin, 1951).

In spite of their significance, the cellulases and hemicellulases
have remained until very recently a relatively little studied group.
Fmthermore, mostly because of the heterogeneous nature of the sub-
strates and enzyme preparations used, much of the earlier information
is sanewhat contradictory. Recently, Whitaker (1953) isolated from
Culture filtrates of the fungus mothecium verrucaria, a cellulase
PPEparation which appears to be homogeneous. 0n the basis of his re—
8‘flts, lhitaker concluded that, at least in the case of this orgnism,

the process of cellulose hydrolysis is unienzymatic. However, the

results reported by Jennyn (1952, 1952a) and by Reese and Gilligan
(1953) indicate that there may be more than one cellulolytic enzyme in
preparations from g. verrucaria and several other fungi.

The object of the present investigation was to study the possible
multiple nature of the cellulose activity of g. verrucaria preparations,
using techniques saewhat different from those employed by Jet-awn and by
Reese and Gilligan, and further, to study the relationships among cellu-

lase, hemicellulase, and fl -glucosidase activities.

Review _o_i_‘ Literature

The cellulases and hemicellulases appear to have a wide and similar
distribution. They have been found in plants, in the digestive secre-
tions of ease invertebrates, and in many species of bacteria, fungi,
actinouwcetes, and in some protozoa. Their occurrence in the digestive
secretions of insects such as termites and roaches is open to some
question (Pigman,-l950).

In Table I a list of some of the sources of cellulase is presented.
This compilation has been restricted to those sources from which ex—
tracts or filtered culture media have been tested for cellulolytic
acti‘rity, thus excluding a great many species of bacteria, fungi, and
actinonwcetes which have been shown to be cellulolytic in live culture.
This is an arbitrary restriction and is not meant to imply that the
excluded organisms do not possess the enzyme. Sin (1951) has compiled

tables of these organisms containing 357 3990193 01' fungi, 103 Spades

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of bacteria, and 32 species of actinomycetesl. Although the criteria
for classification of microorganisms as either cellulolytic or non-
cellulolytic are quite definite (either the demonstration of growth on
media containing cellulose as the only source of carbon, or the demon-
stration of cellulose utilization, or preferably both), Siu (1951)
criticized some of the methods applied to bacteria and stated that
some of the species classified as cellulolytic are highly suspect.
On the other hand, Norman (1937) has pointed out that the usual cri-
teria for demonstrating cellulolytic activity may be too rigid and
the methods too exclusive. No examples are given in which certain
microorganisms, although not considered cellulolytic by the usual
tests using native cellulose, nevertheless are capable of decomposing
the cellulose is well as some of the other polysaccharides when they
are offered simultaneously in such natural substrates as straw.
Further, Norman stated that instances are known in which one organism
is able to utilize cellulose only in the presence of certain others.
In the same connection, it is interesting to note that although
Siu (1951) lists Amrgillus ni er, 5. mg, g. £13123, and g. m
among the noncellulolytic fungi, all have been shown to elaborate
enzymes capable of hydrolyzing the fi -l,h-glucosidic linkages of
Various modified celluloses and'cellulose derivatives, and are thus
1n¢1uded in Table I. A possible explanation of these discrepancies

has been advanced by Reese 33 g. (1950) and 1:111 be discussed in

later paragraphs.

‘

1

Among the bacteria and fungi, Siu distinguished beheen those
alreacv isolated from eXposed cotton fabrics and those not yet recorded
38 isolated from cotton fabrics. All the actinouwcetes species listed

3" Siu came from the soil.

Cell-free cellulolytic preparations are more difficult to obtain
from bacteria than from the fungi. With the latter, crude active pre-
parations are obtained simply by filtering the liquid culture medium
to obtain the culture filtrate, which contains nearly all the activity,
or by pressing the liquid from moist solid substrates on which the
fungus has grown. Bacterial culture filtrates, on the other hand,
contain very little of the activity possessed by the intact culture
(Hungate, 19112; Kitts and Underkofler, 1951:). Failure to demonstrate
the activity in the culture filtrate could be eXplained in several ways.
Complete adsorption of the active protein on the filter could account
for it (Zaremska, 1936), as would the postulation of some workers
(Meyer, 19113; Khouvine, 1923) that these enzymes are strictly intra-
cellular. However, it is difficult to conceive of a mechanism whereby
the insoluble cellulose substrate could be taken into the bacterial
cell.

At present, there seems to be little doubt that bacterial as well
as the fungal cellulases are extracellular (Siu, 1951). Perhaps the
most widely accepted explanation of the failure to detect appreciable
amounts of activity in bacterial culture filtrates is that the bacter-
ial cell liberates the enzyme only on very close approach or actual
contact with the substrate (Hungate, 19112). One could further postu-
late that the enzyme is tightly adsorbed on the outer surface of the
Gen membrane. That the bacterial cellulases are apparently adaptive
enzymes (Siu, 1951) can be used as an argument in favor of the first
suggestion. Direct microscopic studies of bacterial attack on natural

and regenerated cellulosic fibers, showing "corrosion figures" and

"enzymic cavities" immediately surrounding the firmly attached bacterial

cells, also lend support to these interpretations (Baker and Harriss,

191:7).
A list of some of the demonstrated sources of hemicellulases is

presented in Table II. The substrates used in these investigations are

also given. It should be noted that in most instances, even when given

a definite designation such as xylan, the substrates used were either

impure or contained more than one type of sugar residue (Pigman, 1950).

The hadcellulases, like the cellulases, are demonstrably extra-

cellular, at least in the case of the fungi and the snail. They are

also obtained by the same procedures used for the preparation of cellu-
lass.

Comparison of the sources of celltflases and hemicellulases (Tables
I and II) brings out the provoking, if not entirely surprising observa-

tion, that in every instance in which a hemicellulase has been found,

cellulase activity is also present. In other cases, hemicellulase

activity has not been sought. From the ecological point of view, this
Situation is perhaps not at all unexpected, since the natural substrates

of the cellulolytic microorganisms are nearly always intimate mixtures

01‘ cellulose and one or more hemicellulases.

The occurrence of discrete sources of enzyme preparations which

will act on several different but related substrates is of course rather

common. An example appropriate for the present discussion is the diges-

tive juice found in the crop of the snail, Helix pomatia. Holden and

TI‘a cey (1950) have compiled from the literature a list of 30 enzymes

reported to have been found in this single source. Twenty of these are

10

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12

carbom'drases. The list of substrates includes cellulose, lichenin, the
starches, glycogen, fructosans, arabans, xylans, mannans, mannogalactans,
chitin, polygalacturonic acid, several naturally occurring di- and tri-
saccharides, stachyose and d- and fi-glycosides. The question which
arises in this and in similar cases is whether the activities are pos-
sessed by a few enzymes of relatively low specificity, or by many en-
zymes of absolute or nearly absolute specificities.

There have been very few investigations specifically designed to
establish the identity or nonidentity of the cellulases and hemicellu—
lases. Grassman and co-workers (1932, 1933) found that crude commer-
cial enzyme preparations from A. m hydrolyzed cellulose and a
xylan, and had some action on a mannan. The latter activity was lost
on dialysis, and treatment with animal charcoal removed the xylanase,
leaving only the cellulase. They concluded that mannanase, xylanase,
and cellulase are separate specific enzymes.

In the absence of similar studies on different preparations, it is
perhaps unwise to extend Grassman's conclusions to other organisms.

The possibility of the identity of cellulase with fi-glucosidsse
has been mentioned by Veibel (1950) on the basis of experiments reported
by Helferich and Thieman (1911b). The latter found that glycol 3-D-
cel1<>b:loside anhydride was slowly twdrolyzed by the fi -g1ucosidase of
Sweet almond emulsin. T'ney admit, however, that this type of evidence
13 only suggestive.

According to Pigman and Goepp (19118), the fi -glucosidases and the
P°1y8accharidases should be considered separately, not necessarily on

the basis of differences in Specificity, but rather because of the great

13

quantitative differences in rates of the reactions that they catalyze.
The possibility of a close relationship or even identity was not im-

plicitly denied.
Jermn (1952, 1952a) has reported the results of an investigation

of the cellulose and fl -g1ucosidase activities of A. 21233 prepara-
tions. Filter paper electrophoresis revealed the presence of at least
eight separate protein components in the crude enzyme preparations.

The distribution of activity along the paper, against various substrates,
was determined by spraying each developed paper with one of the buffered
substrates, incubating, drying, and spraying again with a reducing sugar

reagent. The substrates used were salicin, aesculin, para-nitrophenyl-

fi-glucoside, and carboxymethylcellulose. At least seven of the eight
components were found to be active against both salicin and carboxy-
methylcellulose. The distribution of "aesculinase" activity was not
so well resolved, but again was spread the length of the paper. Only
with para-nitrophenyl- fl -glucoside was there but a single well defined
area of activityi.

Attempts by Jermyn to fractionate the crude material by solvent
and salt precipitation methods gave erratic results. In one experiment,
the enzyme active against carboxymetm'lcellulose remained in solution
"hen alcohol was added up to a concentration of forty percent. Addition

01' Belid ammonium nitrate to the alcoholic solution, held at a temperature

E
_.__

1

It should be mentioned that with this substrate, a sugar reagent
Spray was not used, since free para-nitrophenol is itself a colored com-
?O‘mde If this procedure is not as sensitive as the detection of reduc-
1mg sugars, it seems possible that other areas of hydrolysis might have

been found with the sugar reagent spray.

1b

of 10° 0., brought about an immediate precipitation of some of the
active material. Active protein continued to be precipitated with each
small increment of amonium nitrate up to about 85 g. per 100 m1. of
original solution} There were only slight differences between the
activities of the various fractions.

These results, along with others to be mentioned later, led Jermyn
to conclude "that in the 5. 21215 system the enzymes breaking down
monomeric (3 -glucosides do not differ qualitatively from those break-
ing down polymeric fl -glucosides", and that there is no need to assune
the existence of a specific cellulase to explain the hydrolysis of '
soluble cellulose derivatives. Jermm instead thinks that any estima-
tion of cellulase or H -glucosidase activity is the summation of the
separate activities of a whole battery of 3 -glucosidases, each with
its own ratio of activities against any one substrate.

Although obtaining evidence of similarly complex systems in almond
emulsin and Stachybotris £93 preparations (1953), Jermn is cautious
about applying the results generally.

It will be noted that nearly all the substrates listed in Table I
are cellulases modified to some degree from native cellulose. Most
investigators in this field have found it necessary, or at least de-
sirable, to modify cellulose from its native form in order to obtain
sufficient Mdrolysis for accurate measurement.

M

1 .

After the addition of each increment of salt, one hour was allowed
for completion of precipitation. The adequacy of this period of time
might be questioned.

15

SeilliEre (1906), after confirming earlier observations that cotton
and filter paper seem to be very resistant to attack by the snail enzyme,
found that cotton cellulose reprecipitated from solution in Schweizer's
reagent (cuprammonium hydroxide) was much more susceptible to hydrolysis.
The reaction did not go to completion even after the addition of more
enzyme. It was also found that swelling cotton with zinc chloride or
sodium hydroxide had somewhat the same effect (SeilliEre, 1910).

These Observations have since been repeatedly confirmed and extended.
Karrer and Illing (1925) found that by repeated treatment of reprecipi-
tated cotton cellulose with fresh snail enzyme, 96 percent hydrolysis
could finally be attained. Others have not been able to attain such an
extent of breakdown. There is always a resistant residue.

Karrer _e_t El' (1925a) reported that filter paper and mercerized
cotton are less resistant to enzymatic attack than native cotton, but
alkali celluloses are strongly attacked (Karrer and Schubert, 1926).

Pringsheim and Baur (1928) studied the differences in rate of
hydrolysis, by barley malt enzymes, of cotton cellulose swollen by
different means. Cellulose treated with calcium thiocyanate was most
rapidly attacked, lithium chloride was somewhat less effective, and
treatment with cold dilute sulfuric acid produced the least susceptible
substrate.

Syrupy phOSphoric acid has also been used successfully for the
Preparation of cellulosic substrates susceptible to enzymatic attack.
Walseth_(19h8) found that ground, dewaxed cotton linters were only about
five percent hydrolyzed by'Q, gigs: enzymes after six days incubation.

In the same time, an equal concentration of enzyme hydrolyzed a

16

phosphoric acid-treated sample of cotton linters to the extent of about
80 percent. This effect can be graded by control of the time of swell-
ing treatment.

Since all of the treatments listed above result in a shortening of
the cellulose chains, the simplest explanation for their effectiveness
in increasing the enzymatic susceptibility of native cellulose would be
that the rate of hydrolysis is a function of the degree of polymeriza-
tion. This idea has been proposed by Hajo (l9h2). However, the exten-
sive work of Karrer and his associates indicates that the degree of
polymerization is not as important as another variable prOperty, namely,
the degree of crystallinity. Karrer and.Schubert (1928) could find no
constant relation between the degree of polymerization, as determined
viscometrically on cuprammonium solutions of different celluloses, and
the rate of enzymatic hydrolysis byuflglix enzymes. Instead, a constant
inverse relationship between the amount of breakdown and the degree of
crystallinity was noted (Karrer and Schubert, 1926).

In further eXperiments, Karrer (1930) and Faust gt a}, (1928) com—
pared the action of snail cellulase on two series of viscose rayons,
prepared under identical conditions up to the spinning stage. One
series was set with tension, the other with no tension. The samples
Spun without tension were hydrolyzed more rapidly and to almost twice
the extent. X-ray diagrams of the samples revealed that tension applied
during the spinning process increased the orientation of the cellulose
micelles.

Karrer (1930) concluded that the effect of swelling and of regen-

eration treatments on the susceptibility of cellulose to the action of

1?

enzymes can be ascribed to an increase in eXposed surface due to a
change in the physical arrangement of the micelles.

More recently, Ialseth (19b8) has provided further good evidence
that the degree of crystallinity1 is the more important factor. Differ-
ent samples of phosphoric acid-swollen cotton linters which varied
markedly in their enzymatic susceptibility (g, 2353;), varied only
slightly in their degree of polymerization. In another series of ex-
periments, a sample of swollen cotton linters was about 28 percent
hydrolyzed by an a. gigs: preparation in 2b hours. ‘When the residual
cellulose was recovered, washed, and treated with fresh enzyme, very
little further breakdown was obtained. However, when it was treated
again with phosphoric acid, it was rapidly hydrolyzed by the enzyme
(about 50 percent in 2b hours). walseth also found a direct correlation
between the extent of hydrolysis in a given time and the equilibrium
moisture contents of various samples of swollen linters. The equilibrium
moisture content has been found to reflect the degree of accessibility
(Howsmon and Sisson, 195h).

Among the substrates listed in Table I are three soluble cellulose
derivatives: carboxymethylcellulose, cellulose sulfate, and hydroxy-
ethyl cellulose. Others which have been used are methylcellulose (Reese
§§_§l,, 1950; and Tracey, 1953) and ethylmethylcellulose (Tracey, 1953).

Soluble substrates have obvious advantages. Solutions of these

xHowsmon and Sisson (195h) favor the term "accessibility" rather
than the older term "degree of crystallinity", because the crystalline
and amorphous areas of cellulose fibers cannot be strictly delineated,
Whereas accessibility to any single reagent can be accurately measured.

\{1‘

b‘

18

derivatives form homogeneous solutions: consequently substrate concen-
tration becomes a more meaningful term, and the uncertainties of sub-
strate surface area and degree of crystallinity are of less concern.
The initial rate of hydrolysis of these substrates is much more rapid
because of the great increase in surface area in going from a solid to
a colloidally dispersed substrate.

As Tracey (1953) has pointed out, however technically advantageous
the use of soluble substituted celluloses may be, it should be kept in
mind that these substrates are not cellulose. To be satisfactory, any
substitute should faithfully reflect, by some known function, the sus—
ceptibility of the natural substrate. This is by no means true. of
carboxymethylcellulose, as has been shown by Reese 3.1. a_l_. (1950) and
Reese and Levinson (1952). The latter determined the activity of
several fungal metabolic filtrates toward ground cotton and carboxy-
methylcellulose. The ratio of these activities varied considerably,
the range being from about five to about forty. Tracey (1953) has
supplied confirmatory evidence of the same nature.

As with native or modified cellulose, the degree of polymerization
(DP) seems to have little effect on the rate or extent of enzymatic
hydrolysis of substituted cellulases. Reese at. a}: (1950) could demon-
strate no differences in the rate of hydrolysis of carboxymethylcellu—

lose with DP values of 125, 150, and 200 by Merginus fumigatus

1Jenny-n (1952) finds that solutions of carboxymethylcellulose of
more than about 0.5 percent are not homogeneous; centrifugation separ-
ates Part of the material as a gel-like mass. Furthermore, the rate
°f hydrolysis of these solutions by A. game cellulase is not appre-
Ciably influenced by changes in concentration.

 

19

culture filtrates. However, as pointed out by the authors, this range
is rather narrow. V

Siu (19h9) and Greathouse (1950) have shown that the nature of the
substituent in substituted cellulases is apparently of only secondary
importance in determining the resistance to attack by live microorganisms.
'lhat is more important, they found, is the degree of substitution (DS)
i.e., the average number of substituent groups per anhydroglucose residue.

Reese, Siu, and Levinson (1950), using four samples of carboxymethyl-
cellulose of about the same DP (150), but varying in BS from 0.52 to 1.2,
found that the resistance of these substrates to attack by active meta-
bolic filtrates from cultures of several fungi is an inverse function of
the DS. The sample of US 0.52 was about eight percent hydrolyzed in two
hours (as indicated by the appearance of reducing sugar); the sample of
US 1.0, about 3.5 percent; whereas the sample of DS 1.2 was not hydrolyzed
to any measureable extent in this time. They concluded that no enzymatic
hydrolysis is possible if each anhydroglucose unit of the polymer bears
a single substituent, i.e., if the D6 is unity. The small amount of
hydrolysis Observed with the sample of US 1.0 was probably due to a
nonhomogeneity of substitution.

Holden and Tracey (1950), using a somewhat different approach and
snail instead of fungal enzymes, Obtained evidence which suggests that
both members of a pair of anhydroglucose residues in carboxymethylcellu-
lose of'DS 0.h5 must be unsubstituted to render the linkage between
them suwceptible to hydrolysis. 'With ethylmethylcellulose (D5 1.2),
°nIY'one of’a pair of contiguous residues need be unsubstituted. These

conclusions were based on the limited extent of hydrolysis achieved by

2O

adding fresh enzyme to the substrate until no more increase in reducing
sugars could be detected. The limiting extent of kwdrolysis (ho percent
for the carboxymetlwlcellulose and 17 percent for ethylmethvlcellulose)
was then compared with calculated estimates of the frequency of occurr-
ence of two consecutive or of a single unsubstituted residue in the two
substrates. Calculations were based on Spurlin's (1939) statistical
treatment of the arrangement of substituents in partially substituted
cellulose derivatives, which takes into account the degree of substitu-
tion and the relative reactivities of the primary and secondary hydroxyl
groups of cellulose.

In considering the early and most of the recent work on cellulases
and hemicellulases, the nature of the active preparations should be kept
in mind. Most of the enzyme solutions used have been crude, unfraction-
ated extracts or culture filtrates. Another possible source of confusion
is the multiplicity of sources used. In the absence of a pure cellulase
from more than one source, it is difficult to make comparisons between
enzymes from different sources. Part of the seemingly contradictory
evidence concerning the end products of cellulose breakdown by cellulase
is due to this circumstance.

One of the end products of the action of crude cellulase prepara-
tions on cellulose and its derivatives has been shown, beyond a doubt,
to be D-glucose. This has been shown for preparations from plant tissue
(Pringsheim and Baur, 1928), snail enzyme (Seilliere, 1910; and Karrer
and Illing, 1925), protozoa (Trager, 1932; and Hungate, 19142), bacteria
(Simola, 1931) and fungi (Saunders st 31., 19148; Whitaker, 1953; and

Whistler and Smart, 1953) by the isolation of D-glucose as the osazone

21

from the hydrolysates. With the snail enzyme, Karrer and Illing (1925)
found that the conversion to glucose was almost complete. Walseth (19148),
using 1_\._. n_i_g_e_1_‘_ preparations, was also able to account quantitatively for
the loss in weight of swollen cotton linters by the appearance of glucose
alone.

In contrast to these findings, Pringsheim (1912) was able to demon-
strate the accmulation of cellobiose as well as glucose in cultures of
anaerobic cellulolytic bacteria after growth was arrested by addition of
such metabolic poisons as toluene, chloroform, or iodoform. He also
found that by heating the arrested cultures to about 70° 0., cellobiose
accumulated to an even greater extent. These findings were interpreted
to mean that cellobiose is the end product of the action of cellulase
and that glucose is liberated from the disaccharide by a second enzyme,
cellobiase.

This has been the prevailing theory until very recently, in Spite
of the fact that later workers (until recently) were unable to detect
cellobiose in other systems. This failure was attributed to the pre-
sence of a very active cellobiase, an assumption which gained credence
with the work of Grassman e_t 91. (1932, 1933), who were able to separate,
at least partially, the cellobiase from the cellulase of fungal prepara-
tions by adsorption of the former on aluminum metahydroxide at pH 3.5.
The cellobiase was eluted with 0.021 molar sodium bicarbonate. The two
fractions thus obtained were tested for their activity against cello—
dextrin and the fi-oligosaccharides of two, three, four, and six
residueS. The cellobiase fraction readily hydrolyzed not only cello-

biose but also the other oligosaccharides as well; however, it had

22

little effect on the cellodextrin. The cellulase fraction had no effect
on celldbiose (in 2b hours), and hydrolyzed cellotriose and cellotetraose
only to the slightest extent. It attacked cellohexaose to a certain
extent and cellodextrin fairly rapidly. Thus, there was some overlapping
of the domains of each enzyme at the cellohexaose level.

More recently, Whistler and Smart (1953) provided the first unequi-
vocal proof of the formation of cellobiose as an intermediate in the
enzymatic degradation of cellulose. They pointed out that the cello-
biose found by Pringsheim in arrested bacterial cultures might be a
metabolic product rather than an intermediate or end product. ‘Hhistler
and Smart were able to detect only D-glucose in the hydrolysates of
swollen cellulose produced by (l) the crude extract of g, niggg, (2)
the preparations obtained from ammonium sulfate or acetone fractiona-
tions, (3) the crude extract held for various periods at elevated
temperatures or in strong acid or alkali, or (b) the fractions from
calumns of ion—exchange resins, charcoal, wheat starch, Kaolin-Supercel,
bauxite, or aluminum hydroxide. Finally, a fraction was obtained by
sorption of the crude extract at pH 3 on a powdered cellulose column
and elution with a pH 9 borate buffer which hydrolyzed swollen cellue
lose to D-glucose and cellobiose in detectable amounts. The latter
was isolated both by paper and column chromatography and identified
by its melting point, X-ray diffraction pattern, and by the formation
of its phenylosazone. Apparently the cellobiase was not entirely eluted
from the cellulose powder, thus allowing cellobiose to accumulate in
the hydrolysates. A search for other oligosaccharide intermediates and
phosphorylated sugars, using paper chromatographic techniques, produced

negative results.

23

Reese, Siu, and Levinson (1950), using metabolic culture filtrates
from several different fungi, were unable to detect any cellobiase acti-
vity. Although no Specific search for cellobiose in the hydrolysates of
ground cotton cellulose was made, these workers postulated that glucose
can be liberated directly from the cellulose chains without the inter-
mediation of a cellobiase.

In a subsequent publication, Levinson, Mandela, and Reese (1951)
reversed their stand and reported, after using enzymatic techniques for
the simultaneous and specific determination of both glucose and cellobiose,
that cellobiose appeared in the hydrolysates before glucose could be de-
tected. 'With the culture filtrates of some fungi, the amounts of cello-
biose produced exceeded the amounts of glucose. The cellobiose was
hydrolyzed very slowly, which indicated that there is little, if any,

fl -glucosidase in these filtrates. The results were qualitatively
confirmed by paper chromatography. No other intermediates were detected
when powdered cellulose and alkali cellulose were hydrolyzed, but at
least two Spots other than those representing glucose and cellobiose
‘were noted when cellulose sulfate was used. These unidentified products
'were probably sulfate-substituted glucose or oligosaccharides, since
they did not appear in the hydrolysates of unsubstituted cellulose.
Furthermore, incubation of the m'drolysates with fl-glucosidase before
chromatography did not eliminate these spots.

0n the basis of the results just described, Levinson gt_al, (1951)
hypothesized that the principal final product of the enzyme which Splits

the F—l,h linkages of cellulose is cellobiose.

2h

As mentioned previously, it has been found by Reese gt El! (1950)
that all the cellulolytic fungi tested could utilize both native cellu-
lose and carboxymethylcellulose (of low US), and that culture filtrates
from these organisms could also attack these substrates. UneXpectedly,
it was also found that certain noncellulolytic fungi, while not having

the ability to utilize solid cellulose, did grow when carboxymethyl-

'7’“?

cellulose was used as the substrate. Their culture filtrates reflected

 

these characteristics. These results led the authors to suggest that
all cellulolytic organisms possess two enzymes. One of these, which
they designated C1, and which Siu (1951) later suggested should be
called cellulase, supposedly brings about some change in native cellu-
lose making it amenable to hydrolysis of the fi-l, h linkages by the
second enzyme, Ox. 0;, according to Reese gt 31., occurs in all cellu-
lolytic and in many noncellulolytic fungi as well. The precise action
of 01 was not stated, but the authors speculated about the possibility
that it splits the nonglycosidic cross-linkages postulated to be present
in native cellulose. Siu (1951) even suggested that the action of C1
may be that of a "hydrogenbondase" which would serve to separate the
chains in the relatively more crystalline areas of the native fibers.
In any event, the result of its action is supposedly the production of
straight polyanhydroglucose chains available to the Cx enzyme.

The extreme variations in the ratio of activity on cellulose sul-
fate or carboxymethylcellulose and solid cellulose substrates from one
enzyme source to another (Reese and Levinson, 1953; and Tracey, 1953)
can be explained in terms of this hypothesis. It is only necessary to

assume differences in the proportions of C1 to Cx in different fungi.

 

25

The validity of Reese's multiple enzyme hypothesis is difficult to
assess in the absence of pure enzyme preparations. Final proof or dis-
proof of its general validity can only await the isolation of one or
both of the enzymes in a high state of purity from several sources.

'lhitaker (1953) has succeeded in isolating an active cellulase
from culture filtrates of Myrothecium verrucaria, apparently in a high

state of purity. The preparation appears to be homogeneous at pH levels

" 4—.~_>‘7:1

above, near and below the isoelectric point, and shows only one peak in
the ultracentrifuge at pH 5.03. The procedure used for the isolation
involved concentrating the culture filtrates by vacuum evaporation and
by slow partial freezing, followed by precipitation of the proteins by
saturation with ammonium sulfate at l0 C. The Specific activity (units
of cellulase activity per unit of protein) at this stage was little
changed.from the original culture filtrate, but some undesirable im-
purities of unspecified nature were removed at 30 percent saturation
‘with ammonium sulfate. The next step, fractionation with alcohol at
low temperature, low ionic strength, and pH b.95, yielded a fraction
at 25 percent alcohol which contained about two-thirds of the protein
and had a somewhat higher Specific activity; The final step was a
fractional precipitation with polymethacrylic acid at pH b.35. The
‘protein liberated from the complex by addition of barium chloride had
a specific activity about 2.25 timesias great as the starting material.
The ammonium sulfate precipitates were analyzed electrophoretically
land.found.to contain five components, two major slowbmoving peaks and
three minor ones migrating at successively faster rates. These runs

‘were made in phoSphate buffer of pH 6.8 with an ionic strength of 0.2.

26

Protein concentrations for the electrophoretic analyses had to be kept
low because of the dark color of the material.

Whitaker, Calvin, and Cook (l95h) determined the diffusion constant
(5.61 x 10'"7 at 20° C. in water) and the sedimentation constant (3.72 x

- O
13 at 20 c. in water) of the purified cellulase. In the diffusion

10
measurements, no evidence of deviation from a Gaussian distribution was pa
found, another indication of the homogeneity of the preparation. From
these values and an assumed partial specific volume of 0.7h ml. per gram,
the molecular weight was calculated to be 63,000t1600.

During the purification procedure, the enrichment in Specific
activities against all substrates (phosphoric acid swollen cotton lin—
ters, reprecipitated cellulose, unswollen linters, carboxymethylcellu-
lose, and cellobiose) was approximately the same. Although this find-
ing, coupled with the demonstrated homogeneity of the final product, is
considered by lhitaker to suggest a unienzymatic mechanism of cellulose
hydrolysis, he suggested that it is not necessarily at variance with the
work of Reese. According to Ihitaker (1953), Reese's findings can be
interpreted as an indication that the cellulase of the cellulolytic
fungi has the capacity to form an effective complex with all types of
cellulose, whereas the enzyme of the noncellulolytic fungi loses its
ability to hydrolyze the ,6 -l,14 linkages when it is adsorbed on a
solid substrate.

The end products of cellulose hydrolysis by the purified cellulase
were shown to be glucose and cellobiose in about equimolecular amounts.
The enzyme has so low a cellobiase activity that the amount of glucose
produced could not originate from cellobiose. Ihitaker (1953) concluded

that the appearance of glucose is not dependent on formation via cellobiose.

27

Whitaker (1952) found that the presence of any one of several pro-
teins in the cellulase assay medium has a decided stimulatony effect on
the action of partially purified cellulase preparations on insoluble
substrates. The effect was not so marked when carboxymethylcellulose
was used as the substrate. ‘Bvidence was presented showing that the
effect is not one of neutralization of an inhibitor nor of protection
of the enzyme against denaturation.

Basu and Whitaker (1953) found that at low pH levels, basic dyes
and neutral salts are also stimulatory, whereas acid dyes are inhibitory.
At higher pH levels, the reverse effect was found. It was suggested
that the effect of proteins and dyes may be due to their effect on the
surface potential of the solid cellulose substrate.

Whitaker's work seems to indicate that the cellulolytic activity
of Myrotheciumlggrrucaria filtrates is that of a single protein come
ponent. These results are in distinct contrast to those of two other
investigators.

Jermyn's (1952) findings indicating the presence of perhaps seven
different components in 5, 252333 preparations active against carboxy-
methylcellulose have been mentioned previously. Reese and Gilligan
(1953) also obtained evidence suggestive of a multiple nature of their
Cx enzyme. Their procedure was somewhat like that of Jermyn. They
used chromatographic separation of the components on zein-treated paper,
rather than paper electrophoresis, and determined the Cx activity dis-
tribution along the length of the developed strip by cutting it into
short sections and incubating these sections with carboxymethylcellur

lose in buffered solution. Plotting the activity contained in the cut

_ v "--M~\.fi

28

sections against their location on the original chromatograms gave
curves showing at least two maxima for seven of the eight culture fil-

trates tested. The exception, a culture filtrate from Tgichoderma viride,

 

contained only one active component. ’Ihe curve for g. verrucaria had two
different maxima, a slow-moving component barely migrating from the start-
ing point and a faster moving component migrating somewhat less than half
the distance of the solvent front. With other culture filtrates, the
fast-moving component almost kept pace with the solvent front. In all
cases but one, every section of the developed strip had some activity,

and the curves did not reach the base line (zero activity) at any point.

Further evidence that the maxima on the curves represented distinct
protein Species was afforded by successful attempts to separate them by
other means. The single, slow-moving component of I. 3.13.329. could be
almost completely adsorbed on cellulose (Solka Floc). Only the slow
component, representing about 30 percent of the CI activity of g. m-
ga_r_i_a_ filtrates was adsorbed. The other component could be removed by
adsorption on kaolin, leaving the slower one in solution. The two
enzymes thus must have distinct pm'sical prOperties. Furthermore, with
!. verrucaria, the relative amount of the fast-moving component can be
influenced by changes in the conditions under which the organism is
cultured.

The question of whether the. action of cellulase on the cellulose
chain is an endwise attack or proceeds in a random manner has received
some attention. The most convincing evidence in favor of the random
cleavage mechanism would be the isolation of an oligosaccharide inter-

mediate in enzymatic hydrolysates of cellulose. On the other hand,

29

failure to find any such intermediates, in Spite of concerted efforts to
do so (Whistler and Smart, 1953; Levinson, Mandels, and Reese, 1951) does

not constitute a repudiation of the random cleavage hypothesis, in the

light of a recent report by Whitaker (1951;). Using his purified g.

verrucaria cellulase, Whitaker found that cellotetraose, cellopentaose,

and cellohexaose are very rapidly hydrolyzed. Cellobiose is very slowly ,

attacked, and cellotriose occupies an intermediate level of suscepti-

.l.
bility. All possible hydrolysis products of each substrate were deter-

mined at short intervals. The results suggest that with cellotetraose

the enzyme shows a preference for splitting the central rather than the
terminal linkages, but with the higher members of the series, there is
a tendency toward increasing randomness of chain-Splitting with increas-

ing chain length.
Whitaker suggested that the detection of the higher soluble oligo-

saccharide intermediates is likely to be very difficult, since their
rate of hydrolysis greatly exceeds the rate at which they could be
fGranted from an insoluble. cellulose substrate.

At the same time that the above paper appeared, Hash and King (1951;)
Published a short account of the demonstration of an oligosaccharide
intermediate in the hydrolysis of cellulose by g. verrucaria cellulase.
The 1r technique was to remove possible diffusible intermediates from

ct>ntact with the enzyme by carrying out the reaction in a collodion sac

\

1Turnover numbers (moles of substrate degraded in one minute by one
$1019 of enzyme) were: cellobiose 5-6, cellotriose 50-200 (depending on

rVitial concentration of substrate), cellotetraose 1:00, and both cello-
p ehtaose and cellohexaose, at least 1450.

30

suspended in a large volume of buffer. After a suitable incubation
period the outer solution was concentrated and chromatographed on paper.
Besides spots corresponding to cellobiose and glucose, a third Spot
with a lower Rf value was noted. A small amount of the material iso-
lated from.this area was tentatively identified as cellotetraose by
estimating its molecular weight through comparison of its reducing
power with that of cellobiose and determining total glucose in the
molecule by the anthrone method. That the oligosaccharide is an inter-
mediate and not an end product was indicated by its rapid hydrolysis to

cellobiose and glucose on incubation with the !, verrucaria enzyme.

 

Other evidence supporting the hypothesis of random attack on
cellulose by this enzyme is the finding that samples of cellulose
differing only in degree of polymerization are hydrolyzed at about
the same rate by fungal cellulase (Halseth, l9h8, 1952). If the mechan-
ism were primarily an endwise process, the rate of hydrolysis would
logically be eXpected to increase with a decrease in the degree of
polymerization.

With the soluble cellulose derivatives, it has been found that the
viscosity of the solutions being hydrolyzed by snail or plant cellu-
lases falls appreciably before a significant increase in reducing end-
groups occurs (Holden and Tracey, 1950). Similar results were obtained
by Levinson and Reese (1950), using fungal cellulases. These findings
can be interpreted in favor of an extension of the random cleavage
mechanism to the soluble derivatives.

Other investigators, notably Clayson (l9h3), have eXpressed the

belief that the process necessarily proceeds endwise. Kitts and

31

Underkofler (195k), the first to extract a cell-free cellulase from
rumen bacteria, were unable to detect any products of cellulose degra-
dation by this crude preparation other than glucose and cellobiose, and
believed that this tends to confirm Clayson's hypothesis of endwise
attack.

It seems to be generally assumed that the enzymatic breakdown of
cellulose is a hydrolytic process. However, according to Siu (1951),
this question is not yet settled. Simola (1931) reported that the
cellulolytic activity of a cell-free bacterial preparation was increased
almost three times over that of the controls by 0.033 n phosphate.
Greater concentrations were inhibitory. Norkrans (1950) reported that
the stimulatory effect of phosphate on Tricholomawgudum enzymes occurred
only in the presence of calcium. Lane and Greenfield (1953) found that
the cell-free cellulase system obtained from the gut of the mollusc
.Iggggg_pedicellata is stimulated by the presence of adenosinetriphos-
phate, the production of reducing sugar being increased ten times.
Nevertheless, no phosphorylated sugars have been found in enzymatic
hydrolysates of cellulose (Whistler and Smart, 1953).

Table I includes a column giving the pH optimum for the particular
system and conditions used. It can be seen that there are considerable
differences between the optima for different enzyme sources and even
with the same source as determined by different investigators. In his
study of the stimulating effect of inactive proteins on the action of

£5 gerrucaria cellulase, lhitaker (1952) found that the added protein

 

also caused a relatively great shift in the optimum.pH. He also found

that the shift depends on the particular substrate used. This may have

32

some general bearing on the lack of agreement noted in Table I. The
possibility of the existence of more than one cellulase in a given
source (Jermyn, and Reese and Gilligan, 1i_d_e m) might also explain
some of these differences. This might also be pertinent to the obser-
vation that most of the pH reSponse curves seen in the literature are
rather broad and flat. In contrast to the latter curves, showing a

single optimum (or optimal range), Freudenberg and Ploetz (1939) found

._._ ._ ul’

three distinct pH optima (MI, 6.1, and 6.9) for a commercial ft. m:
preparation.

The kinetics of the enzymatic degradation of insoluble celluloses
have been studied in a few instances. Most investigators agree that
the experimental results are difficult to interpret because of the in-
homogeneity of the substrate (areas of different accessibility) and the
impurity of the enzyme solutions. Karrer and Illing (1925) found that
With viscose rayon and snail cellulase preparations, the reaction at
first follows a monomolecular course, changing later to coincide with
the Shutz rulel. Walseth (19118) obtained similar results with swollen
cellulose and 11. gigs}; enzymes. This type of rate curve, a relatively
rapid primary phase followed by a much slower secondary phase, with a
rapid transition between the two phases, can be interpreted to be the
result of the nonhomogeneity of the substrate, the rapid part repre—
senting the hydrolysis of the more accessible regions of the substrate.

With both carboxymethylcellulose and cellodextrin, a linear rela-

tionship between concentration of enzyme (5. mae) and the initial

K

1
The quantity of substrate changed is proportional to the square
root of the reaction time.

1

rate of reaction was found by Jermyn (1952). When the enzyme concentra-
tion was held constant and the substrate concentrations varied, Lineweaver-
Burk plots of the data yielded straight lines for both substrates. The
Michealis constants obtained from these data were 3.2 and h.h x 10'”3 for

cellodextrin and carboxymethylcellulose, reSpectively.

"E

Norman's (1937) definition of the hemicellulases is the one employed

in the present work. Since this definition includes the hexosans, pento-

.._. ._4-

To

sane and hexo~pentosans, as well as the polyuronide hemicelluloses, any
enzyme hydrolyzing any linkage of these substrates might be called a
hemicellulase. This is perhaps unfortunate, since the term is not des-
criptive of the specificity which may be involved. 0n the other hand,
until the Specificity relationships for a member of the group are
thoroughly worked out, a specific name is not in order.

The Specificity requirements of the hemicellulases have not been
rigidly defined in any case, since none has been isolated in a condition
pure enough to warrant such a project. As mentioned before, Grassman
$2.21! (1933) presented evidence which seems to indicate that the xylanase
and mannanase activities of an a, gryggg_preparation are due to separate
enzymes, and that both are different from cellulase.

The early observations of Bierry and Giaja (191?) afford further
evidence that the hemicellulases are a group of enzymes with different
Specificities. These workers found that although the digestive juice
of flsliz’pomatia hydrolyzes the mannogalactans of alfalfa seed and
fenugreek and the mannans of the date palm and the ivory nut, that of
certain crabs and lobsters attack only the ivory nut mannan. Action of
the digestive secretion of the crayfish on the mannogalactans produced

much more galactose than mannose.

314

Such observations as the latter are not surprising, considering the
wide variety of sugars and types of linkage to be found among the hemi-
celluloses. In the absence of precise knowledge of the composition and
structure of a hemicellulose substrate, end products of the reaction
should be at least qualitatively identified in order to give signifi-
cance to the results. g-

Pringsheim and Genin (l92h), using a two-months old malt extract

a.‘ ._ a“- as
.b

as the source of hemicellulase, observed an almost quantitative cleavage
of salep mannan to mannose. When a six-months old malt extract was used,
the reducing power of the final hydrolysate was only one-half as great.
Mannobiose was identified by means of its phenylhydrazone. The authors
suggested that there are two enzymes involved, a fairly stable mannanase
and a less stable mannobiase. This observation is of particular interest
since it is entirely analogous to the mechanism of cellulase action post-
ulated by Pringsheim (1912), and to the action of fi-amylase on starch.
Xylose has been repeatedly identified as the main end product of
hydrolysis of various xylans by snail (Ehrenstein, 1926; and V055 and
Butter, 1938), malt (Luers and Volkamer, 1928), and fungal (Grassman
53 31,, 1932; and Yundt, 19h?) preparations. To the author's knowledge,
xylobiose or higher oligosaccharides of xylose alone have never been
reported. Hydrolysis of the xylans seems never to go to completion,
even with the addition of fresh enzyme solution. The limiting extent
of hydrolysis varies not only with the xylan, as might be eXpected, but
also with the enzyme (Fhrenstein, 1926) and with the method of diaper-

sion (Yundt, 19b9).

35

The nature of the resistant residues has been investigated in a
few cases. O'Dwyer (1937, 1939, l9h0) found that the portion of several
oak hemicellulases resistant to at 251333 enzymes had a composition
corresponding to six xylose residues to one of a methylhexuronic acid.

The linkages involved were not determined.

1

Some pH optima for various hemicellulase preparations acting on

various hemicelluloses are given in Table II. It can be seen that the

#1

range reported (b.65 to 5.28) is much narrower than for the cellulases.
Fhrenstein (1926) found that the nature of the buffer affected the pH
optimum of snail xylanase. In citrate buffer it was 11.65, in phosphate,
5.28. No further reference to this phenomenon could be found in the
literature.

There have been few studies of the kinetics of hemicellulase action.
Fhrenstein (1926), using a wheat straw xylan and snail enzyme, found
that the reaction followed neither a monomolecular course nor Shutz's
rule. V085 and Butter (1938) reported that the hydrolysis of xylans
by both snail and if 251332 enzymes did proceed according to Shutz's

rule over an intermediate range.

Statement 2f the Problem

As mentioned previously, Veibel (1950) has discussed very briefly,
and left open, the question of the identity of p -glucosidase and
cellulase. Although Pieman and Goepp (l9b8) separate the two activities
completely, at least for purposes of discussion, the question seems
never to have been Specifically answered one way or the other. Whitaker's

purified cellulase would seem to present a good opportunity to investigate

36

this problem, but up to the present time, no investigations pertinent
to the question, other than the demonstration of a relatively slow rate
of attack on cellobiose 13 the purified cellulase, have appeared.

Jermyn's work, suggesting the identity of not one but several
fla-glucosidase and cellulase components in g. oryzae preparations,
should stimulate further research along these lines. His results,
along with those of heese and Gilligan, have emphasized the complexity
of the problem.

If the possibility is conceded that cellulase and /9 -g1ucosidase
activities can be possessed by a single enzyme, at least in some cases,
a discussion of the known Specificity requirements of the )9 -glucosi-
dases would be pertinent. Veibel (1950) has recently reviewed this
subject, with Special emphasis on the fi -glucosidase of sweet almond
emulsin..

The investigations of several authors, references to whose work
will be found in Veibel's review, may be summarized briefly as follows.
The Specificity with regard to carbon atoms 1, 2, and 3 seems to be
absolute. In other words, the substrate must have the fia-configura-
tion, and substitution or epimerization of carbons 2 and 3 (to a con-
figuration Opposite to that of D—glucose) gives a product which is
resistant to attack by 3 -glucosidase.

With regard to the rest of the molecule, the specificity is more
relative. For example, (3 -methylmaltoside (which may be regarded as
a fi-methylglucoside substituted in the ’4 position with an d.-g1uco-
pyranose unit) is readily hydrolyzed to methanol and maltose by‘ l3-

glucosidase. This is of interest to the present discussion since the

37

constituent residues of cellulose and of many of the residues in the
hemicelluloses are linked at the b position.

At carbon atom S, the only change which seems to be tolerated is
substitution of the entire -CH20H group by hydrogen, yielding a A3-D-
xyloside. It must be pointed out, however, that this type of glycoside
is hydrolyzed by' fi3—glucosidase of almond emulsin at a much slower rate
than are the fi-glucosides.

Certain substitutions at carbon atom 6 may be made without complete
loss of hydrolyzability. The volume and the relative electrical charge
of the substituent appear to be the important factors. A positively
charged group is inhibitory.

The aglycon Specificity requirements of the a -glucosidase of
almond emulsin have received.a great deal of attention. It is sufficient
for the present discussion to repeat Veibel's statement that no case of
real absolute Specificity caused by the aglycon has been reported, and
also to point out that this generality extends to various disaccharides
such as cellobiose, gentiobiose, celtrobiose, and phenyl- aL-cellobioside.

None of these specificity requirements rules out cellulose and some
(3 -glycosidic linkages of many hemicelluloses as suitable substrates
for 3 ~glucosidase. The work of Grassman it; ill. showing that the cello-
biase (a fi-glucosidase) and cellulase activities of 1;. m prepara-
tions can be partially separated suggests that cellulase and [3 -gluco-
sidase are not identical. Whitaker's demonstration of the relatively

weak cellObiase activity of his purified g. verrucaria cellulase may

 

also be cited. In partial answer, it can be pointed out that the A3.-

glucosidases of different origin are known to differ considerably in

38

their Spectrum of relative specificities toward various substrates
(Veibel, 1950). Furthermore, Jermyn's results and those of Reese and
Gilligan demonstrating the multiplicity of the £3 —glucosidase and
cellulase activities of fungal preparations leave the question still
open, in the opinion of the author.

If the Specificity requirements of cellulase resemble those of
’3 -glucosidase, cellulase would be eXpected to have some action on
certain hemicelluloses, particularly those containing 3 -D—xylosidic
linkages. The nature of the aglycon side of the molecule and the
position and nature of the substituent would be eXpected to have some
influence on the rate of the process. Thus, there occurs the question
of whether it is necessary to invoke a Special hemicellulase (or
xylanase) in those cases in which both cellulase and hemicellulase
(xylanase) activities are known to occur. As far as the author is
aware, this question has been Specifically investigated only once:
Grassman §t_al. were able to remove the xylanase activity of a fungal
preparation by treatment with animal charcoal. The cellulase activity
remained in solution. On the basis of this single observation, the
question cannot be considered settled.

at the beginning of the investigation to be described in the re-
maining pages, the object was to isolate, purify, and characterize the
extracellular cellulase of Myrothecium verrucaria. However, after the
appearance of Whitaker's work on the purification of the same cellulase,
and of Jermyn's and Reese and Gilligan's demonstrations of the possible
multiple nature of the activity, a different approach was thought to be

possibly more fruitful. The final plan was to isolate from the original

 

tw_ 7

39

multicomponent culture filtrate of g, verrucaria a series of fractions
containing different ratios of the various protein components and to
determine, if possible, which of the components is active against
various monomeric and polymeric fl -glycosidic substrates. Greatest
emphasis was placed on the relationships among cellulase, hemicellulase,
and fi -glucosidase, with a view toward establishing their identity or

nonidentity.

EL liRD-TENTAL

hl

EXPERIMENTAL

The course taken for the investigation of the problem stated pre-
viously may be outlined briefly as follows. Culture filtrates of
gyrothecium verrucaria grown in submerged culture were initially con-
centrated by slow partial freezing. Further concentration of the pro-
teins was accomplished by precipitation with acetone or with ammonium
sulfate. Aqueous extracts of these precipitates served as the starting
materials for fractionation of the protein components. Attempts to
fractionate the concentrates by salt and solvent precipitation and by
chromatography on cellulose columns were generally unsuccessful, but
electrophoresis-convection finally yielded preparations containing
different ratios of the protein components. Fractionations were con-
trolled by estimation of enzymatic activity and determination of pro-
tein nitrogen as well as by electrophoretic analysis. Cellulose, two
different impure hemicelluloses, cellulose sodium sulfate, and salicin
were used as substrates to study and compare the enzymatic properties

of the final preparations.

Materials and Methods

Production of Culture Filtrates

1
The fungus gyrothecigm verrucaria was chosen as the source of
enzyme material because, according to Siu (1951), it is one of the

strongest cellulose decomposers known. Its culture filtrates reflect

 

1
Obtained from the American Type Culture Collection, Washington, D. C.

1:2

this property (Saunders 3t_al. 19h8). Moreover it is not pathogenic,
as are some of the aspergillus species.

Filtrates were obtained from submerged cultures of the organism
grown in the medium devised by Saunders gt 3.1: (19118). This culture
medium contained only mineral salts and cellulose as the sole source

of carbon. The mineral portion of the medium is given in Table III.

TABLE III

COMPOSITION OF THE CULTURF MEDIUM USVD FOR E3 VVRRUCARIA

 

 

Final Final

 

Salt*' concentration Salt concentration

g./l. ug./l.
NaanPo, 1.5 01130, '5H30 2.5
NaH2P0"1-I,O 2 .0 Fe,($0‘)3 M .0
Memo, 3.75 H330, 57.0
Karim. 0.15 21130, 071120 50.0
101,90, 0.20 MnSO‘ 5.5
3:11.110, 0.60 (NH.),M00,** 120.0
ugso, -7H,0 0 .30

 

*

All the chemicals used throughout this investigation
were reagent (or C.P.) grade unless Specifically stated to
be otherwise.

as
Ammonium molybdate was substituted for ammonium phos-
phomolybdate on the basis of its molybdenum content.

h3

Cellulose was supplied at the rate of 20.0 g. for each liter of
culture medium. Solka-Floc1 was used in all but two cultures, in which
it was replaced by Whatman ashless powdered cellulose (medium grade).

The medium was prepared by placing 2h0 g. of cellulose and seven
liters of the mineral salts solution in the incubation vessel illus—
trated in Figure 1. With the t0p assembly in place and all openings
covered with cotton caps, this vessel and five more liters of mineral
salt solution distributed among four two-liter Erlenmeyer flasks were
sterilized in the autoclave at fifteen pounds pressure (1200 C.) for
as minutes. The remainder of the salt solution, while still hot, was
introduced into the incubation vessel by means of suction. Care was
taken during these manipulations to maintain the sterility of the
culture medium.

The inoculum, consisting of a suSpension of ungerminated Spores,
was prepared by growing the fungus on filter paper disks placed on
mineral salts agar2 in Petri dishes. The filter paper disks, cut to
cover about 80 percent of the area of the agar slab, were moistened
with a small amount of the salt solution and sterilized by autoclaving

in a Petri dish. One disk was placed on each solidified agar slab and

 

1Solka-Floc is a wood cellulose product of The Brown Company.

2In accordance with instructions from the American Type Culture
Collection, the composition of this medium was: dibasic potassium
phOSphate, 0.5 g., sodium nitrate, 2.0 g., magnesium sulfate, 0.2 g.,
agar, 17.0 g., and water, one liter. As a precaution against bacterial
contamination, penicillin and streptomycin were added at the rate of
25 units per ml. The antibiotics were dissolved in a small amount of
water, sterilized by filtration, and introduced after the agar had
been autoclaved (1200 0., 30 min.).

 

 

Figure 1. Incubation vessel for the submerged culture of
!. verrucaria.

liberally streaked with spores from a previous culture. Best results
were obtained at room temperatures, under which conditions noticeable
vegetative growth first became visible after three or four days. Sporu-
lation started in about a week and at the end of two weeks nearly the
entire surface of the filter paper disk was covered with the greenish-
black Spores.

The inoculum was obtained by flooding the surfaces of two or three
of the Petri dishes which contained the Sporulated cultures with about
five ml. of sterile salt solution (composition as in Table III) and
scraping the spores into a suspension with a wire loop. Individual
suspensions were transferred first to a sterile test tube and then,
after mixing, to the cooled culture medium in the carboy. Enough of
the spore-suspension was saved to make a spore count.

Spore counts were made on an appropriate dilution of the suSpension
with the aid of a Spencer bright line counting chamber and a microscope.
Although the Spores are small, staining was not necessary because of
their density and dark color. Serious clumping of the Spores was never
encountered so the distribution in the counting chamber was satisfactory.

In some cases, a solution of 300,000 units of penicillin and
300,000 units of streptomycin, sterilized by filtration, was added to
the large culture to prevent bacterial contamination.

Incubation of the large culture was maintained at room temperature
with continuous aeration and stirring. Aeration was provided by means
of a water aspirator. Air entering the system was washed with water

and filtered through a tightly packed sterile cotton column and then

115

introduced.below the surface of the culture (see Figure 1). The air
flow rate was not measured, but was kept rapid at all times.

Vigorous, continuous stirring was provided by means of a stainless
steel rod pivoted in the rubber stopper and connected at the top to a
”rote-stir unit" driven by'a heavy duty motor (See Figure 1). The stirr-
ing rod was fitted very tightly in the rubber stopper, but the entire
thickness of the stopper could not be used as a pivot. Instead, wells
several times the diameter of the rod were sunk from both surfaces of
the stOpper. A hole somewhat smaller than the rod was then cut through

the remaining portion. Autoclaving tended to seal the rubber tightly

to the rod.

In order to follow the increase of cellulase activity, small samples
of the culture were asceptically removed at two or three-day intervals
and assayed, using cellulose sodium sulfate as the substrate. The in-
cubation was terminated when the activity level had reached a plateau,
or was increasing very slowly. The time of incubation varied between
two and three weeks.

Fifteen such cultures were prepared, but two were discarded because
of gross bacterial contamination. Contamination was indicated by cloud-
iness of the filtrates and the appearance of bacterial colonies on dex-
trose. agar streaked with some of the material. The cellulase activity
01' the contaminated culture filtrates could not be estimated before
dflailysis because of the presence of an oxidizing agent which interfered
with the end point. The oxidant could be removed by dialysis, but the

c“linires were nevertheless discarded because of bacterial contamination.

he

Culture filtrates were obtained by filtration through a mat of
glass wool in a large Buchner funnel. (Filter paper is rapidly weakened
by the enzyme). The first pdrtions of the filtrate were returned to the
funnel until the residual cellulose and the fungal mycelium formed an
efficient filter, after which the filtrates were clear.

The color of the filtrates was light amber. In the later fractiona-
tion steps it was found that this color tended, at least in part, to
accompany the protein. It was only partially removed by dialysis and
was nearly completely precipitated along with the proteins by ammonium
sulfate, acetone, and alcohol, but not so completely by trichloroacetic
acid. In some of the more concentrated fractions, the darkness of the
color made it necessary to dilute in order to obtain satisfactory

electrophoretic patterns. Whitaker (1953) reported the same difficulty.

Concentration of Culture Filtrates
The concentration of protein in the filtrates was very low. Since

there was some nonprotein nitrogen present and there was no assurance
that.all the protein could be precipitated, determination of protein
‘nitrogen was not attempted at this point. Total nitrogen, exclusive
of nitrate nitrogen was found to vary'between 0.022 and 0.0hl mg./m1.
.If this were all protein nitrogen and the usual conversion factor of
(5.25 were to apply, then the protein concentration would be from 13.7
t“) 25.6 mg./100 ml. Inasmuch as this concentration is too low for

application of the usual fractionation procedures the culture filtrates

were concentrated .

The volumes of the filtrates were too large (nine to ten liters)

t0 Permit freeze-drying with the apparatus available. Instead, the

117

method of slow partial freezing to remove water with a minimum of enzyme
loss was adopted (Whitaker, 1953). About three liters of filtrate were
placed in each of two four-liter beakers wrapped with a thick layer of
cotton and gauze and then placed in boxes containing about two inches
of cotton. Covers with an inner lining of about two inches of cotton
extending into the beaker close to the surface of the filtrate were
placed on the beakers. When placed in a freezing cabinet, freezing
took place slowly from the outside inward. At the end of 2b to 36
hours, the concentrated solution in the center was poured off and the
ice was chipped and allowed to drain, with frequent stirring, in a
large fannel. The small amount of melt from the ice was combined with
the concentrate, and the process repeated until the volume had been re-
duced at least five times.

Protein nitrogen and cellulase activity (cellulose sodium sulfate)
were determined on the concentrates. The cellulase activity of the
material removed as ice was also estimated. The protein nitrogen con-
tent of the concentrates varied from about 0.08 to 0.21 mg./m1.

Fractionation of the proteins by salt or solvent precipitation was
not very successful, but it was noted during these eXperiments that
(much of the activity of the concentrates could be precipitated by ace-
tone or ammonium sulfate. Consequently some of the concentrates were
further concentrated by these means.

Acetone precipitations were carried out as follows. Three hundred
and fifty ml. of the concentrate was placed in a two-liter Erlenmeyer
flask which was clamped in a large battery jar filled.with ice and water.

The entire assembly was then put on a magnetic stirring unit. When the

b8

temperature of the concentrate reached about 20 C., ILOO m1. of chilled
reagent grade acetone was added down the side of the flask over a period
of three to four hours. As the acetone concentration increased, salt
was added to the ice bath until the temperature of the acetone solution
reached about -100 C. Stirring was continued for one—half hour after
all the acetone had been added, and then the precipitate allowed to
settle for about two hours in a freezing cabinet. The precipitate was
heavy and granular and settled rapidly. Most of the supernatant solu-
tion was siphoned off and the precipitate obtained by centrifugation
washed three times with small amounts of cold acetone and then dried

in a vacuum desiccator. The dried powders were tan in color, and their
weight indicated that they were mostly inorganic salts. They were
stored in this form over calcium chloride in a desiccator.

In an attempt to improve the recovery of activity and proteins,
the acetone precipitation was also applied to a dialyzed concentrate.
Dialysis was against twenty volumes of distilled water for 12 hours at
about ho C., then against the same amount of 0.02 M phOSphate buffer,
pH 6.9 for 2h hours.

‘When the acetone powders were to be used, the proteins were ex-
tracted by slowly adding one part by weight of the powder to five parts
of water with continuous mechanical stirring. The water was cooled to
about 00 C. in an ice and salt bath. This procedure was found to be
necessary because of the heat of solution of the salts. After the pow—
der was added, the ice bath was removed and the mixture allowed.to warm
slowly to about room temperature while stirring was continued. A

'voluminous dark-colored precipitate was removed by centrifugation,

L9

extracted once with a small amount of water, and saved for nitrogen
analysis. An aliquot of this second extract was saved for cellulase
assay and the remainder combined with the first extract. The combined
extracts were then stored in a refrigerator (3. ho C.) overnight, dur-
ing which time a rather large amount of a salt (probably NaZI-IPO‘onO)
separated in the form of large crystals. These were removed by filtra-
tion through glass wool and washed with a small amount of ice-cold
water. The combined filtrate and washings were then dialyzed first
against about twenty volumes of distilled water and then against the
pH 6.9 phosphate buffer used for electrophoresis.

When ammonium sulfate precipitation was used as a means of concen-
trating the proteins, the procedure was as follows. The concentrate
was placed in a two-liter beaker on a magnetic stirrer in the cold
room (ca 140 C.) and an eighteen-inch length of large diameter viscose
cellulose tubing, tied at the bottom and packed with enough ammonium
sulfate to make the solution about 50 percent saturated, was suSpended
in the liquid. Stirring of the outside enzyme solution was continuous,
so that as ammonium sulfate diffused through the membrane it was immed-
iately mixed and diluted. At the same time, water diffused into the
cellulose tubing, bringing about a decrease in the volume of the enzyme
solution. Small samples of the outside solution were removed at inter-
vals and analyzed for ammonium sulfate by steam distillation of the
ammonia released by strong alkali, as in the Kjeldahl procedure to be
described later. When the solution was 30-35 percent saturated, it
was centrifuged if any precipitate was present. The precipitate was

dissolved in a small amount of water and analyzed for cellulase activity

50

(cellulose sodium sulfate as substrate) and protein nitrogen. More
ammonium sulfate was then added to the concentrate in the same way as
before until the solution was 90-100 percent saturated. The precipitate
was collected by centrifugation (in the cold room) and dissolved in a
small volume of water. The small amount of insoluble material was re-
moved by centrifugation. After dialysis first against distilled water
(about 20 volumes for 12 hours) and then phOSphate buffer (pH 6.8,
ionic strength 0.1146) as described in the section on electrophoresis,

the solutions were analyzed for protein nitrogen and cellulase activity.

Fractionation by Salt or Solvent Precipitation

Preliminary experiments to determine the feasibility of fractiona-
tion by precipitation with alcohol or acetone were done in the same
manner. Several 10-ml. portions of a concentrate were measured into
flasks held in an ice bath. Absolute ethanol or acetone was delivered
slowly and with gentle agitation into the flasks until a predetermined
amount had been added to each. Final concentrations of solvent ranged
from 33 to 80 percent by volume. The flasks were stoppered and allowed
to stand at 0° C. for one hour in one eXperiment and for 30 minutes in
a second. The mixtures were then transferred to test tubes and centri-
fuged in the cold room. The supernatant solution was decanted and the
precipitates washed once with about five ml. of an aqueous solutim of
the solvent at the corresponding concentration and then recentrifuged.
The solvent was removed by decantation and allowed to drain for several
minutes; then the precipitate was dissolved in five or ten m1. of cold

phOSphate buffer (pH 6.9). Any insoluble material was removed by

51

centrifugation. The resulting solutions were analyzed for protein
nitrogen and cellulase activity.

In some of these experiments, pH and buffer concentration of the
starting material were controlled by dialyzing some of the concentrate
against an appropriate buffer.

Fractionation by means of ammonium sulfate precipitation was also
attempted. The procedure was the same as that previously described for
the concentration of culture filtrate proteins. The precipitates were
recovered at several intermediate stages of ammonium sulfate saturation
by centrifugation in the cold room and washed once with amonium sulfate
solution of the same or slightly higher concentration. Hash solutions
were prepared by dissolving the required amount of ammonium sulfate in
the pH 6.9 phosphate buffer used for electrophoresis. It was not con-
sidered necessary to add buffer to the enzyme concentrates before the
addition of amonium sulfate because all were strongly buffered at about
pH 7. The washed precipitates were dissolved in a small volume of water
and dialyzed against pH 6.9 phosphate buffer for about six hours to re-
move most of the ammonium sulfate. The dialyzed solutions were then
diluted to a convenient volume and analyzed for protein nitrogen and

cellulase activity.

Fractionation by Chromatography on Cellulose Columns
Whistler and Smart (1953) reported that the cellulase in a crude
g. 3.152: extract could be purified by adsorption of the proteins on
cellulose columns at pH 3 followed by elution of the cellulase with

borate buffer of pH 9. Since none of the methods of fractionation

52

described.above were very successful, this chromatographic technique
was tried.

A column, 150 x 10 mm. was packed'with wet Whatman ashless cellu-
lose powder (medium grade) to deliver about 0.5 ml. per minute when the
receiver was attached to the aspirator. The concentrated filtrate,
first dialyzed against distilled water to remove most of the salts, was
adjusted to pH 3 with dilute hydrochloric acid. Twenty ml. were passed
through the column. ‘Washing with a total of 60 ml. of water adjusted
to pH 3 was begun as soon as the filtrate had passed into the column.
The effluent liquids were collected in several portions. The column
was then eluted with 120 ml. of Clark and Lube borate buffer at pH 9.
Eluates were collected fractionally. All the collected fractions were
assayed for enzymatic activity (cellulose sodium.sulfate) and some for
protein nitrogen.

Variations in the conditions of adsorption and elution are recorded

in the section on results.

Fractionation by Electrophoresis-convection

The only fractionation method which gave any degree of success was
electrophoresis-convection. Since this method has only recently been
developed to the extent that it is a generally useful technique for the
fractionation of proteins and is not yet as familiar a procedure as
electrophoresis, its theory and.application will be discussed in more
detail.

Electrophoresis-convection is an adaptation and refinement of

electrodecantation, a method first used for the partial separation of

53

amylase and amylopectin by Samec and Haerdtl (1920). It also resembles
a method devised by Clusius and Dickel (1938) for the separation of gas
mixtures: In electrodecantation, the colloid mixture to be separated
is placed in a large central compartment separated from electrode com-
partments at either end by semipermeable membranes. When a direct
current is applied, the mobile component accumulates near one end of
the center compartment and, because of the density gradient thereby
created, tends to settle to the bottom. In the Clusius column, the

gas mixture is placed in a long narrow vertical channel, one wall of
‘which is heated, the opposite one cooled. Differences in thermal dif-
fusion rates cause convection currents to carry the heavier gas to the
bottan, the lighter to the top of the channel.

Kirkwood (l9bl) first suggested that protein mixtures might be
separated in somewhat the same manner, proposing however, that density
gradients across the channel be established by the application of a
horizontal electric field through channel walls of semipermeable meme
branes, rather than by'a temperature differential.

The first apparatus designed to perform fractionations in this
manner was developed by Nielsen and.Kirkwocd (19h6) and later improved
by Cann, Kirkwood, Brown and Plescia (19h9). A more recent modifica-

2
tion, designed by'Raymcnd (1952) ,‘was used in the present study.

 

1
It is interesting to note that in their reports on the develcpment

of electrophoresis—convection, only the Clusius column was mentioned by
Kirkwood (19h1, 19h6) and cc-workers.

z
Obtained from the E-C Apparatus Company, New York.

The electrophoresis-canvastion cell in which fractionation takes
place consists of a narrow vertical channel, formed by semipermeable
membranes, with reservoirs at the top and bottom. In Operation, the
reservoirs and channel are filled with the dialyzed protein solution
and imersed in the buffer solution between two large electrodes. 0n
application of a direct current horizontally across the channel, the
mobile proteins migrate toward one of the poles and start to accumulate
at the membrane barrier. The density gradient thus established induces
a convection current which.deposits the mobile proteins in the bottom
reservoir. The concentration of any immobile component which might be
present remains approximately constant in both reservoirs. In theory,
the solution in the top reservoir should be almost completely cleared
of the mObile components. In practice this is seldom achieved in a
single step because of the very long tune required. It is usually
advantageous to perform the fractionation in two or more steps, using
the top fraction in each case for the starting material for the succeed-
ing step.

This fractionation procedure has certain advantages over other
methods. Since it is a purely physical method, losses due to denature-
ticn by excessive salt or solvent concentration are not encountered.
Iflechanical losses are small. Contamination with foreign metals, pre-
cipitants, or solvents is not a prdblem since the solution comes in
contact only with innocuous buffer. In some salt or solvent fractiona-
‘tions, the fractions which do not precipitate at reasonable concentra-
tions are either lost or, at best, are difficult to recover. While

this is a matter of little concern in many cases, it is important in

55

those, like the present, in which several components are suspected of
having enzyme activity. On the other hand, it should be pointed out
that in some cases loss of material due to adsorption on the membranes
may occur.

The theory of transport by electrophoresis-convection in a one-
ccmponent system, or in a two-component system in which one component
is immobile, has been thoroughly worked out in mathematical form by
Kirkwood _e_1_'._ 21. (1950, 1951). Although a complete presentation of this
treatment is beyond the scope of this thesis, it is perhaps desirable
to present the final equations in order to point out the manner in which
certain experimentally controllable variables affect transport.

In this situation (one mobile component), the useful quantity to be
calculated is the time, _,required to transport a fraction, l-y, of the
mobile protein from the top reservoir to the bottom. This time may be
predicted by means of the expression:

t - 6 Hr)
in which 9 , called the characteristic time, is the time in hours re-
quired to reduce the concentration of mobile protein in the top compart-
ment to 0.193 of the original concentration, and 1(y) is an integral1

relating 6 to the time required for the transport of any other fraction.

 

#5] 5h 91. Kirkwocd

1- -
11(1) - (5/3)6/JO X [(21-1) vii-(1+1)

_e__t a_l_. (1950) solved this expression for several values of 1 between
1 -and 0. When I - 0.193, the value of I (y) is 1.00. Consequently,
at this point, 9 - t.

56

The characteristic time is calculated by means of the equation:

- 2(ae g):/‘ DV‘CV‘V

9' (h ’DPF‘ x 1.157%}?

in which up is the density increment produced by one gram of protein
per 100 m1. of solution, 5 is the acceleration of gravity, and ’9 is
the viscosity of the solvent. In the second term, 2 is the diffusion
constant of the mobile protein, 9 is its initial concentration in grams
per 100 m1., and u is its electrophoretic mobility. 1 is the volume
(ml.) of solution in the top reservoir, 2 and l are the width and
height (cm.), respectively, of the channel, and E is the electric field
strength.

The above equation shows that the time required to obtain a given
degree of transport can be varied over a wide range by controlling cer-
tsin variables. For example, since the characteristic time is directly
proportional to the volume, it may be advantageous to concentrate the
solution before subjecting it to electrophoresis-convection. The in-
crease in concentration tends to increase 9, but only in proportion
to its fourth root.

The dimensions of the channel are of some importance, but there
are practical limitations here, and they cannot be varied easily.

The two most important variables affecting the characteristic time
are the electric field strength and the electrophoretic mobility, since
both enter as the inverse square. Thus, an increase in one of these
values by a factor of two should theoretically decrease the time necess-
ary to effect a given degree of tranSport by a factor of 0.25. An in-

crease in mobility may be attained by a change in the pH or ionic

57

strength of the buffer solvent. The magnitude of the field strength is
limited by heat effects, but by providing adequate circulation and cool-
ing, it (an be varied over a considerable range.

The theoretical treatment of transport by electrophoresis-convection
was derived for certain ideal conditions which are not realized in prac-
tice. In testing the theory under practical conditions, Brown at 31.
(1951) found it was necessary to employ an empirically derived correction
for the field strength in order to make the experimental results coincide
satisfactorily with predicted results. It appears to the author that the
use of the empirical correction factor is valid; its necessity is accept-
ably explained on the basis of experimental deviation from the ideal con-
ditions on which the mathematical treatment was based.

The theory has also been extended by Brown at 9.13 (1952) to the
fractionation of two-component systems in which both components are
mobile. The important quantity in this case is the separation factor,
£3, the ratio of the weight fraction of the slow canponent (2) to the
weight fraction of the fast component (1) left in the top reservoir at

the time of sampling. This may be calculated from theory by means of

e.- [ 1+1: (ii-UWY fl

0
in which I, is the initial weight fraction of component 2, f is the

the expression:

fraction of total protein remaining in the top reservoir, and fl - lung/u1
(ua and 91 are the electrophoretic mobilities of components 2 and 1,
respectively) .

It can be seen from this equation that under certain conditions the

separation factor depends to a very large extent on the ratio of the

58

electrOphoretic mobilities of the two components. Solution of the
equation by Brown st 31. (1952) for several different assigned values
of £2, {, p showed that in most circumstances the ratio Liz/m. is the
most important factor in determining the separation factor. The best
separations can be attained when this ratio (hence Us) is as small as
possible. In other words, it is advantageous to work as near the iso-
electric point of the slower component as practicable. These investi-
gators, testing the theory under practical conditions, found reasonably
good agreement between theoretical and eXperimental values of the
separation factors. In this case no correction factors were necessary.

Although the theory of electrophoresis-convection is general and
applies to multicomponent as well as to simple systems, it is not
practical to attempt the necessary calculations for the exact planning
of the fractionation of multicomponent systems. An empirical approach,
guided qualitatively by the theory, is more practical.

The construction and operation of the apparatus used in the frac-
tionation of E. verrucaria culture filtrate concentrates can best be
'described with the aid of Figure 2, which shows the assembled cell and
cell-frame. The cell proper is formed from a suitable length of cellu-
lose tnbing. ‘With the Raymond apparatus, either a single or’a double
channel may be used. For the single channel, one end of the tubing,
softened.in the buffer to be used, is closed tightky‘by'tying a knot.
The tubing, still flattened, is then placed vertically over the slot
in one of the two faceoplates making up the cell-frame. The face-
plates are so constructed that when the second one is placed over the

1
tubing and tightened.into place by means of cap screws, the center

1These screws do not extend all the way through the cell frame.

‘Q
\l ; V

\———

 

Figure 2. Electrophoresis-convection apparatus. One face-plate
is shown in place between the graphite electrodes in the
buffer compartment. A length of cellulose tubing has been
placed over the center slot in the second face-plate.

59

section of the cell, partially eXposed to the external buffer by opposing
slots in the face-plates, will form a flat vertical channel. .The upper
and lower sections of the tubing are left free to expand.and receive the
bulk of the solution.

To use two channels simultaneously, a double length of tubing is
folded in the middle to form a U-tube and.placed in the cell-frame with
the doubled end toward the bottom. This provides two channels and two
top reservoirs with a single bottom reservoir. Double channels sup-
posedly shorten the time required for a given degree of separation by
half.

Besides prodding for the use of double channels, the Raymond cell
has the further advantage of volume adaptability. By'adjusting the
length of the tubing above and below the compressing plates and by
choosing the appropriate diameter tubing (between 2.5 and 11 cm. flat
width), any volume of solution between 20 and 200 m1. may be accommodated.

After assembly, the cell is filled with dialyzed protein solution
and.any entrapped air bubbles are removed from the bottom reservoir and
channel. The assembly is then placed in the buffer compartment between
the two large flat electrodes (Figure 2). The cell frame fits snugly
enough to minimize current leakage around the sides of the cell, but it
does stand on short pegs to allow free circulation of the buffer. Direct
current, up to 35 volts or 2 amperes output, is supplied to the graphite
electrodes by means of a selenium rectifier power pack. The heat evolved
by the passage of the current is dissipated by means of an all-rubber
circulating pump and a cooling coil of stainless steel (see Figure 2).

60

In the first few experiments of this investigation, the cooling
coil was immersed in a mixture of salt and ice or in ethanol cooled with
solid carbon dioxide. In later experiments, it was found more convenient
to use the thermostat tank of the electrophoresis apparatus. In either
case, the temperature of the circulating buffer was maintained between
h and 6° c.

As in electrophoresis, it is important that the protein solution be
in ionic equilibrium with the outside buffer. To accomplish this, the
solutions were dialyzed in collodion sacs in the manner described in the
section on electrOphoresis.

Lack of success in preparing the lengths of collodion tubing re-
quired (70 cm. for a double channel cell) necessitated the use of viscose
cellulose tubing. It was found that the latter could withstand the
action of the enzyme for periods up to 1h hours without leakage, but
extreme care in emptying the cells was necessary, since the membrane
was markedly weakened and could easily be split with a gentle pull after
this length of emosure. Therefore, to prevent loss of material, most
runs were held to periods of less than 10 hours.

Three different pH levels were tried: 6.9, 5.9, and 5.0. The pH
6.9 phosphate buffer used for electrophoretic analysis was found most
suitable, because the time required for dialysis prior to electrophoretic
analysis of the bottom out was much less with this buffer; the progressive
loss of activity with increasing time of dialysis was therefore decreased.
The pH 5.9 buffer was also a phOSphate buffer, with an ionic strength of
0.116, and the pH 5.0 buffer was an acetate buffer of 0.15 ionic strength.

The composition of these buffers is given in Appendix B.

61

The total protein concentration of the solutions was generally
somewhat lower than ideal and varied considerably from one run to
another. Higher concentrations could probably have been obtained by
lyophillization and resolution, but in most cases the dark color of
the bottom out would have required dilution before the electrophoretic
analysis. Then, if this fraction were to be subjected to further
electrophoresis-convection, reconcentration would have been necessary.
It was considered.more practical to use the more dilute solutionS.

The potential gradient in the channels was not calculated since
there is no accurate way to measure current leakage around and under
the cell, and the resistance of the cell membranes is unknown. ‘With
the semi-empirical approach necessitated by the complexity of the
solutions to be fractionated, it was considered sufficient to record
only the current and voltage for each run. These remained fairly con-
stant during the experiment, but when drift was noted it was promptly
corrected.

The values of the experimental variables (time, pH, current and
voltage) are presented in the tables of data on electrophoresis-con-
vection fractionations.

At first, the top and bottom fractions were analyzed for protein
nitrogen and enzyme activity after a single operation with the electro-
phoresis-convection apparatus. Later, however, several runs were per—
formed before these analyses were undertaken. Usually, in these cases,
the bottom fraction of the first run was used as the starting material
for the second run and the resulting top fraction was combtned'with the

first top fraction. Further steps consisted of similar recombinations

62

and runs. These operational details are indicated in the section on
results.

At the end of an experiment, small portions were taken from both
of the final fractions for the determination of protein nitrogen and
enzyme activities. Electrophoretic analyses were usually performed
only on the original starting material and on the bottom out. The com-
position of the top cut was calculated from these results. In some
cases, the top cuts were concentrated by freeze-drying and analyzed
electrophoretically. These control data were used to determine the
mamer in which fractions were canbined.

During the course of fractionation it became apparent that the
primary objective - the isolation of one or more pure fractions ac-
counting for all the enzyme activity - was not attainable with the
amounts of material on hand. Consequently, the secondary objective --
the obtaining of several fractions containing the suspected components
in widely different ratios -- had to be accepted. Fractionations were
continued until this objective had been attained.

Although valuable information would have been gained by determining
the activity of each fraction against several different (3 -glycosidic
substrates, time did not permit such extensive control of the fractiona-
tion procedure. Instead, reliance was placed on a few early eXperiments
which showed that most of the activity against cellulose, two different
impure hemicelluloses, and cellulose sodium sulfate, is confined to the
three components having the lowest electrophoretic mobilities. These
data suggested that determinations of the activity against cellulose
sodium sulfate and occasional electrophoretic analyses would afford

sufficient control.

63

Estimation of Enzymatic Activity

Although several proposals for the establishment of a standard unit
of cellulase activity have been made in the past, none has become univer-
sally accepted. Bach group of investigators has used a different cellu-
losic substrate and different experimental conditions. For example,
Karrer 23; g. (1925) defined a unit as the amount of enzyme which will
hydrolyze 200 mg. of fibrous cuprammoniun cellulose in 96 hours at 360 C.
in 50 m1. of a one percent suspension buffered at pH 5.28 with phosphates.
The unit proposed by Grassman 33 g. (1933) is somewhat larger. It
corresponds to the amount of enzyme required to bring about the splitting
of 320 mg. of hydrocellulose in 20 ml. of solution in eight hours at
300 C. and pH h.5 (0.01M acetate buffer).

The difference in substrates and conditions renders a quantitative
comparison of results obtained in different laboratories difficult and
impractical. Reese at a_l_. (1950) suggested that carboxymethylcellulose
of a low degree of substitution be used as the standard substrate.
Besides being easily standardized as to degree of substitution and
degree of polymerization, carboxymetl'wlcellulose and cellulose sodium
sulfate are initially more highly susceptible to hydrolysis by cellu-
lases than are the insoluble cellulose preparations. The latter pro-
perty is important because it allows the assay period to be greatly
shortened. However, it was later found (Reese and Levinson, 19533
Tracey, 1953) that in spite of these advantages the soluble cellulose
derivatives are not suitable as standard substrates, the reason being,

33 Previously mentioned, that the relation between the rate of hydrolysis

6h

of the soluble derivatives and the rate of hydrolysis of solid cellulose
substrates by different cellulase preparations is not constant.

The point to be emphasized is that, because of the possibility of the
existence (in crude cellulase preparations) of two or more cellulolytic
enzymes, each with its own spectrum of activities toward different sub-
strates, the establishment of a rigidly defined unit of cellulolytic acti-
vity is of doubtful value. It is perhaps better simply to report the loss
in.weight of the substrate or the amount of end product appearing under
stated conditions.

Three different methods have been used to determine the extent of
hydrolysis brought about by cellulases. In addition to the more commonly
used.methods of measuring substrate disappearance or end product accumu-
lation, a viscometric method has been used with the soluble cellulose
derivatives. Although Levinson and.Reese (1950) stated that the vis-
cometric method is not entirely satisfactory because of the abrupt initial
decrease in intrinsic viscosity on adding the cellulase preparation,
Tracey (1951) found this to be a distinct advantage in detecting very
small amounts of cellulase in plant extracts.

‘lith insoluble substrates such as cellulose and some hemicelluloses,
determination of the loss of substrate weight is perhaps more convenient
than deterldnation of end products. .According to Karrer and Illing
(1925) and lhlseth (19b8), the same result is obtained by both methods
when the accumulation of end products is calculated as glucose. This
cannot be true in those cases in which cellobiose accumulates to an
appreciable extent. ‘Iith the hemicellulases, especially those with
branched chains and‘withlmore than one type of residue, the two methods
might be expected to give quite different results because of the possible

accumulation of soluble oligosaccharide end products whose reducing power

65

would be relatively small in comparison to weight. In these cases,
estimation of the increase in reducing sugars would give a better esti-
mate of the number of glycosidic bonds hydrolyzed than would.the deter-
mination of the decrease in weight of the substrate.

For these reasons, since both soluble and insoluble cellulases and
hemicelluloses were to be used as substrates, the estimation of and pro-
ducts seemed to be the method of choice for the investigation reported
here.

The micro method of Somogyi (l9h5) was used for the analysis of
reducing sugars in the hydrolysates. The use of this method, capable
of giving accurate results in the range 0.01 to 0.5 mg. of glucose,
allowed assays to be made at very low enzyme concentrations. (Satis-
factory results were obtained with as little as two micrograms of pro-
tein nitrogen in ten m1. of assay medium, when the most active enzyme
preparation acted on cellulose sodium sulfate). This is an important
consideration because under these conditions the initial rate of re-
action is less apt to be limited by the concentration of the substrate.

The conditions chosen for the enzymatic hydrolysis are compared
in Thble IV with those used by previous investigators. The conditions
employed by Reese gt a}. (1950) served as a guide in choosing the con-
ditions to be used in this work. Reese found that the optimum tempera-

ture for g.'verrucaria enzymes acting on carboxymethylcellulose for one

 

0
hour is near 50 C. However, since longer periods of reaction were to
be used in the work reported here, a lower temperature was considered

to be more suitable.

66

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67

In choosing the initial concentration of substrate to be employed
in the case of cellulose sodium sulfate, an important factor is the vis-
cosity of the sclution. It was found that a one percent solution flows
readily enough to permit accurate sampling by pipette. Solutions of
higher concentrations flow at a noticeably slower rate and the problem
of accurate sampling is encountered. This problem becomes especially
important when many samples of hydrolysates are to be taken at short
intervals. All other substrates except phloridzin were used in one per-
cent concentration.

Reese st 31. (1950) found that the optimum pH for !. verrucaria

 

culture filtrates acting on carboxymethylcellulose is about 5.1. This
is the pH level originally chosen for this work. The stock citrate
buffer solution (pH 5.1, 0.5 l) was incorporated into the assay medium
at a 1:10 dilution. It was later found that the resulting solution has
a pH of 5.35. Reese gt gl_. prepared their assay solutions in the same
manner, the final dilution of the pH 5.0, 0.5 H citrate buffer being
136.67. That the change in reaction was due to dilution and not the
substrate was demonstrated by diluting the stock buffer 6.67 and ten
times with distilled water. The pH's of these dilutions were 5.3 and
5.35, reSpectively. Thus there is reason to believe that Reese gt a}.
(1950) were working at a somewhat higher pH than reported. They did not
indicate that the reaction of the final solutions had been checked.

Substrates. The cellulose sodium sulfate used in this work has a

 

degree of substitution of 0.h, according to information supplied by the

1
manufacturer. The water content of this material, determined by drying

 

i
we wish to thank Dr. C. J. Helm of the Eastman Kodak Company for

his coo ration ifl supplying this substrate (cellulose sodium sulfate
sample 0. CAD-10 5).

68

in the vacutn oven at 650 C., was found on one occasion to be 7.68 per—
cent and on another occasion 7.60 percent. This moisture content was
taken into account when samples were weighed. No other analytical data
were obtained. Levinson, Mandela, and Reese (1951) routinely dialyzed
their cellulose sulfate solutions, finding that some of the sulfate ion
could be removed in this way. Since it was found in this work that
dialysis had no effect on the results, this treatment was omitted.

Salicin (purified, Pfanstiehl) is orthohydroxymethylphenyl-fl-Q—
glucoside. This material was dried in 3322 at 650 C. before use.

Arbutin (mdroquinone fl ~2—glucoside), anorgdalin (Q-mandelonitrile
fl-Q-gentiobioside), and phloridzin (phloretin fl -l_3fglucoside) were used
as supplied, without drying or purification, since they were employed in
only one experiment to determine qualitatively their susceptibility to
attack by !. verrucaria enzymes.

lheat straw polysaccharids fractions were prepared essentially by
the acid chlorite delignification and alkaline extraction methods of
Vise, Murphy and D'Addieco (191:6). These methods were originally de-
veloped for application to wood and wood pulps but have subsequently
been applied to cereal straws and grasses with satisfactory results
(Ely and Moore, 1951:). Three holocellulose samples, presumably with
different lignin contents but otherwise the same, were prepared. From
two other holocellulose preparations, two different mixed hemicellu-
loses and wheat straw cellulose were prepared.

A clean, dry sample of wheat straw was ground to pass a 60-mesh
screen in a Wiley mill, then extracted for 16 hours with an ethanol-

benzene mixture (1:2 v/v) in a Soxhlet apparatus. The air-dried

69

material was then submitted to the chlorite delignification procedure of
‘Iise gt a}. (19h6). A 10-gram sample was suspended in 320 ml. of water
in an Erlenmeyer flask loosely covered with an inverted flask or beaker
and heated to 700 C. in a water bath. The suspension was acidified with
twenty drops of glacial acetic acid, 3 g. of sodium chlorite was added,
and the mixture was heated with occasional gentle agitation for one hour.

One sample was given a single treatment as described above. A
second sample was delignified in two successive steps and a third, in
three steps. The second and third steps were carried out simply by
adding the same amounts of acetic acid and sodium chlorite as in the
first step and continuing the reaction period for another hour.

At the end of the chlorite treatments, the reaction mixture was
cooled in an ice bath and filtered on a canvas filter in a large Buchner
funnel. The residual material was washed repeatedly with small portions
of ice-cold distilled water until the odor of chlorine dioxide was no
longer detectable, and finally with generous portions of acetone. The
holocellulose was then air dried.

For convenience, these three preparations are designated holocellu-
1ose-l, holocellulose-2, and holocellulose-3, the numerals signifying
the number of delignification steps.

There was a distinct gradation in the color of these preparations,
holocellulose-l possessing a moderate tan color, whereas holocellulose-3
was only a shade removed from pure white.

The yields of air-dried holocelluloses-l, -2, and -3 from 10 g. of
extracted wheat straw were 8.5, 8.7, and 8.h g., respectively; 'When

these values are corrected for moisture, ash, and protein (nitrogen x 6.25)

70

content they become 7.5, 7.b and 7.b g. of holocellulose from 8.b g. of
extracted wheat straw.

Moisture was determined by drying in 33239 at 650 C. for four hours.
A second drying period under the same conditions did not further decrease
the weight of the sample. The ash content was determined by first charring
the weighed sample in a Coors crucible over a low Bunsen flame, then heating
in a muffle furnace to 5000 C. for three hours.

The nitrogen content of the holocelluloses was estimated on duplicate
samples (100-200 mg.) by the semimicro Kjeldahl method to be described later.

All these analytical data are recorded in Table V. They were used to
determine the weight of sample to be taken to obtain a Specified amount of

holocellulose for the estimation of enzyme activity.

TABLE V

MOISTURE, ASH, AND NITROGEN IN WHEAT STRAW FRACTIONS

(All values are expressed as percentages)

 

 

 

 

 

Fraction Moisture Ash Nitrogen
Extracted wheat straw 8.0h 5.8h 0.30
Holocellulose-l 7.22 3.75 0.20
Holocellulose-2 7.05 b.11 0.16
Holocellulose-B 6.72 b.03 0.1
Hemicellulose X b.70 6.22 0.05
Hemicellulose x 6.97 h.os 0.0!;
Hemicellulose Y 7.5h 7.10 0.0h
Hemicellulose Y 6.91 b.60 0.05
Cellulose b.05 1.23 0.03

 

71

The lignin content of these holocelluloses was not determined. Ely
and Moore (l95h), examining the application of the same technique to
cereal straws, hays, and grasses, found that a single treatment reduced
the lignin content of wheat straw from 13.0 to about 2.8 percent, a
second step reduced it to about 2.0 percent, and a third to about 1.8
percent. Three more successive acid chlorite treatments reduced the
lignin only to about 1.1 percent and at the same time removed a con-
siderable amount of polysaccharide material. The first three treat-
ments resulted in very little loss of holocellulose components.

Two additional batches of holocellulose, obtained from to g. of
extracted wheat straw by three acid chlorite treatments, were used for
the preparation of two hemicellulose fractions and wheat straw cellulose.

Hemicellulose X was extracted from 30 g. of holocellulose by 750
ml. of 1.5 percent potassium hydroxide for b8 hours at room temperature.
The extraction was carried out in a closed vessel with continuous stirr-
ing. At the end of the extraction period the mixture was filtered with
suction and the residue washed on the filter with 250 ml. of water in
several small portions. The filtrate and washings were then acidified
to pH 5 with glacial acetic acid and, after cooling, poured into four
volumes of absolute ethanol. The voluminous white precipitate was
allowed to settle overnight. Most of the supernatant solution could
then be siphoned off and the precipitate recovered by centrifugation.
The precipitate was washed twice in the centrifuge bottle with 200-ml.
portions of ethanol, then twice with lOO-mfl. portions of ethyl ether.
The air-dried material was almost pure white and was easily ground into

a fine powder.

72

The ash content of the two hemicellulose X preparations was very
high (21.2 percent in the first preparation and 18.5 percent in the
second.) It was found that much of this foreign inorganic material
could be removed by dissolving the preparation in about 200 ml. of 0.2
percent potassium hydroxide, then dialyzing the solution, first against
three changes (one liter each) of distilled water for a total of about
2h hours, then against two changes (two liters each) of one molar acetic
acid for another 2h hour period. The polysaccharides were then precip—
itated with ethanol, centrifuged, washed and dried as previously des-
cribed. These preparations were very nearly white.

Moisture and nitrogen were determined as previously described for
the holocelluloses. The ash determination was modified according to the
procedure recommended by Wise :t_al, (l9h6). After the weighed sample
was charred and cooled, two or three drops of concentrated sulfuric acid
were added.and gentle heating was resumed until all fuming ceased. The
crucible was then heated over a Fisher burner to a dull red glow, cooled
in a desiccator and weighed. According to Wise St El! (l9h6), most of
the inorganic material in the hemicellulase fractions is potassium
acetate, although some of the potassium is probably present as the salt
of uronic acid residues. The weighed ash is mostly potassium sulfate,
so a correction factor of 1.13 was applied.

The results of moisture, ash, and nitrogen determinations are re-
corded in Table V.

The yields of hemicellulase X, corrected for moisture, ash, and
protein (nitrogen x 6.25), from the two 30-g. batches of holocellulose

‘were 5.9 and 6.1 g.

73

Hemicellulose Y was obtained from the residue left by the first
alkaline extraction by further extraction with 500 ml. of 2b percent
potassium hydroxide. The extraction was made at room temperature in a
tightly sealed flask with continuous stirring for four hours. The mix-
ture was filtered and washed on the filter with 300 ml. of water. The
combined filtrate and.washings were acidified and the hemicellulases
precipitated with four volumes of absolute ethanol. The precipitate
was centrifuged, washed and dried as previously described.

The inorganic fraction of these preparations was initially rather
large so the purification procedure used with the hemicellulose X
fractions was again applied. In this case it was necessary to use 10
percent potassium hydroxide to dissolve the material. The purified,
air dry preparations were pure white.

Moisture, ash, and nitrogen were determined as before, and the
results recorded in Table V.

The yields of hemicellulase Y, corrected for moisture, ash, and
protein were 3.h and h.l g.

Wheat straw cellulose was prepared from the residue of the last
alkaline extractions. This residue was washed further on the filter
‘with capious amounts of water, then with 300 m1. of molar acetic acid,
and finally with 500 ml. of water. Residual water was removed by wash-
ing with several small portions of dry acetone. Only one sample of
this residual cellulose was kept, since the first contained small hard
lumps of material which were very difficult to break up. The second

sample was pure white, very light and fluffiy.

7h

Moisture, ash, and nitrogen were determined as before. The results
are recorded in Table V. The corrected yield of wheat straw cellulose
was 11.2 g.

It should be emphasized that no claim concerning the purity of the
wheat straw polysaccharides is made. On the contrary, it is almost
certain that both hemicellulose preparations contained more than one
chemical species. Furthermore, it cannot even be stated with certainty
that the different extraction procedures employed resulted in hemicellue
lose fractions which are chemically different from each other. Accord-
ing to Norman (195k):

It is likely that all hemicellulose preparations so far obtained
have been mixtures, but there is, in fact, reason for believing
that the methods of separation and fractionation that have been
rather generally used may have given a spuriously complex picture.
......... The major separation ordinarily employed in one form or

another distinguishes only between the less-soluble and more—sol-
uble fractions.

This quotation appears to be especially relevant to the relationship
between the two hemicellulose preparations used in this investigation.
Hemicellulose X was completely dispersed in concentrations up to two
percent. The dispersions were cloudy to the point of opacity but were
nevertheless stable. Hemicellulose Y was nearly completely insoluble
in the citrate buffers used. Nevertheless, it will be shown later
that there was no difference in the nature of enzymatic end products
of these two preparations, and little difference in the ratios of dif-
ferent end products in the two hydrolysates. The same is true of the
acid hydrolysates.

Wise, Murphy and DIAddieco (l9h6) termed the cellulose fraction

"alpha-cellulose", but only for convenience. They stated that this

7‘3

fraction invariably contained a small amount of furfuraléyielding material,
possibly xylose or uronic acid residues. This finding was confirmed in
the present investigation by the demonstration of a small amount of xylose
in the enzymatic hydrolysates of the wheat straw cellulose. No uronic
acid was found.

A word about the nomenclature adopted for the hemicellulase fractions
may be appropriate. 'lise st 21. (19h6) used the designations hemicellulose
A and B. It seems to the author that this nomenclature implies that these
fractions are the same as the classical hemicellulases A and B first des-
cribed by O'Dwyer (1926). Norman (19514) has stated that the nomenclature
instituted by O'Dwyer and subsequently used quite generally in reality
indicates the particular method of isolation and does not uniquely define
the composition of the preparations. Inasmuch as the methods used in this
investigation were quite different from those of O'Dwyer, the author pre-
fers to use a different nomenclature.

£2231 procedur . Cellulose sodium sulfate was utilized more than
any other substrate for following the course of fractionation procedures.
The assay method described in the following paragraphs was designed for
this substrate, but was easily adapted to any other substrate by the use
of appropriate modifications.

1
Eight ml. of 1.25 percent cellulose sodium sulfate was mixed with

 

1In the author's experience, this was best prepared by adding hot
distilled water to the powder. The large gummy masses which resulted
'were completely diapersed in 20 to 30 minutes of continuous mechanical
stirring. The clear solution was cooled, diluted to volume and stored
in the refrigerator. A solution was never kept more than one week.

76

one m1. of 0.5 M, pH 5.1, citrate buffer1 in a 20 x 150 mm. Pyrex test
tube. Since aseptic techniques were not used it was found necessary to
include an antiseptic in the assay medium. 'Merthiolate'2 was found to
be entirely satisfactory when included in the buffer at a concentration
of one mg./ml. The stoppered tube was placed in a water bath at hO 1 0.20
C. for about 15 minutes prior to the addition of one m1. of appropriately
diluted enzyme solution. A blank solution was prepared in the same way
except that water was substituted for the enzyme solution. Duplicate
aliquots of the hydrolysate were removed at exactly timed intervals and
delivered directly into five m1. of the sugar reagent (Somogyi, 19h5).
Because of its alkaline reaction and high copper concentration, this
reagent effectively stopped the action of the enzyme. The reaction time
for most determinations of cellulase activity was two hours. When pro-
gress curves were to be constructed the first samples were taken at 15

or 20 minutes, and subsequent samples at increasingly longer intervals

up to 2b hours in most cases.

The same procedure was used with all the monomeric #5 -glucoside
substrates with the exception of phloridzin. This substrate is soluble
at room temperature only to the extent of about.0.l percent, so 10 mg.
was dissolved in eight m1. of water and one m1. of buffer by heating
the closed tube to about 600 C. The tube was cooled in a water bath

0
to ho C. before the enzyme was added.

—‘

1
The preparation of this buffer is described in the appendix.

2
'Merthiolate' is the trade-mark of Eli Lilly and Company for the
8OCH-um ethylmercurithiosalicylate (Thimerosal) made by it.

77

Certain modifications of technique were necessary when the insoluble
substrates were used. Hemicellulose X, although initially soluble, was
included in this group because it was found that a precipitate formed
after the enzyme had.acted on it for a while. A uniform suSpension of
the insoluble substrates was essential, so the reaction was carried out
in plain'Warburg flasks (without side arms or center wells) attached to
the manometers of the Warburg apparatus. The flasks were shaken at the
rate of’about 80 four-centimeter excursions per minute. For activity
determinations the time of incubation was 2h hours. 'When time curves
'were to be constructed, aliquots of the hydrolysate were taken at appro-
priate intervals over a 2b-hour period.

Samples of the hydrolysate were obtained by removing the flask from
the shaker and quickly withdrawing a volume 25-50 percent greater than
the volume to be analyzed. The suspension was then centrifuged briefly
and the supernatant was used for the analysis.

Early in this investigation it was found that even at the low con-
centrations of enzyme employed it was essential, in order that all
activity determinations be reasonably comparable, to use approximatehy
the same amount of enzyme in each assay. This was felt to be particu-
larly important in the estimation of activity against cellulose sulfate,
since this substrate was used to follow the fractionations. To meet
this requirement it was necessary to define a relatively narrow range
of enzyme concentrations in which to make all activity determinations.
The range was defined as the amount of enzyme which would.resu1t in the
production of O.h to 1.5 mg. of glucose (or its reduction equivalent)

per milliliter of cellulose sodium sulfate medium in the two-hour

78

incubation period. At first, this objective was attained either by
using two different dilutions of the enzyme solution or by using two
different volumes of the same dilution. Usually one of the two assays
was within the prescribed range. Later, when with added eXperience the
proper dilution and amount of enzyme could be more accurately predicted,
only one enzyme level was used. The determination was repeated if the
first level was not correctly chosen.

Reducing sugars in the enzyme hydrolysate were determined by the
copper reduction method of Somogyi (19h5). In this method cupric copper
is reduced to cuprous oxide by the reducing sugar on heating the mixture
in a boiling water bath. The amount of copper reduced is directly (but
not stoichiometrically) related to the amount and kind of reducing sugar
present. Because the method is empirical, the time of heating and the
concentration and composition of the reagent must be carefully controlled.
The amount of cuprous oxide formed may be estimated iodometrically or
colorimetrically. In this work, the iodometric method was chosen be-
cause one of the substrates, hemicellulose X, did not form a clear
solution.

Reagents

Somogyi's (lGhS) alkaline copper sugar reagent, prepared
to contain 0.005 N potassium iodate. This concentration
of iodate is sufficient for the analysis of 0.01 to 0.5
mg. of glucose or its equivalent. For the details of
preparation, see Appendix B.

Sodium thiosulfate, 0.005 N, prepared by dilution from a
standardized, approximately 0.1 N stock solution. The
inclusion of about one m1. of 2 N sodium hydroxide in
500 m1. of diluted solution helps to protect the sodium
thiosulfate from atmospheric oxygen.

79

Potassium iodide, 2.5 percent. This solution keeps indef-
initely if made slightly alkaline by the addition of a
knife-tip of potassium carbonate per 100 ml. of solution.
Sulfuric acid, about 2 N.

Soluble starch, one percent.

Duplicate volumes of the test solution and reagent blank were mixed
with accurately measured five-ml. portions of the sugar reagent in
25 x 200 mm. Pyrex test-tubes. The total volume was made up to 10 ml.
with distilled water and the contents mixed. The tubes were covered
with glass bulbs and heated in a boiling water bath for exactly 20
minutes. After cooling for about three minutes in running water, 0.5
ml. of 2.5 percent potassium iodide was carefully layered on top of the
solution, then about 1.5 ml. of 2 N sulfuric acid was added rapidly
with simultaneous agitation. The excess iodine was titrated with the
0.005 N sodium thiosulfate. A few drOps of starch indicator were added
near the end of the titration.

A procedural detail which was not eXplicitly mentioned in the
original paper was found to be of some importance in obtaining precise
and accurate results. It was found that the titrations must be done
within about 15 minutes after the heating period. If the tubes were
allowed to stand longer, the thiosulfate titer became noticeably
greater, probably because of oxidation of the cuprous oxide. When
many determinations were to be made it was found best to start four
tubes every 10 minutes. ‘lith experience, four titrations can easily
be completed in 10 minutes.

In the beginning, the aliquots for the blank determination were

taken at the same time as the test aliquots. Later, it was found that

this was unnecessary because none of the substrates were hydrolyzed to
a detectable extent in the absence of enzyme in 2b hours. Fxperiments
proved this to be true for the whole pH range studied. With this fact
established, it became the practice in later eXperiments involving many
determinations to use a previously determined blank value for each sub-
strate.

Before describing how the substrate blank values were established
it is necessary to digress momentarily. Early in the study, a puzzling
constant increase in the blank reducing titers was noted. It soon be-
came apparent that this increase could.be correlated with only one
factor: the age of the Somogyi reagent. Daily titration of the un-
heated reagent showed that the potassium iodate was not decomposing;
five ml. of the reagent always required 5.00 t 0.05 ml. of the 0.005 N
thiosulfate. 0n the other hand, heated reagent blanks (as opposed to
reagent plus substrate blanks) always required less thiosulfate, and
the difference increased steadily with the age of the reagent. The
blank for a freshly prepared reagent was usually about 0.20 ml. of
0.005 N copper reduced, but after two weeks increased to about 0.h0 m1.

Reducing titers, or blank values, were established for each poly-
saccharide substrate and for salicin by incubating the substrates with-
out enzyme at too C. at a concentration of one percent in citrate
buffers of pH b.20, 5.35 and 6.70. Volumes varying in size from 0.5
to 2.0 ml. were removed at specified times from 0.5 to 2h hours. Their
reducing titers, eXpressed as the milliliters of 0.005 N copper reduced
per milligram of substrate, were determined by substracting the volume

of 0.005 N thiosulfate required from that required by reagent blanks.

The results did not vary in any significant manner either with pH or
with time of incubation. The average and the range of results for each

substrate are given in Table VI.

TABLE VI

REDUCING 'I'ITF‘RS OF SUBSTRATFS

 

_ L l
-—‘—— L n,

M1. of 0.005 N copper per mg. of substrate

 

 

 

Substrate Avera ge Range
Cellulose s odium s ulfate 0 .025 0 .021-0 .02?
Wheat straw cellulose O .013 0 .011-0 .017
Hemicellulose x 0.021 mom-0.021.
Hemicellulose I 0 .015 0 .010-0 .018
Salicin 0.010 0.008-0 .011

 

hizymatic activities were eacpressed as specific activities whenever
the concentration of protein nitrogen was known. Specific activity was
defined as the mg. of reducing substances (as glucose) appearing in the
assay medium per milligram of protein nitrogen: Unless otherwise stated,
specific activity refers to a two-hour assay period when either cellulose
sodium sulfate or salicin was the substrate, and to a Zh-hour period when
cellulose or the hendcelluloses were used.

When the protein nitrogen concentration of the enzyme solution had
not been determined, activities were recorded as a concentration expres—

sion, 1.9., the mg. of glucose or its reducing equivalent released from

1Specific activity was based on protein nitrogen rather than on pro-
tein concentration because the percentage of nitrogen in the proteins

involved was unknown.

82

the substrate into 10 ml. of assay medium by one ml. of the original
(not the diluted) enzyme solution.

In some cases, when samples were taken at incubation times short
enough to permit extrapolation to zero time, the initial rate of re-
action, or the mg. of reducing substances (as glucose) appearing per
minute per milligram of protein nitrogen, was also calculated.

Somogyi (1915) found that 0.135 mg. of glucose reduced one ml. of
0.005 N copper when the mixture was heated in a boiling water bath for
10 minutes. He. found this time to be adequate for glucose, but for
some other sugars, including arabinose, a longer time was necessary. A
reaction period of 20 minutes was chosen for the work reported here
because of the anticipated presence of sugars other than glucose in
some of the hydrolysates. Under these conditions one m1. of 0.005 N
copper was the equivalent of 0.131; mg. of glucose. Each new batch of
reagent was calibrated with three replicates each of three levels of
glucose: 0.05, 0.10, and 0.20 mg. If the average reduction equivalent
of the three levels did not fall in the range 0.133-o.135 (inclusive),
the reagent was adJusted by dilution (with the alkaline-copper reagent
without iodate) or by addition of potassium iodate, then recalibrated.

An example may help to make clear the method of calculation of
results. A certain enzyme solution had a protein nitrogen concentration
of 0.51 mg. per m1. It was diluted 25-fold and one m1. of the dilute
solution was used in an assay with cellulose sodium sulfate. The final
volume of the assay medium was 10 m1. Duplicate 0.2 m1. portions were
taken for sugar determination after two hours of incubation at 140° C.

The volume of 0.005 N sodium thiosulfate required for the reagent plus

83

substrate blank was b.65 m1. and for the assay, 3.11 ml. The difference,
1.5h m1., multiplied by the glucose reduction equivalent (0.13h), gave
the amount of reducing substances (as glucose) in the aliquot of hydroly-
sate: 0.206. Therefore, in 10 ml. of the hydrolysate there were 50 x
0.206, or 10.3 mg. of glucose or its equivalent. Since the original
enzyme solution had been diluted 25 times, it contained sufficient
activity in one ml. to release 10.3 x 25, or 257 mg. of glucose from

the substrate under the conditions of the test. The specific activity
in this case was 2S7/0.Sl, or 50h mg. of glucose per milligram of pro-

tein nitrogen.
Plectrophoretic Analysis

Electrophoretic analyses were performed with a Klett apparatus
which follows closely the design of Longsworth (1939, 19116). The light
source was a type H-h mercury vapor lamp. A Wratten No. 22 light filter
was found to be satisfactory. Visual observation of the progress of
electrophoresis was made with the aid of a diagonal straight edge,
cylindrical lens, and a ground glass screen. Patterns were recorded
‘photographically by means of'a schlieren scanning device.

Three different analytical cells of rectangular cross section and
ll mu. capacity were used throughout this study. In order to calculate
xnobilities, it is necessary to know the cross-sectional area of the
center sections of the cell, and the magnification of’the optical system.
The former constant was calculated, for both limbs of'each cell, from the
volume, determined by calibration with clean, dry mercury, and the length,

measured with an accurate caliper. All measurements were carried out in

8h

duplicate and the average used. The cross-sectional areas of the three
cells were: cell No. b, 0.768 for both limbs; cell No. S, 0.71:8 for
the left limb, 0.756 for the right limb; and cell No. 6, 0.756 cm2 for
both limbs.

The magnification of the optical system was determined by mounting
a transparent ruler vertically in the filled thermostat tank in the
place usually occupied by the cell. After adjusting the components of
the optical system to obtain a sharp image on the screen, a photograph
of the image was taken. Calculated as length of image/length of object,
the vertical magnification was found to be 1.05 at two different times.

Samples to be analyzed were in most cases dialyzed against two
changes of phosphate buffer of pH 6.9 and ionic strength 0.1146. The
details of preparation of this and other buffers are given in Appendix
B. This phosphate buffer was the only buffer used for electrophoretic
analysis. Consequently, subsequent statements concerning the number of
components refer only to these conditions and are to be regarded as
minimum.

The duration of dialysis was not less than 12 hours for each per-
iod. The sample was placed in a collodion sac of suitable size and the
sac was tied securely on the eXpanded end of a glass tube extending
through a rubber stopper. This assembly was then placed in a one- or
two-liter Erlenmeyer flask. Stirring of the outside buffer was pro-
vided by a magnetic stirrer; the agitation was vigorous enough to cause
moderate swaying of the collodion sac and mixing of its contents. All ‘
dialyses were performed in a cold room at a temperature of about 34° C.

For samples up to 50 ml. , one liter of buffer was used for the

first period, two liters for the second.

85

In several of the early eXperiments, the conductivities of the
buffer and sample were measured at the end of 12 hours of dialysis, and
the dialysis allowed to continue three more hours, after which conduc-
tivities were again determined. Lack of significant change in these
values showed that 12 hours of dialysis was sufficient in most cases.
However, the second period of dialysis against fresh buffer was neces-
sary in many instances because the conductivity of the outside buffer
was significantly different from the original.

This procedure was changed when circumstances demanded, for example,
'when solutions of acetone or ammonium sulfate precipitates containing
relatively large amounts of salts were used. In these cases, a pre-
liminary dialysis against about lO volumes of distilled water for 10
to 12 hours removed much of the salt. To avoid excessive dilution of
these samples due to their high initial osmotic pressure, the outlet
of the glass tube was closed tightly with a section of rubber tubing
and a screw clamp. If the internal pressure appeared to be excessive
at.any time, it was partially relieved by loosening the screw clamp.
‘After this preliminary treatment, the samples were transferred to two
Iliters of phOSphate buffer for the first dialysis, and two liters for
the second.

Solutions from the electrOphoresis-convection experiments, when
'the buffer was the same as that used for electrophoretic analysis, were
(iialyzed only once against two liters of buffer. In two instances, the
sanmfles were not dialyzed at all prior to electrophoresis. ,Although
the conductivities of these solutions were lower than those of dialyzed

solutions, the only adverse effect noted was a large boundary anomaly.

86

The mobilities were satisfactory, symmetry was unaffected, and no new
anomalies appeared.

Collodion sacs were used for dialysis, since it was found early in
the investigation that commercial viscose cellulose tubing was unsuit-
able. Ehzyme solutions of high activity weaken cellulose tubing to the
extent that it is apt to break when removed from the buffer. Moreover,
loss of activity, possibly due to adsorption on the membrane or to actual
leakage, was greater with viscose tubing than with collodion. The sacs
were prepared on the inner surface of test tubes of appropriate size, or
200 ml. centrifuge bottles, and were stored in distilled water at least
22; hours before use to remove residual alcohol.

The specific conductances of both buffer and sample were determined
after dialysis. A conductivity cell of the Shedlovslqr type (Longsworth,
Shedlovsky, and naclnnes, 1939) was used in conjunction with a Leeds and
Northrup Iheatstone bridge. The cell was suspended in the constant-
temperature bath of the electrophoresis apparatus and allowed to attain
temperature equilibrium before the final measurement was taken. This
procedure was always followed to make sure that the temperature of the
conductivity determination and the electrophoretic analysis was the same.

The cell constant was determined, at 20 C. , with a 0.1000 N potas-
sium chloride solution prepared from the dried C.P. salt in double-
distilled water. The water was freed from carbon dioxide by heating
to about 80° C. and drawing carbon dioxide-free air through at a rapid
rate for about 30 minutes. The resistance of water prepared in this
manner could not be accurately determined with the apparatus available.

The resistance of the cell filled with this water was greater than

87

1,000,000 ohms; when filled with ordinary distilled water it was about
LO0,000 ohms.

The temperature of most electrophoretic analyses was 2.0 z 0.10 C.
Certain exceptions are noted in the mobility tables.

Potential gradients varied from 6.26 to 6.93 volts/cm., depending
on the current and the conductivities of buffer and sample. In the
majority of cases, a potential gradient of close to 6.5 volts/cm. was
used. The current, about 20 milliamperes, remained uniform, rarely
varying more than 0.1 milliampere throughout the course of a run. Cur-
rent measurements were performed at intervals, using a Leeds and Northrup
potentiometer with a sensitive galvonometer. At the start of each ex-
periment, the potentiometer circuit was calibrated against a standard
weston cell.

The duration of current application for each analysis was the re-
sult of a compromise between the attainment of good resolution and the
maintenance of adequate height of each maximum. The protein concentra-
tion of some samples was necessarily quite low. In these cases, the
peaks representing minor components tended to decrease in height and
approach the base line with increasing time. This circumstance made it
necessary to stop some runs short of the best resolution of components.

Base lines and initial boundaries for both the ascending and des-
cending sides were photographed before the current was started and the
final patterns were taken on the same 9 x 12 cm. Kodak Panchromatic
IProcess or M plates by means of the schlieren scanning device. The
nexposed plates were developed with D-19 develOper in a semi-automatic

gilate processor in complete darkness.

88

The patterns were then projected and carefully traced on graph
paper at an enlargement factor of 2.5 times by means of an Omega D—II
photographic enlarger. Measurements for mobility and relative concen-
tration determinations were taken from these tracings.

Mobilities were calculated from measurements on the descending
patterns by means of the following equation:

u - qus/mit
where 2;is the mobility in cm? volt-1 second:1 d.is the distance in
centimeters on the enlarged tracing between the center of the initial
boundary and the particular maximum under consideration, q is the cross-
section of the descending side of the cell,‘1(_8 the Specific conductance
of the system, 5 the combined magnification factors of the electrophore-
sis apparatus and the photographic enlarger, i_is the current in amperes,
and E is the time of electrolysis in seconds (Longsworth and.MacInnes,
19ho).

The relative concentrations of the individual components were
determined by comparing the area under each peak with the total area
of the complete pattern minus boundary anomalies. areas were delineated
by the minimum ordinate method (Longsworth, 19b2). Area measurements
were taken from.the enlarged tracings with the aid of a planimeter.

The average of three separate measurements for each component was used.
The deviation of individual measurements from the average seldom ex-
ceeded :82 percent for the major components. The results from ascend-

ing and descending patterns were averaged.

89

Identification of bid Products

Paper chromatography was the only method used to identify the end
products of enzymatic hydrolysis. Identification of sugars by this
means alone is admittedly not positive, but it can be quite satisfactory
if the sugars which are likely to be encountered are definitely known
and if the eXperiments are properly controlled by the use of known sugars
on each chromatogram.

The hydrolysates of cellulose sulfate and of hemicellulase X con-
tained some soluble polysaccharide residues even after exhaustive treat-
ment with the enzyme. Although the possibility was not investigated,
there also may have been some soluble polysaccharide residues in the
centrifuged enzymatic mdrolysates of hemicellulose I. Levinson 32 fl.
(1951) found that the polysaccharide residues of cellulose sulfate inter-
fered with the migration of the sugars during chromatography, causing
undesirable trailing or comet formation. These workers removed the
larger molecules by precipitation with 50 percent ethanol and then
evaporating the filtrate to dryness either by lyophilization or on the
steam bath. The solids were then reconstituted to one-tenth of the
original volume. They stated that none of these procedures significantly
affected the amount of glucose or cellobiose present.

In this investigation, all hydrolysates which were to be used for
the chromatographic detection of sugars were centrifuged, when necessary,
to remove the insoluble residual substrate. Four volumes of absolute
ethanol were then added and the mixture was allowed to stand at ho C.
for at least three days. This long period was found to be necessary

to allow for the aggregation and settling of the very finely diapersed

9O

salts. The precipitate was removed by filtration (sintered glass filters )
and the filtrate was evaporated to dryness on the steam bath. The residue
was extracted with several small volumes of 80 percent alcohol and the
remaining solids were taken up in a small amount of water and saved. me
alcoholic extract was again evaporated to dryness on the steam bath and
the solids dissolved in a small volume of water. Reducing sugar analyses
of both of these solutions showed that extraction of the first residue
was not quantitative; only 65 to 85 percent of the reducing sugars was
extracted: Chromatograms of both sugar solutions later showed, however,
that the extraction removed approximately the same percentage of each
component.

Chromatograms obtained with these solutions were unsatisfactory be-
cause of extensive comet formation. This was probably due to the salts
which persisted after the extraction of the first residue with 80 percent
alcohol. lost of the remaining salts were removed by treatment with
Amberlite ion-exchange resins.

The cation-exchange resin, Ill-120, was prepared for use by treatment
with five percent hydrochloric acid followed by washing on a filter with
water until the filtrate was free of chloride. The anion-exchanger,
IRA-1:00, was regenerated before use by washing with 0.5 percent sodium
carbonate and then with water. These resins have been recommended by
Phillipps and Pollard (1953).

Deionizing was done by the batch process. One-half gram of the
Amberlite IR-l20 was shaken for five minutes in a test tube with 2 ml.

of the sugar solution. The mixture was filtered and the resin washed

‘- A

1Extraction of the residues with dry pyridine according to the
technique described by Malpress (19149) was not satisfactory.

91

on the filter with 1 ml. of water. One gram of Amberlite IRA—hOO was
added to the combined filtrate and washings and the mixture was allowed
to stand with frequent shaking until the resin no longer floated to the
top with adherent bubbles of carbon dioxide. If no more carbon dioxide
was evolved after the addition of a small amount of fresh anion-exchange
resin, the mixture was filtered and the resin washed with .3 small volume
of water.

The effectiveness of the deionising treatment was tested by applying
it to a 0.1 percent solution of glucose in 0.1 M sodium chloride. Glucose
analyses before and after treatment showed that there was no loss of the
sugar and conductivity measurements of the final solution (after boiling
briefly to remove the excess carbon dioxide) indicated that the sodium
and chloride ions had been almost completely removed.

The deionized sugar solutions were analyzed for total reducing
sugars (as glucose). On the basis of the results, small volumes were
evaporated to dryness E 32932 and reconstituted to a concentration of
approximately 25 micrograms per microliter.

The chromatographic technique described below was a combination of
features taken from several methods reported in the literature (Block,
Darwin, and Zweig, 1955).

Appropriate volumes of the concentrated sugar solutions were trans-
ferred to a sheet of Whatman No. 1 filter paper along a pencilled line
3-5 cm. from one short edge. In most cases the paper measured 10 by 15
1Belles. The distance between the individual spots was not less than 2 cm.

The Volme of sample deposited on the paper varied between 2 and 15

 

92

microliters: depending on the concentration of reducing sugars.

Known reference sugars (ho micrograms) were used on every chromato-
gram. For the hemicellulose hydrolysates, D-glucose, D-xylose, and
L-arabinose were always used, and in some cases D-galactose, D-mannose,
D—fructose, D—glucuronic acid and D-galacturonic acid were also included.
Cellobiose and D-glucose were the only reference sugars used for the
hydrolysates of cellulose sodium sulfate and wheat straw cellulose.

lhen the spots were dry, the filter paper was shaped into a cylinder I
by sewing the long edges together with cotton thread so that the edges
did not quite touch. Since the cylinders supported their own weight
even when wet with solvent, no supporting racks were necessary for as-
cending chromatography. The paper cylinders were simply allowed to
stand upright in the solvent covering the bottom of the chromatograph
chambers.

A large desiccator topped with a bell jar of the same diameter
served satisfactorily as a chromatograph chamber. It provided adequate
height to accounodate the cylinders, and when the ground glass surfaces
of the desiccator and bell Jar were greased, the joint was airtight. A
second chamber which proved to be useful was a large glass jar 10 inches
in diameter and 18 inches in height, with a screw cap 5.5 inches in dia-
meter. Occasionally, three chromatograms were developed simultaneously
in this chamber.

The paper cylinders were allowed to stand in the solvent-saturated

atmosphere of the chromatograph chambers for 21: hours before development

1To keep the area of the spots small, no more than it microliters
were delivered at one time. The first such spot was dried under an
infrared lamp before the next was superimposed. A microburette cali-
brated in microliters was used for this work.

93

was begun. They were held in l-liter tall-form beakers standing in the
solvent in the desiccator-bell jar containers, or suspended above the
solvent by threads held in place by the screw cap of the other chamber.

Two different solvent mixtures were tried. The chromtograms pro-
duced by deve10pment with the alcoholic phase of g-butanolmcetic acid:
water in the proportion hzlzs (v/v) (Bayly et 21., 1951) were found to
be unsatisfactory because of trailing of the spots and low Rf values.
The second solvent system tried, n—butanol:pyridine:water in the pro-
portion 6:133 (v/v) (Whistler and Smart, 1953), gave better results;
the spots were more nearly round and the sugars migrated at a faster
rate. All the chromatograms shown in the section on results were de-
veloped with this solvent.

The components of the hemicellulose hydrolysates were not resolved
sufficiently by a single ascent of the solvent. Consequently, the
multiple development technique used by Jeanes (1951) and by Pasur and
French (1952) was adopted. After the solvent had made one ascent
nearly to the top of the chromatogram, the cylinder was removed, dried,
and then returned to the chamber for a second passage of the solvent.
The time required for each ascent varied from 10 to 12 hours. In this
way the effective distance of the solvent front was doubled and the
efficiency of the resolution was proportionately increased.

The chromatogams were developed at room temperature, but were pro-
tected against extreme variations in temperature.

After the developed paper cylinders were dried in a gentle current

of air in a hood, they were unrolled and sprayed with an alcoholic

9h

1
p—anisidine phosphate color reagent (lukherjes and Srivastava, 1952).
The spots containing the reducing sugars became visible after heating
0
at 95-100 0. for five minutes.

The spots were identified by their R values, i.e., the ratio of

8
the distance traveled by the unknown spot to the distance traveled by
glucose (on the same chromatogram). The use of R8 rather than Rf values
obviates the necessity for strictly controlled conditions; variations in
the rate of migration due to temperature or other variables are auto-
matically taken into account (Hirst and Jones, 1919). Distances were
measured from the starting line to the center of the spot. The color
of the spots also helped to identify the sugars. Xylose and arabinose
gave dark brown spots, the aldohexoses and cellobiose light brown, and
the uronic acids reddish-brown areas. Fructose was easily identified
by the bright yellow color it produced with the reagent.

Sons of the end products of cellulose sulfate and hemicellulase
hydrolysis could not be identified by their R8 values. The unidentified
products migrated on the chromatograms at a slower rate than any of the
known sugars, except the uronic acids. To test the possibility that the
unidentified comments of the hemicellulose hydrolysates were disac-
charides or higher oligosaccharides and also to determine their compo-
sition, a small amount of each component was isolated and studied in

the following manner. Streak chromatograms were prepared by applying

0.07 to 0.12 ml. of the concentrated anddeionized hydrolysates in a

1
The preparation of this reagent is described in Appendix B.

 

 

95

streak along the starting line of a paper cylinder and developing twice
as described before. Narrow, vertical control strips were cut from both
sides, treated with the color reagent, and heated as before. Horizontal
sections corresponding to the spots on the control strips were cut from
the rest of the chromatogram and eluted with water. The elution tech-
nique was essentially the same as that described by Dent (1914?). One
end of a strip was placed between two glass plates resting on tin bottom
and the edge of a Petri dish filled with water. The other end, cut to a
point, was suspended over a small beaker. Water reached the paper by
capillary attraction, irrigated the strip and dropped into the beaker.
Five or six elution assemblies were placed under a large bell jar to
prevent evaporation. A period of 8 to 12 hours was required to obtain

1 to 3 m1. of eluate.

A small portion of the eluate was analysed for reducing sugar be-
fore hydrolysis, and a second portion was analyzed after hydrolysis with
0.2 N hydrochloric acid at 120° c. (autoclave) for us minutes. (Before
the addition of Somogyi's reagent, the hydrolysate was neutralized with
sodium hydroxide solution and diluted to 5 ml. with water.) A third
volume of the eluate was concentrated in m and transferred to a
chromatOgram. A fourth sample was hydrolyzed with 0.2 N hydrochloric
acid in the autoclave, concentrated, and chromatographed with some of
the unhydrolysed sample.

The unidentified components in the cellulose sulfate hydrolysates
were isolated in the same manner, but in this case the eluates were
analyzed for reducing sugars before and after hydrolysis and tested
qualitatively for sulfate ion (barium chloride) before and after hydroly-

sis. No chromatograms of the eluates were made.

Two-dimensional chromatography was employed in two cases to separ-
ate compounds which were not adequately resolved by the one-dimensional
treatment. The solutions, concentrated eluates obtained from streak
chromatograms, were spotted in a corner of a lO-inch square of filter
paper. The paper was fixed in the shape of a cylinder and developed
twice with the gfbutanolapyridineawater solvent system as previously
described. The cylinder was then unrolled and made into a cylinder
whose long axis was perpendicular to that of the first cylinder. It
was then developed twice with water-saturated phenol as the second
solvent.

The sugars present in the acid hydrolysates of the two hemicellulase
preparations were also identified by paper chromatography. The prepara-
tions were hydrolyzed with 1.0 N hydrochloric acid in the autoclave
(120° C. for 2 hours). The hydrolysates, perfectly clear but slightly

colored, were concentrated in vacuo and then chromatographed as before.

DETERMINATION OF PROTEIN NITROGEN

Protein nitrogen was estimated by a semimicro Kjeldahl analysis of
the precipitate formed when the enzyme solution was treated with tri-
Chloroacetic acid. Direct determination of the protein nitrogen in this
manner gave more precise results than did the indirect method of determ-
ining total nitrogen and non-protein nitrogen. Duplicate analyses of
total nitrogen usually gave results in good agreement, but duplicate
amilyses of the non-protein nitrogen in the trichloroacetic acid fil—
tMites yielded results in such poor agreement that they were considered

mu"Enable .

97

The final concentration of trichloroacetic acid was 2.5 percent.
According to Hiller and Van Slyke (1922), trichloroacetic acid in this
concentration is superior to all the other reagents they tested in
separating protein from non-protein nitrogen. 0n the other hand, Kirk
(19h?) stated that in all probability, all protein precipitants pre-
cipitate some non-protein nitrogenous substances along with the protein.
Strictly speaking, then, the results should be reported as trichloro-
acetic acid-precipitable nitrogen rather than protein nitrogen. How-
ever, for the sake of convenience, the latter term is retained, with
the understanding that the results may include a small amount of non-
protein nitrogen.

Duplicate volumes of the enzyme solution containing 0.2 - 2.0 mg.
of protein nitrogen were delivered into test tubes and 20 percent tri-
chloroacetic acid was added to a final concentration of 2.5 percent.
Heating the tubes in a hot water bath (22, 90° C.) for 5-10 minutes to
coagulate the proteins became routine practice. In many cases in which
the protein concentration was low, the solution merely became cloudy
‘when trichloroacetic acid.was added; only by heating could the protein
be made to coagulate. The tubes were centrifuged for 20 minutes, the
supernatant solution carefully decanted, and the tube inverted and
drained for at least 5 minutes. The precipitates were triturated with
5 ml. of 2.5 percent trichloroacetic acid, centrifuged, drained, and
washed a second time in the same way. After the final washing the
precipitates were dissolved in 2 ml. of 0.1 N sodium hydroxide and the
solutions transferred by pipette into semimicro Kjeldahl flasks. The

tubes were rinsed three times with 2 ml. of water and the washings

98

were added to the Kjeldahl flasks.

The samples were digested for 2 hours (after clearing) with 2.5
ml. of sulfuric acid, one gram of potassium sulfate and 1.0 ml. of 10
percent copper sulfate (pentahydrate). The ammonia liberated by making
the digest alkaline was distilled.with steam into 10.00 ml. of 0.02 N
sulfuric acid. The excess sulfuric acid was titrated with approximately
0.01 N sodium bydroxide‘. A mixed indicator prepared by combining 5
parts of 0.02 percent methyl red (sodium salt) and 2 parts of 0.025
percent methylene blue was used? Blank analyses were done with 2 ml.

of 0.1 N sodium hydroxide replacing the protein solution.

Results

Before describing the results of this investigation, it may be well
to review the definitions of activity and specific activity. .Activity
is a concentration term‘and is eXpressed as the milligrams of reducing
substances (calculated as glucose) which would be produced by one
milliliter of enzyme solution acting at too 0. for a specified period
on 100 mg. of substrate in 10.0 ml. of reaction mixture buffered at
pH 5.35 with 0.05 N citrate buffer. The reaction time for cellulose
sodium sulfate and salicin was 2 hours, for the hemicellulases and
cellulose, 2h hours.

The more meaningful term, specific activity, is defined as the
milligrams of reducing substances (as glucose) released under the con-

ditions specified above per milligram of protein nitrogen.

 

1The sodium hydroxide was standardized by titration against the
sulfuric acid each time it was used.

2The range of the color change from purple (in acid) to green was

99

Production and Concentration of Culture Filtrates

Fifteen l2-liter cultures of g} verrucaria were started. Two of

 

the cultures were discarded because of bacterial contamination. The
final activities (088)1 of the filtrates, the size of the inocula, and
the times of incubation of the thirteen successful cultures are given

in Table VII. It will be noted that the final activities cover a three-
fold range: 7.26 to 21.8. The controlled variables which might be
expected to influence the final activity are the size of the inoculum
and the nature of the cellulose substrate:

The results in Table VII show that within the limits used, there
was apparently no direct relation between the size of the inoculum and
the final activity. The smallest inoculum was 1.7 billion spores. In
one case this number of spores produced a culture filtrate having the
highest activity and in a second culture an average activity. One of
the largest inocula (1h.0 billion Spores) produced the lowest activity.
This inverse relationship was not general.

Although the activities of cultures 1h and 15, in which whatman

ashless powdered cellulose replaced Solka-Floc as the substrate, are

 

1For convenience, the substrates used to determine activity will
be indicated parenthetically, using the following abbreviations for
cellulose sodium sulfate, hemicellulase x, hemicellulase I, wheat straw
cellulose, and salicin: 088, x, Y, 0, SW

2The time of incubation is more of an affect than a cause since
each culture was continued until the activity was no longer increasing
rapidly or was actually decreasing. .

100

TABLE VII

THE INOQJLUM SPORE COUNT, TIME OF INCUBATION AND
FINAL ACTIVITY OF CULTURES OF iflROTHECIUM VERRUCARIA

 

Culture Inoculus Time Final ,
no. spore of activity (088)
count incubation
billions days Th
1 1.7 lb 21.8
2 1.7 19 12.3
s 1h.o 26 7.26
6 9.1 16 111.7
7 10.2 1h 13.6
8 7.6 18 12.7
9 7.9 21 12.5
10 5.7 17 13.1:
11 7.3 17 10.11
12 11.1. 11: 11.1
13 114.6 15 10.1
u.“ the 17 20.8
15" 5.6 16 18.2

4*
Activity ((3) is expressed as the milligrams of

reducing substances (calculated as glucose) released in

2 hours at 110° C. from 100 mg. of cellulose sodium sulfate

in 10.0 ml. of reaction mixture buffered at pH 5.35 with

0.05 I citrate by one ml. of the enzyme solution.

'l-l

lhatman powdered cellulose for chromatography was
the substrate for cultures 1b and 15; Solka-Floc was used
for all others.

101

both considerably higher than the average activity (12.7) of the first
eleven culture filtrates, it cannot be stated with certainty that the
nature of the substrate influences the production of the enzyme. The
data are too few and the occurrence among the Solka-Floc cultures of
one filtrate having an activity greater than either culture lb or 15
makes any conclusion highly tentative.

Variations in other conditions, such as the supply of oxygen and
the rate of stirring cannot be invoked because, although they were not
rigidly controlled, they were kept reasonably constant from culture to
culture.

The rate at which the activity accumulated in four of the cultures
is illustrated in Figure 3; complete data for all the cultures are pre-
sented in Table VIII. Three of the four curves in Figure 3 are diphasic;
a more or less pronounced increase in the rate of accumulation of
activity occurred at 10 to 12 days. The fourth curve is smooth. The
inflection also occurs (at the same time interval) in the curves (not
shown) for cultures 1, 2, 10, and 11, but not in those for cultures 5,
7, and 12. The possible significance of this phenomenon‘will be dis-

cussed later in connection with other observations to be described.

1

The culture filtrates, ranging in volume from 9 to 11 liters, were

concentrated by slow partial freezing to 0.1-0.2 of their original
volumes. The activity (035) and protein nitrogen content of the con-
centrates were determined; only the activity of the solution removed

‘8 ice was estimated. These results, as well as the specific activities

E

1Part of culture filtrate 5 was lost.

 

2C)

l5

5

(D

 

ACTIVITY OF CULTURE FILTRATE

 

 

 

l I l
C) 5 IC) l5 2!)

AGE OF CULTURE, DAYS

0 1

 

Figure 3. Accumulation of cellulase activity in cultures
of M. verrucaria. The substrate for determination
of enzyme activity was cellulose sodium sulfate.
A. Substrate for cultures 1h and 15: powdered filter
paper (Whatman ashless). B. Substrate for cultures
8 and 9: Solka-Floc.

 

102

TABLE VIII

THE ACCUMULATION OF CELLULASE ACTIVITY IN CULTURES OF !, VERRUCARIA

 

(Substrate: cellulose sodium sulfate)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Days Activity Days Activity’ Days Activity
Culture 1 Culture 7 Culture 11
3 7.80 3 3.10 3 2.2h
6 12.1 7 8.10 7 6.93
10 16.6 10 11.h 11 8.08
11 17.6 13 13.5 15 10.8
13 21.6 11‘ 13 06 17 10'"
1h 21.8 _¥
Culture 8 Culture 12
Culture 2
3 5038 h 60"?
3 8.72 6 8.68 8 9.65
6 10.5 9 10.3 12 10.9
9 10.9 12 11.2 1h 11.1
12 12.8 15 12.9 .11
15 12.8 18 12.7
19 12 03 - A ‘ Culture 13
“’ *‘ “‘ Culture 9 12 10.8
Culture 5 15 10.1
3 b.0h ___ _ ___
5 3.61 7 6.56
9 S 095 12 9020 Culture ”4
13 7.20 19 12.3
19 8.20 21 12.5 h 8.25
22 8.07 ____ _ 8 15.2
Culture 10 1h 21.3
17 20.8
Culture 6 h 5072 __*___._
7 8.86
6 5.68 15 13.5
16 1b.? 17 13.b 3 h.93
__h_‘___ _‘_‘;~ 7 13.8
"" ' 10 15.0
13 18.7

16 18.2

103

of the concentrates and the fraction of the original activity recovered

in the concentrates and in the ice, are presented in Table IX.
The specific activity (CSS) of the concentrates varied from 853 to

6&7. There was no apparent correlation between the activity of the
original culture filtrate and the Specific activity of the concentrate,
although the specific activities of concentrates lb and 15 were both
somewhat higher than any of the others, indicating that the relative
proportions of the proteins synthesized and excreted by g, verrucaria
may be influenced by the nature of the substrate.

An interesting and puzzling feature of the results in Table 11 is
the recovery of activity. This was calculated from the total activity
of the original filtrate (the product of the volume, in m1., and the
activity). The total recovery of activity ranged from 79.9 to an
apparent total recovery of 199 percent. In only one case did the total
recovery amount to less than 100 percent. The calculated recovery of
activity in the concentrates varied from 62.9 to lh3 percent of the
original. The ice contained 17.0-55.7 percent of the original activity.
The distribution of activity appears in a more favorable light if the
percentage recovery of activity in the two fractions is based on the
total activity recovered rather than on the activity in the original
filtrate. Calculated in this manner, the recovery in the concentrates
was 65.8 to 78.7 percent. In general, the greatest percentage loss of
activity in the ice occurred with filtrates which were concentrated to
the greatest extent.

Using the same method of concentration,'lhitaker (1953) reported

a recovery of about 95 percent of the activity, but did not state whether

TABLEIX

CONCENTRATION OF CULTURE FILTRATES BY

SLOW PARTIAL FREEZING

1011

 

 

 

Culture Activity Recovery Prot . Specific
no . Fraction Vol . (CSS ) of N . activity
activity (CSS)
1 o % mg 0 /m1 0

1 Original filtrate 9.10 21.8

I“ 7 015 9 062 3h 07

Concentra te 1.90 112 107 0 .210 533
2 Original filtrate 9.09 12 .3

Ice 7 063 5 J10 3608

Cmcentrate 10,85 5205 67 09 00116 1‘53
5 Original filtrate 7 .70 7.26

Ice

Concentrate 0.825 117.1 69.6 0.083 567
6 Original filtrate 9.16 18.7

ICC 7 0 3h 3 em 17 .0

Concentrate 1.75 118.5 62 .9 0.095 511
7 Original filtrate 9.01: 13 .6

Ice 7.21 6.21 36.11

Concentrate 1.66 65 .3 87 .8 0.131 500
8 Original filtrate 10 .00 12 .7

ICC 8 .22 8 .60 55 c7

Concentrate 1.65 110 1&3 0.206 5311
9 Original filtrate 9.65 12.5

Ice 8.12 5.00 33 .6

Concentrate 1 .145 95 .0 11).: 0 . 189 503

10 Original filtrate 9.98 131
Ice 8.76 7.0!. 1.6.0

105

 

 

 

TABLE IX concluded
Culture Activity Recovery Prot. Specific
no. Fraction Vol. (OS) of N. activity
activity was)“
1 . 5 ago. .
11 Original filtrate 10.63 10.1:
108 9005 S 0’83 111802
Concentrate 1.h7 85.5 11h 0.1h7 582
12 01-131mm filtrate 10.36 11.1
Ice 8072 18.26 32 03
Concentrate 1.61 5h.8 76.7 0.089 616
13 Original filtrate 9.62 10.1
10° 8 o 29 6 o 35 96 01
Concentrate 1.30 89.5 119 0.162 552
1h Original filtrate 9.93 20.8
10. 8020 1102 M03
Concentrate 1.70 123. 101 0.190 6h?
15 01'1ng 1 filtrate 10 .95 18 .2
Ice 9.15 10.3 10.3
Concentrate 1.70 120 103 0.187 6&1

P

a
Specific activity (CSS) is the mg. of reducing substances (calcu-

lated as glucose) released from cellulose sodium sulfate per milligram
of protein nitrogen under the standard conditions.

106

this was the total recovery. The impression is left that this amount
was recovered in the concentrates, and that the ice was not analyzed.

The increase in total activity accompanying the concentration pro—
cess cannot be explained on the basis of a stimulatory effect of in-
creased salt content. The dilutions necessary to bring the activity
of the concentrates into the range specified for all activity determ-
inations reduced the salt content to about the same level as that used
in assaying the original filtrates. Furthermore, neither sodium chloride
nor a sodium phosphate buffer (pH 5.3) had an appreciable effect on
the activity of a filtrate when they were included in the assay medium
in concentrations of 0.02 to 0.1 M. The activity of the filtrate with-
out added salt was 12.h, and 0.02, 0.05, and 0.1 M sodium chloride added
it was 12.6, 12.3, and 12.h, reapectively. The corresponding values
‘with ph03phate buffer in the same concentrations were 12.h, 12.3, and
12.3. These variations are within eXperimental error}

Basu and Whitaker (1953) demonstrated a slight (8 percent) stimu-
lation of the cellulase of !, verrucaria by 0.02 M sodium chloride at
pH 5.0. The effect was only 3 percent at 0.05 M.and disappeared entirely
at 0.1 I sodium chloride.

In a second eXperiment, a small amount of the original filtrate
of culture 9 was saved until the remainder had been concentrated. Its
activity (053) was then determined with and without the addition of

some of the concentrate which was diluted to the same extent required

 

lln a series of ten separate activity (CSS) determinations done in
duplicate on the same enzyme solution and with the same conditions, five
were within e2 percent of the mean, and nine within at percent. The
greatest variation from the mean.was 5.7 percent.

107

for estimation of its activity and inactivated by autoclaving for 15
minutes at 120° C. The activity of the original filtrate was 12.3,

that of the filtrate plus inactivated concentrate l2.h. The increase is
not significant. Thus no eXplanation based on eXperimental evidence

can be advanced for the effect of concentration by freezing.

Fractionation by Solvent and Salt Precipitation

The effect of the ethanol and acetone concentration on the pre-
cipitation of the activity of one of the concentrates is presented
graphically in Figure h. Each point represents the fraction of the
original activity (088) which was precipitated from a separate portion
of the concentrate by the slow addition of a predetermined volume of the
solvent. The pH of this undialyzed concentrate was 6.95, the protein
nitrogen content 0.21 mg./ml., and the specific activity 533.

Only about one-fourth of the activity was precipitated by 71 percent
(v/v) ethanol. The amount was less below and above this concentration.
With acetone, the results were better, 6h percent of the activity being
precipitated at an acetone concentration of 75 percent.

The protein nitrogen content of the precipitates was determined when
it was present in sufficient amount. The data, calculated as percentage
of total protein nitrogen, are presented in Table X. It will be noted
that with both acetone and ethanol, the fraction of total protein nitrogen
precipitated was greater in every case than the fraction of total activity.
Consequently, the specific activity of the precipitated proteins was always
less than that of the starting material, and generally decreased pro-

gressively with increasing concentration of solvent. More important, the

PERCENTAGE RECOVERY OF ACTIVITY

 

  

ACETONE

 

8

.5
O
I

0‘
O
l

3
I

ETHANOL

 

 

 

 

o . l l l l

30 4O 5O 50 70 80
CONCENTRATION OF SOLVENT, ‘79 0%)
Figure h. Effect of concentration of solvent in the precipi-

tation of the cellulase activity of concentrated culture
filtrates with acetone and alcohol.

 

 

TABLE X

108

EFFECT OF SOLVENT CONCENTRATION ON THE PRECIPITATION OF THE

_ __ _ __.__—_ *‘Ww

Ethanol precipitate

. M-~-__ -____

PROTEINS AND ACTIVITY (CSS) OF CULTURE FIlTRATES 0F .11. VERRUCARIA

 

 

Acetone precipitate

 

 

3°1vent Protein Activity' Specific Protein Activity ,speoiric
Concentration nitrogen activity nitrogen activity
$(v/v)‘ % of original % of original
33.3 1.5 2.9
h2.9 2.0 2b.2 22.8 502
b7.h 3.h h1.2 39.3 508
50.0 6.1 53.3 b7.o h71
52J4 55.7 b9.3 h71
Sh.5 1h.3 11.3 uzu
60.0 22.5 16.1 381 66.7 57.7 1163
66.7 36.h 23.2 3&0 7h.3 61.h hh2
71.h h1.o 25.1 326 76.2 63.6 hb6
75.0 77.1 6h.o hhz
77.7 h6.6 2b.6 282
80 .0 b7 .0 23 .0 261 63 .6 1429

E

79-0

calculated specific activities of the fractions precipitated between

Successive levels of ethanol were less than 533 in each case.

Wi th

a("Stone as the precipitant, the calculated Specific activity of the addi-

tional proteins precipitated by increasing the acetone concentration from

6607 to 7l.h percent was 615. However, since the amount of protein was

only (3.9 percent of the total, isolation of this fraction would be un-

Pr°fi table .

The calculated specific activity of the proteins precipitated

109

between all other successive acetone levels was less than 533.

The influence of salt concentration on the precipitation of the
active proteins was determined by dialyzing a portion of the concentrate
against 20 volumes of distilled water for 12 hours, then against the same
amount of 0.02 n phOSphate buffer (pH 6.9) for 21. hours before the addi-
tion of 3 volumes of acetone. Twenty-eight percent of the activity was
recovered in the precipitate. Protein nitrogen was not determined. In
a second experiment, the concentrate was dialyzed against 5.5 volumes of
distilled water for 21. hours prior to precipitation with 1. volumes of
acetone. In this case only 8.1 percent of the original activity was re-
covered. It appears that a high concentration of salts favors a greater
recovery of activity.

In an attempt to improve the total recovery of activity, the effect
of pH on solvent precipitation was studied. Portions of the concentrate
were adjusted to various pH levels by dialysis against 0.5 M acetate
(DH 11.0) or phosphate buffers until the pH of the concentrate was close
to that of the buffer. The final concentration of ethanol and acetone
'38 75 percent (v/v). The results, shown in Figure. 5, indicate that the

bes t recoveries with both solvents were obtained at pH 6.7 to 7.0. At
more alkaline reactions, the amount of activity precipitated fell off

r“PIT-(fly. The rate of decrease with change of pH toward more acid con-
ditions was less rapid.

The results of an attempted fractionation by means of ammonium sul-
fate precipitation are given in Table XI. The starting material was .250 ml.
Of ‘1 Concentrate having a protein nitrogen content of 0.116 m./ml, a

BPOC11‘1c activity of 1453, and an initial pH of 6.8. The precipitates

PERCENTAGE RECOVERY OF ACTIVITY

 

7° I I I I

   
 

ACETONE, 75 °/.

 

50

4O

20 ETHANOL,75 °/. _

 

 

 

pH

Figure 5. Effect of initial pH on the precipitation of the
cellulase activity of concentrated culture filtrate with
75 percent acetone and ethanol.

110

TABLE XI

ATTEMPTED AWZONIUM SULFATE FRACTIONATION
or g. VERRUCARIA CULTURE FILTRATE

 

 

 

 

Ammonium Protein Activity Specific
sulfate nitrogen Activity
3 saturation % of original
30 ' 1.1

52 30.5 26.0 387

69 13.2 11.9 1108

90 32.0 20.1 286

100 14.1 2.2 243

 

obtained at several levels of amonium sulfate saturation were analyzed
for activity ((38) and protein nitrogen. None of the fractions had a
specific activity as great as the starting material, although the pre-
cipitate removed at 69 percent saturation approached this value. Ninety-
three percent of the activity recovered, and 97 percent of the protein
nitrogen recovered, were precipitated by the time the ammonium sulfate
saturation reached 0.9.

Whitaker (1953 ), also working with E. verrucaria filtrates, found
that some enrichment of enzyme activity could be achieved by fractionation
with ammonium sulfate. Best results were obtained with a 1 percent protein
solution and an initial pH of 5.6. The precipitate obtained between 50
PBPCGnt and complete saturation had a higher specific activity than the
original material (no data given), but refractionation of this material

at. intermediate saturation levels was not successful. The same author

111

also reported some success with ethanol fractionation. The starting
material contained 3 percent protein in 3a. 0.01 ll phosphate , and had a

pH of 14.95. The precipitate removed at 5 percent (v/v) ethanol, con-
stituting a little less than one-fourth of the total protein, had a
specific activity about one-seventh that of the starting solution. The
protein which precipitated between 5 percent and 25 percent ethanol con-
stituted 63 percent of the total protein and had a specific activity 25
percent greater than the original. Jermm (1952b) reported erratic results
in attempts to concentrate or fractionate the enzymes of A. m
preparations by salt or solvent precipitation.

Attempts to increase specific activity by salt and solvent precip-
itation were unsuccessful. It is possible that the use of greater pro-
tein concentrations or different conditions would have given more favor-
able results. It is also possible that the explanation offered by Jermrn,
namely, the presence of more than one enzyme with the. same type of

activity, applies to these studies.

Concentration by Precipitation with Acetone and Ammonium Sulfate
The failure of fractionation by salt or solvent precipitation may
have been due in part to the low concentration of protein. Further con-
centration was achieved by precipitation with 80 percent acetone and
90-100 percent saturated ammonium sulfate.
The results with acetone precipitation, presented in Table III,
were erratic. The recovery of protein nitrogen varied from 135.5 to 90.2,
and the activity from 37.2 to 96.3 percent. The specific activity of the

extract was significantly decreased in two cases and substantially in-

creased in three others.

112

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113

Further precipitation of protein could not be induced by addition of
more acetone to the supernatant solution after removal of the precipitate
obtained at 80 percent acetone. Some of the protein loss was probably
caused by denaturation, for in each case an insoluble residue remained
after two extractions of the acetone precipitate with water. The nitrogen
remaining in three of the residues after two extractions with 2.5 percent
trichloroacetic acid was determined. Twenty-five percent of the protein
nitrogen of concentrate 1, 16 percent of concentrate 2, and 9 percent of
concentrate 11 were accounted for in this way.

With ammonium sulfate precipitation (Table XIII), the recovery of
protein nitrogen (50-80 percent) was generally less than with the acetone
method. The percentage recovery of activity (32-77) was less than that
of protein nitrogen in every case, resulting in a reduction of the Specific
activity. The precipitates obtained at 30-35 percent saturation with
ammonium‘sulfate in four cases contained 2-3 percent of the total protein
nitrogen and 0.5-2 percent of the total activity. The very small amount .
of insoluble material in the precipitates obtained by saturation with
amonium sulfate was not analyzed. Further precipitation of proteins
from the final supernatant solutions could not be obtained either by
complete saturation with ammonium sulfate or by addition of sulfuric
acid or sodium hydroxide.

Using the same method of concentration and practically the same
conditions, Whitaker (1953) recovered 86 percent of the protein and 91
Percent of the activity in the saturated ammonium sulfate precipitate,
and aceounted for an additional 12 percent of the protein and 2 percent

of the activity in the precipitate obtained at 30 percent saturation and

111;

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115

the insoluble residue from the saturated ammonium sulfate precipitate.
The protein nitrogen content of the enzyme solution before precipitation
was 0.3 mg./m1.

The protein nitrogen concentration of six of the starting solutions

in this investigation was less than 0.2h mg./ml. In the seventh (5,11A)

it was 0.6 mg./m1.

Fractionation by Chromatography on Cellulose Columns

Three eXperiments were performed to determine the feasibility of
fractionation of the cellulase solutions by chromatography on cellulose
columns. The procedure, described in the section on methods followed
closely the one outlined by Whistler and Smart (1953) for the purification
of £3 ggzzgg_preparations. .

The solution used for eXperiment A was 20 ml. of a dialyzed concentrate
adjusted to pH 3 with dilute hydrochloric acid. The protein nitrogen
content of this solution was 0.95 mg./ml., and the specific activity was
h07. The wash solution was distilled water adjusted to pH 3 with hydro-
chloric acid, and the eluant was a Clark and Lubs borate buffer, pH 9.0.

EXperiment B was the same as the first with the exception that the
concentrate was not dialyzed. The protein nitrogen concentration was
1.03 m2./ml. and the Specific activity'was bl7.

The enzyme solution used for eXperiment C was a dialyzed concentrate
adjusted to ionic strength 23. 0.1 with sodium chloride and to pH 3 with
hydrochloric acid. Twenty ml. of this solution, having a protein nitrogen
content of 0.90 mg./ml. and a specific activity of bOO, was used. The
wash solution was 0.1 I sodium chloride adJusted to pH 3 as before. The

eluant was a 0.05 M Clark and Lubs ph08phate buffer, pH 8.0.

116

The solutions percolating through the column were collected fraction-
ally, adjusted to pH 5.3 and analyzed for cellulase activity (088) and,
when possible, for protein nitrogen. The results are presented in Table
XIV.

In the first experiment, only 35.2 percent of the protein nitrogen
and 28 .6 percent of the activity was adsorbed on the column. A larger
proportion of the inactive (or less active) components appears to have
been adsorbed, since the Specific activity of the unadsorbed material
was greater than the original Specific activity. Such a small degree
of purification was considered unprofitable in view of the losses of
both protein nitrogen and activity. Slightly more than half of the
adsorbed activity was recovered in the eluates. One of the fractions
of the eluate had a Specific activity a little greater than the original.
Eighty-one percent of the protein nitrogen and 87 percent of the activity
were accounted for.

The greater ionic strength of the enzyme solution used in the
second eXperiment (B) apparently favored increased adsorption of the more
active proteins. The specific activity of the unadsorbed fraction was
reduced to about one-half of the original. However, the increased ad-
sorption of activity was of no advantage, because only 15.1 percent of
the total, or about one-fifth of the adsorbed activity was eluted. The
pattern of elution was similar to that of the first eXperiment. In this
run, 55 .6 percent of the protein nitrogen and 37.1: percent of the activity
were recovered.

Part of the loss of activity was probably due to inactivation of the

enzymes at pH 9. A loss of 114 percent of the activity was observed when

117

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115

a small portion of dialyzed concentrate was mixed with an equal volume
of the pH 9 borate buffer and allowed to stand one hour at room temper-
ature. Similar treatment with a neutralized (pH 7) borate buffer caused
no loss of activity, and the effect of a reaction of pH 3 for 30 minutes
was also negligible.

The third experiment (C) was designed to test the effect of an inter-
mediate ionic strength on the adsorption of the proteins, and to determine
whether a different buffer would elute a greater fraction of the adsorbed
activity.

The amount of protein nitrogen adsorbed was practically the same as
in the first experiment, but the activity retained by the column was
substantially greater. The pH 8 phOSphate buffer was not as efficient
as the borate buffer, only 10.2 percent of the protein and 10.7 percent
of the activity being recovered in the eluates. There was no indication
of any purification.

The degree of purification of Al. gigs}; cellulase preparations achieved
by Whistler and Smart (1953) cannot be assessed quantitatively for compar-
ison with the results obtained in this investigation because they re-
ported. no protein data. Qualitative evidence of some purification was
afforded by detection of cellobiose in cellulose hydrolysates produced
by the purified material. Cellobiose was not found in hydrolysates pro-

duced by the crude preparations,

Fractionation by ElectrOphoresis-Convection
Whereas the previous fractionation methods failed to yield the de-
sired results with consistency, electrophoresiSoconvection almost invariably

resulted in a substantial separation of the active from the inactive (or

 

119

less active) components, nearly every run yielding one fraction with a
significantly higher Specific activity and a second fraction with a

lower specific activity than that of the starting material. Large losses
of material were encountered with the other methods, but with electro-
phoresis-convection total recoveries of protein nitrogen and activity
were good, both being more than 90 percent in most cases. In some of
the cases in which recoveries were less, a precipitate was noted in the
bottom fraction.

The procedure has been described in the section on methods; the
results are presented in Table XV, which gives the volumes, protein
nitrogen concentrations, activities and Specific activities of the tap
and bottom fractions after electrOphoresis convection, and of the starting
solution before electrOphoreSis convection. Also recorded in this table
are the percentage recoveries of protein nitrogen and activity in the
final fractions, and in the last column, the ratios of their Specific
activities to that of the original solution.

It will be observed that the increase in Specific activity always
occurred in the top fraction, indicating that the mobility of the active
component (or components) was lower than the mobilities of the inactive
components. It will also be observed that the activity as well as the
protein nitrogen of the bottom cut increased in most cases} whereas these
values decreased in the top fraction. However, because the rate of change
of protein concentration was greater than the rate of change of activity

1111 some instances, the increased volume of the bottom out caused
by rinsing the bottom reservoir with buffer obscured the results in this

reSpect.

120

TABLE XV

THE FRACTIONATION OF CRUDE CELLULASE PREPARATIONS
BY ELECTROPHORESIS—CONVECTION

(The conditions and starting materials used for each
run appear in Table XVI.)

Run Free; Volume Protein nitrogen Activity (088) Sp. A'F
no. tim

 

 

Cone. Recov. Cone. Recov. spefcific S . Ls
.1 e .8 old 0 1 ‘

1"" s 35.0 620

T 21.7 89 e3 8 e9

8 32.5 363 58.8
2 s 60.0 710

‘r 38 .0 536 87.9

B 22.5 900 87.8
3 s 125.0 0.501 328 655

T 80.0 0.288 36.3 250 88.7 880 1.38

s 89.0 0.881 65.8 380 85.8 852 0.69
8 s 65.0 0.898 303 608

'r 32.0 0.336 33.3 256 81.6 758 1.25

a 37.0 0.507 58.0 285 53.3 562 0.92
5 s 85.0 0.870 300 638

'r 59.0 0.382 50.5 237 58.8 693 1.09

B 33 .0 0.812 38. 236 31.3 573 0.90
6 s 100.0 1.03 538 522

'r 71.0 0.558 38.2 853 59.9 819 1.57

B 3605 1.9:. 510“ 560 3709 386 007‘!-
7 s 87.0 1.92 750 391

'r 86.5 1.09 30.8 713 50.8 655 1.66

B 85.0 2.86 66.5 783 53.9 317 0.81
8 s 100.0 1.79 805 850

'r 89.5 0.930 25.1 708 83 .5 778 1.73

B 5605 2012 67 .0 763 5305 359 00m
9 s 106.5 2.00 768 388

'r 56.0 0.986 28.9 625 82.8 660 1.72

B 56.0 2.68 70.8 768 52.6 287 0.75

 

121

TABLE XV continued

 

 

Run Frac; Volume Protein nitrogen Activity (ass) Sp. 1.1. "
3°. ‘10“ Cone. Recov. Conc. Recov. Specific Sp. 1.3
‘10 ago/m1. if f

10 s 116.0 0.900 690 767

T 65.5 0.889 56.0 685 56.1 770 1.00

B 89.5 0.871 81.8 677 81.9 777 1.01
11 s 198.0 0.760 331 837

T 130.5 0.330 29.3 271 55.2 821 1.88

a 80.0 1.22 66.8 379 87.2 311 0.71
12 3 75.0 1.22 379 311

T 52.0 0.51 29.0 262 88.0 518 1.65

a 35.0 1.79 68.5 366 88.9 208 0.66
13 s 395.0 0.301 203 678

T 271.0 0.185 82.1 179 60.6 968 1.88

a 123.0 0.515 53.2 276 82.3 536 0.80
18 s 120.0 0.515 276 536

T 75.0 0.330 80.1 259 58.6 782 1.86

s 85.0 0.750 58.7 305 81.8 805 0.76
15 s 82.0 0.750 305 805

T 22.0 0.818 29.2 258 83.6 608 1.50

a 21.0 0.976 65.1 313 51.3 321 0.79
17 s 92.5

T 58.5 1.28 317 287

B 38.0 3.58 621 173
18 s 58.0 1.28 317 287

T 35.0 0.626 31.7 286 50.3 393 1.59

B 25.0 1.69 61.2 350 50.9 206 0.83
19 s 38.5 1.61 930 578

T 17.0 0.885 28.2 817 38.8 923 1.60

B 22.0 2.17 76.9 950 58.8 838 0.76
20 s 62.0 1.28 1080 813

T 88.5 0.770 83.2 985 65.3 1230 1.51

s 20.0 2.23 56.2 1100 38.1 893 0.61
21 s 80.0 1.10 531 883

T 26.3 0.538 31.8 363 85.0 679 1.81

s 16.0 1.88 68.8 703 52.8 378 0.77

 

Wm
B Frac- Volme Protein nitrogen

un

TABLE XV

concluded

 

Activity (w)

122

Sp. A.F

 

E" tics)" Conc . Recov. Conc. Recov. Specific Sp . A .5
n1 . mg ./ml. 5 i
22 s 117 .5 1 .37 501 366
T 102.0 0.863 58.7 868 80.3 538 1.87
B 23 .8 2.90 82.9 586 23 .6 202 0.55
23 s 101.0 0.863 868 538
T 81.5 0.885 85.3 880 76.5 903 1.68
B 22.0 1.96 89.8 622 29.2 317 0.59
28 s 81.3 2.00 2080 1080
T 31.5 0.980 35.6 1500 55.1 1600 1.58
s 26.5 1.99 63.3 1750 58.0 879 0.85
25 s 118 1.00 853 853
T 139 0.812 88.6 285 63.7 595 1.31
s 30.5 0.886 21.9 305 17.8 361 0.80
26 s 88.0 1.87 585 313
T 120.0 0.685 89.8 298 68.5 839 , 1.80
a 15.0 8.99 85.8 867 25.3 173 0.55
27 s 60.0 0.399 180 351
T 55.0 0.182 82.6 76.7 50.2 822 1.20
a 30.0 0.336 82.9 53.8 19.1 159 0.85
28 s 75 .0 0.761 282 320
T 61.0 0.388 81.1 189 63.2 892 1.58
a 16.0 1.07 30.0 216 19.0 202 0.63
29 s 138.0 1.13 510 851
‘1' 110 .0 0.636 51 .3 382 53 .8 538 1.19
B 31.0 0.987 18.8 312 13 .7 329 0.73

bottom fractions, respectively.

activity of the starting material.

*Tno letters S, T, and B indicate the starting material, top, and

“Ratio of the specific activity of the final fraction to the Specific

“*8 collodion membrane was used for the first eXperiment; viscose
cellulose dialysis tubing was used in all subsequent runs.

123

concentration, changes of Specific activity occurred. In other words,
although a portion of the active components was carried into the bottom
reservoir, their rate of tranSport was less than that of the inactive
components. The Specific activity of the top fraction increased 80
percent or more in 18 of the 25 runs for which complete data were ob-
tained. The entire range of increase was from 9 to 88 percent.

Factors which, according to theory, influence the degree of separ—
ation achieved in the top reservoir are pH and ionic strength (conditions
which determine the ratio of the mobility of one component to that of a
second; hence their differential rate of electrOphoretic tranSport), the
potential gradient across the channel and the length of time it is applied,
the initial concentration of total protein and of the various components,
and the volume of solution in the top reservoir. The conditions used for
each run are listed in Table XVI.

The first two runs were pilot experiments to determine whether
fractionation by electrophoresis convection was feasible. A collodion
membrane was used for the first trial. When the cell was emptied, a
relatively large amount of precipitate was found in the bottom reservoir
and on the anode wall of the channel. The activity of the solution in
the top reservoir decreased to about one-seventh of the original and
only part of the loss was accounted for by an increased total activity
in the bottom reservoir. It appeared that the protein was inactivated
and precipitated in some way as it passed through the channel. Whether
this was a specific effect of the collodion membrane or was due to some
other cause was not established since the second eXperiment, in which

Visking cellulose tubing was used, indicated that the use of collodion

membranes was not necessary.

 

TABLE XVI

CONDITIONS AND STARTING MATERIALS USED IN THE
ELECTROPHURESIS-CONVECTION FRACTIONATIONS

m

12h

 

fl—

 

 

Run no. Starting material. pH Tine Current;
hr. anp. *
1" Ce, 0,1oAcAs 6.9 11.0 0.50
2 Cs,s,1oAcAs 6.9 6.5 0.75
3 00,7 ,e,o,1oAcAs 6.9 114.0 0.50
h 00,? ,s,0,1oAcAs 5.0 7.0 0.50
5 00,7 ,s, s,1oAcAs 5.9 7.0 0.50
6 0.2.1.» 6.9 7.5 0.50
7 014“ 6.9 7.0 0.50
8 C1sAs 6.9 7.0 ‘ 0.50
9 m7,sB 6.9 7.0 0.50
10 scan"? 5.0 7.0 0.50
11 ms .4 .s,sB 6.9 7.0 0.50
12 sans 6.9 7.0 0.75
13 ms“ ,6 ,0,11 ,1sT 6.9 7.0 0.75
1!: sons 6.? 7.0 0.75
15 some 6.9 7.0 0.75
16 M12 .1158 6.9 7.0 0.75
17 $0,108 6.9 9.5 0.60
18 EMT 6.9 10.5 0.80
19 m1oT,Bm 6.9 7.0 0.75
20 mi: ,14 ,1s ,10 ,1sTL 6.9 7.0 0.80
21 ECsoB 6.9 7.5 0.75
22 E01? ,1 9,218 6.9 7.0 0.75
23 3023? 6.9 7.0 0.75

125

TABLE XVI continued

 

 

Bin no. Starting material* pH Time Current
hr. amp. —‘=

211a 3010.20.21 ,u T-L 6.9 6.0 0.75
b 3034 a8 6.9 6.5 0.75

c 3020 a ,bT 6.9 6.0 0.75
25s $22 ”1,108 6.9 h.0 0.65
b mesa? 6.9 3.0 0.65
mesa,bB 6.9 11.0 0.65

d mascB 6.9 11.0 0.65
26. Ge ,11 AcAs 6.9 7.0 0.75
b macaB 6.9 7.0 0.75

c saws 6.9 7.0 0.75
278 ”263 5.9 700 0060
b 300703 5 .9 11.0 0.50

0 cents 5 .9 h.0 0.50
28a -# 6.9 7.0 0.75
b E02008 6.9 7.0 0.75
29a ECss,ss,ssT 6.9 11.0 0.75
b sonar 6.9 . 5.0 0.75

c ECsea,bB 6.9 6.0 0.75

 

*The designations used to identify the starting materials have the
following meanings: C indicates that the material is a concentrate, the
minerals following specify the number of the concentrate“), Ac and As
indicate previous precipitation of the proteins with acetone and ammonium
sulfate, respectively, and EC followed by subscripts and either _T_ or B
shows that the material is the top (‘1‘) or bottom (2) fraction of. the
previous electrophoresis-convection BC) fractionation(s ) specified by
the numbers. E indicates that the so ution has been lyophillised and
reconstituted.

126

TABLE XVI concluded

a
A collodion membrane was used for the first experiment; viscose
cellulose dialysis tubing was used in all subsequent runs.

as
The top and bottom fractions of run No. 10 were combined and
concentrated by ultrafiltration.

“The starting material for run No. 28 was a mixture of several
residual fractions, some of which had been stored in the refrigerator
for several months.

127

The results of the third, fourth and fifth runs, all done with
portions of the same solution} showed that apparently a high pH (6.9,
run 3) would give better results than a low (5.0, run 1;) or an inter-
mediate pH (5.9, run 5). Conditions other than pH were not strictly
comparable, but the greater volume of the third run was partially com-
pensated by the longer time used (lb instead of 7 hours as in the other
runs). A later run at pH 5.0 (run 10) ended in failure to achieve any
separation whatever. According to the theory experimentally confirmed
by Brown St; 2.]: (1952), the best separations should be obtained when
the ratio of the lower to the higher mobility is small, i.e., when the
pH is near the isoelectric point of one component. Whitaker (1953) found
that the isoelectric point of his purified g. verrucaria cellulase was
near pH 5.0. Thus, the results obtained in this investigation were
contrary to that was expected. However, Brown .e_t_ a_l_. worked with a two-
component system of plasma albumin and gamma globulin, proteins with
widely different mobilities. In the present investigation there were
at least six components with fairly close mobilities at pH 6.9. Further-
more, the manner in which the mobilities changed with pH was not known.

The effect of ionic strength on the degree of separation was not
studied. All three buffers had nearly the same ionic strength (0.15).

Amcng runs reasonably comparable with respect to other conditions,

increasing the current from 0.5 to 0.75 ampere (and the voltage from

 

1The small but significant differences in activity and protein
nitrogen were caused by differences in the time of dialysis. The solutions
used for the fourth and fifth runs were dialyzed 2h additionalhours
against the new buffers. Some loss of activity was always found on
prolonged dialysis at all pH levels used.

128

22—23 to 30-33) seemed to have little or no effect on the results. Vari-
ations in the time of electrophoresis were not used often enough in com-
parable situations to allow any conclusions. ‘

Comparing runs 3, 6-9, and 11, in which the pH and current were the
same, it appears that there was a reciprocal relation between the initial
Specific activity and the degree of purification. Usually, when the
Specific activity of the starting solution was high, the separation of
inactive from active components was low; the converse was also true. It
also appears that the initial protein concentration influenced the re-
sults, a higher protein nitrogen concentration apparently favoring a
better separation. The same tendency is not so apparent in runs 12-23,
in which the current was usually 0.75. It is possible that the eXplana-
tion for this difference is that with the stronger current the same per-
centage clearance of the faster, inactive components was reached in a
shorter time, and during the remainder of the period, because of their
increased relative concentration in the top reservoir, a constantly
increasing proportion of the slower, more active components was trans-
ported to the bottom reservoir.

The highest specific activity (CSS) attained was 1600 in the top
out of the twenty-fourth run. This represented an increase of more than
three times the average specific activity of the unfractionated starting
solutions. Because of the decreased volume of the top cuts and the
modest separations achieved in each run, it was necessary at several
points to pool the top fractions of previous runs and use the combined

solutions for further fractionation. In some cases it was also necessary

to 1yophilize the combined top cuts and reconstitute at a smaller volume

129

because of the decreased protein nitrogen concentrations. Thus, the
starting solution for run No. 2h represented portions of the top cuts of

all previous runs. Some of these had been previously refractionated three

times.
Electrophoretic Analysis of Fractions
Obtained by'ElectrOphoresis-Convection

Fractionation by electrophoresis convection was controlled both by
activity determinations and electrophoretic analysis. Twentyefive of
the enzyme solutions were analyzed electrophoretically. The mobilities
of the components were determined to help in their identification, and
relative concentrations were calculated for use in conjunction with
activity data for the purpose of determining which component was most
active with a given substrate. All analyses were carried out with a
pH 6.9 ionic strength 0.1h6 phoSphate buffer. Other conditions are given
with the mobility data in Table XVII. Protein distribution data are
given in Table XVIII, and several electrophoretic patterns are repro-
duced by direct tracing in Figures 6 through 9.

The mobilities shown in Table XVII were all anodic. Only the values
for the four slowest components are shown since these seemed to possess
nearly all the activity. The mobilities of components 5 and 6 were
calculated in several cases. The average mobility of component 5 was 5.7
and of component 6, 7.2. There was considerable variation in the mobility
of each component. This might be eXpected on the basis of the wide
variation in the concentration of a component in the different solutions.
Differences in the duration of current application may also have influenced

the results. In Spite of the variations encountered, the mobility results

130

TABLE XVII

THE RECTROPHORETIC MOBILITIES OF FOUR OF THE PROTEIN
COMPONENTS OF E. VERRUCARIA CULTURE FILTRATES IN pH 6.9
PHOSPHATE BUFFER, 0.1h6 IONIC STRENGTH

 

 

 

 

 

32,... Conditions liabilities
solution Time Temper— Potential Components
ature gradient 1 2 3 h
min. 0 c volts/cm. cmz/volt/sec. x 10'5

was 180 1.9 5.99 0.63 1.3 2.5

sc-rs 220 1.8 5.70 0.98 1.5 2.8 3.h
20.3 220 2.0 6.56 0.86 1.); 2.5 ‘ 3.8
E78 270 2.0 6.26 0.69 1.2 2.1 3.2
150.3 270 2.0 6.32 0.70 1.2 2.1 3.1;
307,6,“ 180 2.0 6.62 0.85 1.5 2.5 3.7
mun 130 1.8 6.75 1.h 2.8 h.5
songs 180 2.0 6.79 0.70 1.1. 2.9 3.9
macs 180 2.0 6.53 0.87 1.6 3.1 11.2
some 130 2.0 6.57 1.5

30:23 150 2.9 6.53 0.77 , 1.5 3.0 11.0
H3228 150 2.0 6.53 1.3 2.7 3.7
100233 150 2.0 6.53 0.52 1.); 2.9 11.!)
sous 200 2.0 6.5!; 0.68 1.h 2.6 3.9
ECaaT 200 2.0 6.91 0.79 1.5 3.0

£0248 180 2.0 6.51; 0.72 1.6 3.0 h.1
mass 130 2.0 6.62 0.65 1.8 3.1 h.5
scum 160 1.9 6.614 0.59 1.3 2.7 14.2

131

TABLE XVII concluded

 

 

 

‘ ———:—7—
_—‘ I

 

 

 

 

 

Mayne Conditions liabilities
solution. Time Temper- Potential Components
ature gradient 1 2 ‘ 3 h
min. 0 C volts/cm. cmz/volt/sec. x 105
38203 150 2.0 6.69 0.85 1.1 2.5 3.8
ICQQT 130 2.0 6.618 0.8).! 1.3 2.6 11.1
1002.8 130 2.0 - 6.69 1 .3 2.7 3.9
some 130 1.9 6.53 3.0
1002.1 170 2.0 6.89 , 0.59 1.3 2.5 3.6
H3883 120 1 .9 6 .62 l .6 3 .1
30293 120 1.9 6.91 1.6 2.8 3.9
Mean 0.73 1.1. 2.7 3.9

Standard Deviation 0.122 0.17 0.29 0.31

132

TABLE XVIII

RELATIVE CONCENTRATIONS OF THE ELECTROPHORETIC COMPONENTS OF
SEVERAL FRACTIONS PREPARED FROM 11. VERRUCARIA CULTURE FILTRATES

___ A _—

 

 

Enzyme

 

 

Specific Relative concentration (2) Figure
solution activity Component no. no.
__¥ (CSS) 1 2 3 h <‘55 & 6__
sous 522 13.6 25.5 25.1 - 35.8 -
cows 391 211.6 12.7 21.8 28.8 12.3 611
Ross 1.50 20.6 18.0 28.). 23.3 13.8
save 317 1h.0 111.1. 21.6 26.5 23.5 6b
no.3 359 1t.8 16.6 22.1. 317.0 1h.1
1101, s, .1" 770 23.9 25.0 17.0 32.1. 1.5 6c
1801.3 2014 0.0 7.3 16.9 — 75.8 --
some 813 16.0 1.3.7 20.11 12.2 6.7
30208 1493 8.1 30.5 30.1 17.1 13.8
some 37h 0.0 21.8 - 59.6 - 18.1
was 366 9.9 13.1 3h.1 21.0 21.9 7a
scan 202 0.0 5.7 29.0 23.2 81.9 7b
was 317 3.2 10.1 1.2.0 22.9 21.6
sous 101.0 19.2 1.8.8 27.3 8.0 0.0 8a
1802.1 1600 30.7 63.3 6.1 0.0 0.0 (lb
1302.13 879 8.8 39.9 1.0.). 10.3 0.0 86
E0268 2.53 h.2 17.5 33.2 21.5 23 .3
FCst 595 7.8 25.1. 1.11.1. 13.8 8.6
813206 313 5.5 10.9 23.1 11.7 118.7
scar 1.39 11.6 15.9 33.3 17.6 21.5
313sz 173 0.0 6.0 12.3 7.1 714.5
was 159 0.0 0.0 3.8 - 96.2 — 9b
sour 1.92 9.1 37.0 26.9 10.2 17.0
was 202 0.0 6.1. 214.1 -— 69.5 --

302.8 329 0.0 19.8 38.3 25.1: 16.6 9.

 

133

were valuable for identification purposes, for there was no overlapping
of the ranges covered by each component.

The electrophoretic pattern of an unfractionated preparation ob-
tained by ammonium sulfate precipitation is shown in Figure 6a. Six
components, four major and two minor, are evident. In this reapect,
the pattern presented is typical of the original solutions before frac-
tionation. That the distribution of protein differed somewhat in the
various solutions may be seen by comparison of the relative concentrations
of components in the starting solutions for electr0phoresisaconvection
runs 6, 7, 8, and 26 in Table XVIII. Whitaker (1953), uSing similar
conditions for his analyses, found only two major and two minor com-
ponents in his preparations. He used a pH 6.8 ionic strength 0.2 phos-
phate buffer and a time of 120 minutes. His patterns, as reproduced for
publication, had no boundary anomalies.

After the solution whose pattern is shown in Figure 6a was subjected
to electrophoresis-convection (run No. 7), the bottom fraction had the
pattern shown in Figure 6b. It can be seen by visual inSpection and by
the results in Table XVIII that components 5 and 6 were increased and
component 1 decreased in this fraction. The large boundary anomalies
can be attributed to the fact that the solution was not redialyzed before
analysis. Figure 6c is the electrOphoretic pattern of the combined top
fractions of runs 7, 8, and 9. Components 5 and 6 almost disappeared,
and.the relative concentrations of the first two components increased
significantly. From the results in Tables XVII and XVIII it is evident
that most of the activity (033) was associated with the no... moving
components, since an.increase or decrease in these components was

accompanied by a similar change in the specific activity.

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The electrophoretic patterns in Figure 7 are shown in order to
illustrate the nonhomogeneity of two of the components. In Figure 7a,
component b apparently comprises two distinct parts. After electro-
phoresis convection, the Split was even more apparent in the bottom
fraction (Figure 7b), and component 6 also showed two distinct peaks.
These were the only components which showed this tendency; it was noted
in five other instances.

Both the tap and bottom fractions as well as the starting material
of “0 runs (2b and 26) were analyzed electrophoretically, affording an
opportunity to calculate the percentage recoveries of individual com-
ponents and at the same time to examine the accuracy of the electro-
phoretic analyses. The recoveries, calculated as the sum of the per-

centages recovered in the two final fractions, are presented in Table XIX.

TABLE XIX

PERCENTAGE RECOVERY OF INDIVIDUAL COMPONHVTS
IN RECTROPHORESIS CONVECTION F RACTIONATIONS

W

 

 

Run Component

N00 1 2 3 ll 5 and 6
21; 86.1 107 .3 102.7 81.8

26 106.5 98.3 96.3 103 .1 91.8

 

The results are of the order to be eXpected, taking into account the

overlapping of adjacent components in the electrophoretic patterns . A
small error in assigning areas to the minor components for calculation
of relative concentration would be magnified in the calculation of re—

coveries in Table XIX.

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135

The objective of the fractionation originally was to obtain several
of the components in highly purified condition in order to determine
which ones were active against several substrates. When it became
apparent that this was impractical with the amount of material available,
three solutions containing the suspected components in distinctly differ-
ent proportions were prepared. The electrOphoretic patterns of these
preparations are shown in Figures 8b, 9a, and 9b. Their sources were,
respectively, the tap fraction of run No. 2’4, and the bottom fractions
of runs 29 and 27. The three solutions are subsequently designated as
Preparations I, II, and III, reapectively. The proteins of Preparation I
were made up mostly of components 1 and 2, with only a small amount of
component 3. Preparation II contained all the components except com-
ponent 1, and Preparation III contained components 3-6, but no component
1 or 2. The resolution of components h-6 in Preparation III was not
satisfactory, but component 3 was well isolated and its mobility definitely
distinguished it from components 2 and b. Although the presence of a
given component can usually be definitely established, the same confidence
cannot be attached to statements concerning its absence. In this investi-
gation, this was especially true of component 1, because very small
amounts of this slow-moving component might be obscured by the boundary
anomaly. With other components, for example 2 and h in Preparations III
and I, respectively, amounts much less than 1 percent of the total protein
probably would not be detected by the Optical system of the apparatus,
eSpecially if the total protein concentration is low.

Inspection of the data presented in Table XVIII shows that most of

the activity (088) was associated with the three proteins possessing the

 

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~

 

 

 

 

[—9 ascending descending e—i

Figure 9a. Electrophoretic pattern of Preparation II.

4+:

 

 

[—9 ascending descending {—4

Figure 9b. Electrophoretic pattern of Preparation III.

136

lowest mobilities, and it appears that, of these, component 2 was the
most powerful. The direct relationship between the relative concentration
of component 2 and the specific activity is obvious. It also appears that
components 1;, S, and 6 had very little, if an action on cellulose sodium
sulfate. Although the results with Preparation III indicate that component
3 may be active, it is difficult to decide by casual inspection of the
results whether component 1 had any twdrolytic action on the substrate.

The coefficients of correlation between the relative concentration
of the components and the specific activity of the mixtures were calcu-

lated (Snedecor, 19116). The values are presented in Table XX. The

TABLE XX

COEFFICIENTS OF CORRELATION BETWEEN SPECIFIC ACTIVITY (CSS)
AND RELATIVE CONCENTRATION OF COMPONENTS

fl

 

 

Coefficient Signficance at
Component of

No. Correlation 5% level 1% level

1 0.71 + +

2 0.92 e +

1 + 2 0.911 + +

3 ' 0.10 + -

1 4- 3 OJJS * '-

2 e 3 0.72 + +

1 + 2 “f 3 0.82 h- +

h —O.Sh : +

5 ‘F' 6 .0067 4' §

 

137

conclusions stated previously concerning components 2, h, S, and 6 are

confirmed by the coefficients. The high positive correlation between

the relative concentration of component 2 and specific activity indicates

that this protein was more active than any of the others. The negative

coefficients for components 11, S, and 6 indicate that they had little,

. if any, activity.
A coefficient of 0.71, such as the one for component 1, would ordinarily

be interpreted to mean that there is a direct relationship between the

two variables, especially since it satisfied the test for significance

at the 1 percent level (Snedecor, 19116). However, interpretations in

this case must be made with caution. It must be remembered that because

of the similarity of the mobilities of components 1 and 2, any change in

the relative concentration of the first component during electr0phoresis

convection was reflected by a qualitatively similar change in the relative

concentration of the second. Thus, the relationship between relative

concentration of component 1 and specific activity may be more apparent

than real. On the other hand, when components 1 and 2 are considered

together, the coefficient of correlation, 0.9h, is higher than for either

one alone. If component 1 were completely inactive, the coefficient of

the combined proteins would be eXpected to be somewhat lower than that of

component 2.
For the same reasons stated before, component 3 cannot be said to be
active on the basis of the coefficient (0.10) even though the value is

significant at the 5 percent level. In this case, treating components

2 and 3 together resulted in an intermediate coefficient, and adding

components 1 and 3 gave a coefficient of 0.145, not significantly different

138

from thatcufcomponent 3 alone. However, the results with Preparation

III, whiChcrmtained neither of the two slowest components, indicate that

component 3 probably exerted weak hydrolytic action on cellulose sodium
sulfate.

Action of g. verrucaria Enzymes onflaGlycosides,
Extracted Wheat Straw, and Wheat Straw Holocellulose Preparations

The action of one enzyme preparation on arbutin, amygdalin, and

phloridzin was studied. The eXperiment was undertaken to compare the

susceptibility of thesel3-glycosides to the hydrolytic action of the

enzymes with that of cellulose sodium sulfate. The preparation chosen,

20,,T, contained all the original protein components (see electrOphoretic

The Specific activity with cellulose sodium sulfate
The

data, Table XVIII).
was 1439. The results with thefl—glycosides are given in Table XXI.

TABLE XXI

HYDROLYSIS ow -rucosmss WITH
{1, VERRUCARIA ENZYMES

(Enzyme preparation: BCasT) -.‘
Specific activity

 

 

 

 

Substrate 2 hours 7 hours
Arbutin 8.8 20.0
Amygdalin 2.0 7.7
Phloridzin 0.6 1.6

3119.

Cellulose sodium sulfate

 

139

results with arbutin were corrected for the presence of hydroquinone,
0.935 mg. of which was found to be equivalent in reducing power in
Somogyi's reagent to 1.0 mg. of glucose. In the case of amygdalin, no
attempt was made to determine whether the reducing sugar end product
was glucose or gentiobiose, or whether benzaldehyde contributed to the
reduction of copper. The concentration of phloridzin was 0.1 percent;
that of the other substrates was one percent. With this exception,
the conditions were the same as usual.

The results show that, under the conditions employed, thelg-glyccsidic
activity of the preparation was very low in comparison with its action
on cellulose sodium sulfate. The highest specific activity at two hours
incubation time was only 2 percent of the specific activity with cellulose
sodium sulfate. Because of the low results, differences of the specific
activities obtained with the threeIG-glycosides are probably of little
significance.

The results of an experiment comparing the action of two enzyme
preparations on extracted wheat straw and three wheat straw holocellulose
preparations are presented in Table XXII. The purpose was to determine
the effect of delignification on the availability of the substrates.

The enzyme preparations employed were the tOp and bottom fractions of
electrophoresis convection fractionation 19. The substrates have been
described previously. all conditions were standard. For comparison,
the results with cellulose sodium sulfate are included in the results.

.Although the three holocelluloses supposedly contain different

percentages of lignin (the designations l, 2, 3 refer to the number of

acid chlorite delignification steps each underwent), no consistent or

1&0

TABLE XXII

ACTION OF E. VERRUCARIA ENZYMES ON
EXTRACTED WHEAT STRAW AND WHEAT
STRAW HOLOCELLULOSE PREPARATIONS

r‘
‘

Specific activity

 

 

Substrate F019“? FTClgE'
EYtracted wheat straw 6.h 3.6
Holocellulose 1 13.9 q.g
Holocellu ose 2 114.8 5.?
Holocellulcse 3 12.9 5.9

923 . 1438 .

Cellulose sodium sulfate

significant differences in their enzymatic hydrolysis could be detected.
However, the extracted (ethanol-benzene) wheat straw was noticeably more
resistant than the holocellulOSes. Although other changes may have
been involved, the removal of most of the lignin probably was an important
factor in making the polysaccharides more available to the enzymes.
Comparison of Cellulase, Hemicellulase, anng-Glucosidase
Activities of Three Enzyme Preparations

Comparative activities with standard conditions. The Specific activ-
ities of Preparations I, II, and III with five substrates were estimated
and compared.to determine whether one, or more than one, enzyme was in-
volved in each.case. Standard conditions were used throughout; it will
be recalled that the standard incubation time for cellulose sodium sulfate

and salicin was 2 hours, and for cellulose and the hemicelluloses, 2h hours.

The results are shown in Table XXIII.

lhl

TABLE XXIII

COMPARISON OF SPECIFIC ACTIVITIES CF
PREPARATIONS I, II, AND III ACTING ON
FIVE SUBSTRATES

 

 

Specific activity Relative specific activity

 

 

substrate I II AIII_' I II III ‘__
Cellulose sodium sulfate 1600. 329. 159. 1.00 0.21 0.099
Hemicellulose x 912. 177. 106. 1.00 0.19 0.12
Hemicellulose Y Lab. 163. 109. 1.00 0.3h 0.23
Wheat straw cellulose 93.0 58.0 15.8 1.00 0.62 0.17
Salicin 231. 20.1 13.2 1.00 0.087 0.057

For easier comparison of the results with the three enzyme prepar-
ations acting on the same substrate, relative values have been calculated
by arbitrarily assigning Preparation I a Specific activity of 1.00 for
each substrate and calculating the relative values of the other two.
These ratios are also presented in Table XXIII.

The electrOphoretic composition of the three enzyme preparations has
been taken from the table presented earlier and brought together in
Table XXIV for greater convenience in correlating the electrophoretic and
activity'data.

The activity data with reapect to cellulose sodium sulfate were taken
from Table XVIII. Standing alone, the results agree substantially with
the main bodyof data presented in Table XVIII. Component 2 was the most
active in hydrolyzing cellulose sodium sulfate, but components 1 and 3

also probably had some hydrolytic action on the substrate.

1&2

TABLE XXIV

ELECTROPHORETIC COMPOSITION OF PREPARATIONS I, II, AND III

 

 

Relative concentration of component

 

 

Preparation
N .
° 1 2 3 u Sandé
percent of total protein nitrogen
I 30.7 63.3 6.1 0.0 0.0
II 0.0 19.8 38.3 25.b 16.6
111 0.0 0.0 3.8 96.2

 

The specific activities of the three preparations acting on hemi-
cellulose X were in nearly the same ratio as with cellulose sodium sul-
fate, indicating that the same enzymes hydrolyzed the two substrates. In
the case of'hemicellulose Y, the ratio was somewhat changed, making it
appear that components I and 3 were not as active with this substrate.

The relatively high Specific activity of Preparation III is difficult
to interpret without assuming that components h-6 possessed some activity.

With wheat straw cellulose as the substrate, the results were again
quite different from those with cellulose sodium sulfate. In this case,
component I probably had little activity. The distribution of activity
among the remaining components is not clear from the results. Preparation
II had a specific activity of 6b percent of that of Preparation I, where-
as its content of component 2 was only about one-third as great. The
activity of component 3 alone could not have made up the difference because
Preparation III had a specific activity nearly one-half as large as that
of II, but contained only about one-tenth as much component 3. Assuming
that component 2 was probably the most active, it must be tentatively con-

cluded that components b-6 contributed a substantial share.

lh3

The results with salicin were more clear-cut. Preparation I had a
high specific activity, whereas those of Preparations II and III were both
very low and differed from each other only slightly. It appears that
component 1 was the only really effective "salicinase". The activity of
the other components was very low with this substrate.

In summary, the results indicate that component 2 was the most active
enzyme with the four polysaccharide substrates. It was relatively more
active with cellulose and hemicellulose Y than with cellulose sodium sul-
fate and hemicellulase X. Components 1 and 3 evidently had some action
on the latter two substrates, but relatively less action on cellulose and
hemicellulose Y. Evidence that component b, S, or 6 had some hydrolytic
effect on all the polysaccharide substrates, especially cellulose and
hemicellulose Y, is fairly convincing. The results with salicin were
unique in that they indicated that a single component, 1, possessed almost
all the activity.

It is interesting to note the similarity in behavior of the three
enzyme preparations toward the two soluble polysaccharide substrates
(cellulose sodium sulfate and hemicellulose X). The results with the in-
soluble substrates (cellulose and hemicellulose Y) were similar to each
other but different from those with the soluble substrates.

Although it is not possible to assign specific activities to each
enzyme with the data available, the evidence is good that more than one
enzyme was involved with each substrate. It is also obvious that at
least one enzyme acted on both cellulose and hemicellulose. In other

words, at least one cellulase was identical with a hemicellulase.

1M:

‘Effect of pH on the rate of enzymatic hydrolysis. The effect of pH
on the rate of hydrolysis of the five substrates by the three enzyme
preparations was studied. In most cases, seven or eight pH levels, ranging
from pH h to pH 7, were used. Samples of the hydrolysates were analyzed
for reducing sugars at intervals over a 2h—hour period. The original
data, presented in Tables XXXVIII-XLII in Appendix A, were used to con-
struct progress curves. The curves for three of the pH levels used for
each combination of enzyme preparation and substrate are presented in
Figures lO-2b. For the sake of comparison, one of the curves representing
the hydrolysis of each substrate by Preparations II and III was reproduced
on the corresponding graph for Preparation I. The curves chosen for
presentation were usually the ones for the lowest and highest pH's and
for the pH which gave maximum activity. Because of the heterogeneous
nature of some of the substrates and the possible multiple nature of the
enzyme preparations, reaction rate constants were not calculated.

The curves representing the hydrolysis of a single substrate by the
three enzyme preparations are very much alike, but those illustrating the
action of'a single enzyme preparation on different substrates are quite
different. Whereas the curves for the hydrolysis of cellulose sodium
sulfate (Figures 10-12) showed a steadily decreasing rate of hydrolysis,
those for the hydrolysis of cellulose (Figures 13-15), hemicellulose 1
(Figures 16-18), and hemicellulose Y (Figures 19-21) were biphasic. In
all three cases, the relatively high initial rate of hydrolysis was fol-
lowed by'a rapid change to a long-continued, slower rate of reaction.

The effect*was most pronounced with hemicellulose Y.

“Elseth (IQhB) obtained the same type of results for the hydrolysis

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of ph08phoric acid-swollen cellulose by if gigs: cellulase preparations.
His evidence suggested that the rapid primary phase was due to hydrolysis
of the more accessible amorphous regions, and the slower secondary phase
to the hydrolysis of the more crystalline areas of the substrate. The
same explanation probably does not apply to the hydrolysis of the hemd-
cellulose preparations. In the first place, hemicellulose X was com-

pletely diSpersed. Secondly, both hemicellulose preparations used in this

 

study were probably mixtures. Furthermore, they were made up of several 2
different Sugar residues and very likely contained more than one type of
linkage. Branched chain structures were probably present. Some of the
polysaccharides in the mixture, and certain portions of other components
would probably be more susceptible to enzymatic hydrolysis than others.

The curves for the hydrolysis of salicin (Figures 22-2h) resembled
none of the others. The initial rate of hydrolysis decreased so slowly
that the curves approach the straight line characteristic of zero-order
reactions.

To study the effect of pH more effectively, the milligrams of re-
ducing substances per milligram of protein nitrogen at Specified times
‘were taken from the curves presented in Fisures lO-Qb and from similar
curves (not shown) for every pH level. These values are presented in
Tables XXV-XXIX. Because of the relatively large differences between
the absolute values for each of the enzyme preparations, graphical com-
parisons of the results in absolute terms would be difficult. To make
comparison easier, the maximum value for each enzyme preparation with
each.substrate was arbitrarily given a value of 100 percent and the

specific activities at the other pH levels in relation to the maximum

TABLE XXV

THE EFFECT OF pH ON THE ACCUMULATION OF END PRODUCTS
FROM THE DEGRADATION OF CELLULOSE SODIUM SULFATE
BY THREE DIFFERENT ENZYME PREPARATIONS

1116

 

 

 

mule pH Reducing substances (as glucose)
preparation 20 minutes 2 hours 211 hours
Isa/.80 ‘ 0f Mo/llgo x or mg./ng. % Of
prOtvo N. ”I. prOto N. m. prOto N. m.
I 3.78 110 30.6 630 38.11 3120 57.6
11.211 2110 66.7 1150 70.1 11th 81.9
b.80 320 88.9 11180 90.2 5280 97.1.
5.10 360 100.0 161.0 100.0 51.20 100.0
5.311 3115 95.8 1550 9105 5370 99.1
5.95 297 82.5 11110 86.0 1.8110 89.3
6.145 290 80.6 1360 82.9 11380 80.8
6.98 160 MM. 920 56.1 3260 60.1
7.37 125 3107 615 37.5 21.20 1114.6
7.55 90.0 25.0 3150 27.11 2200 110.6
II 11.15 67.0 62.0 263 77.8
11.75 9h .0 87 .0 33b 98. 8
5.05 108 100.0 338 100 .o
5.35 106 98.1 325 96.1
S .90 103 95.11 322 95 .3
6.115 89.0 82.1. 285 88.3
6.95 72.6 67.2 239 70.7
111 1015 25.0 511.2 101. 6h.6 280 72.2
11.75 38.0 82.1: 156 96.9 1.02 10!.
5.08 116 .1 100 .o 161 100 .o 388 100.0
5.37 115.5 98.7 157 97.5 378 97.1:
5.65 115.0 97.6 151 93.8 358 92.3
6.00 hb.o 95.11 1h8 91.9 318 81.9
6.51 1.2.6 92.1. 1111 87.6 295 76.0
6.90 39.9 86.6 1121 70.8 231 59.5

_

TABLE XXVI

lb?

THE EFFECT OF pH ON THE ACCUMULATION OF END PRODUCTS

FROM THE DEwDATION OF WHEAT STRAW CELLULOSE
BY THREE DIFFERENT ENZYI~IE PREPARATIONS

 

; # ————.—

 

 

 

 

Enzyme pH Reducing substances (as glucose)
preparation 2 hours 8 hours 211 hours
mg./mg. % of mg./hg. i of mg./mg. % of
prOto N. m. prOto N. m. prOt. N mx.
I 11.15 2806 7503 .3505 6605 14806 50.).
11075 37 e? 99 02 67 o3 98 I" 9006 9h00
S .10 3 8 on 100 .0 68 a" 100 .0 96 oh 1m .0
5.33 37.3 98.1 68.0 99oh 98.7 98.2
5.95 27.2 71.6 h8.9 71.5 55.9 58d0
6.85 28.0 63.1 89.7 72.7 61.9 68.2
6.81 19.1. 51.0 1411.0 614.3 51.1 53.0
II 8.18 12.3 78.1 23.7 66.3 39.2 68.3
13072 11107 8805 3209 9207 5700 93.0,}
5.10 16.6 100.0 35.5 100.0 61.0 100.0
5.0 1501 9100 3202 90.7 56.1 92.0
6.80 9.8 59 22.6 63.7 33.3 62.8
III 8.20 3.0 71 7.0 78 11.9 73.9
8.80 11.0 95' 8.7 97' 15.8 98.1
5.10 8.2 100.0 9.0 100 .0 16.1 100.0
5.10 11.0 95 8.7 97 15 .5 96.3
6600 3.6 86 8.h 93 13.7 85.1
6.50 2.9 69 7.0 78 11.6 72.0
6.85 2.5 60 6.0 67 10.h 65.0

0‘4-

 

TABLE XXVII

THE EFFECT OF pH ON THE ACCUMULATION OF END PRODUCTS
FROM THE DEGRADATION OF HIMICELLULOSE X
BY THREE DIFFERENT ENZYME PREPARATIONS

1118

 

A

 

 

 

mums pH Reducing substances (as glucosej
preparation 2 hours 8 hours 21; hours
mg Jug . 5 of mg.,/mg. % of mg./mg. 5 of
Profit No We probe N. m. PI‘OIM No max.
I [1.05 20!. 68.2 368 62 .h 1.80 52 .3
14.61: 286 95.6 580 98.3 9811 107
h .91! 296 99.0 583 98 .8 998 109
5.25 299 100.0 590 100.0 918 1.00.0
5.55 281 911.0 570 96.6 9014 98.5
5 .36 290 97 .0 575 97 J: 961 105
6.22 275 92.0 561 95.1 91.8 103
6.197 271 90.6 51.6 92.5 816 88 .9
II 15.09 31'. 03 6206 we" 5309 93.2 ”908
11.65 116.5 81.8 85 .3 76.2 159 85.0
31.95 118 .6 88.7 91.0 81 .3 1.66 88. 8
5 .25 50.2 91 .6 97 .2 86.8 173 92 .5
5.80 58.8 100.0 112 100.0 187 100.0
6.20 148 .0 87 .6 97 .0 86.6 179 95 .7
6 .110 116.6 85 .0 93.0 83 .0 168 89 .8
III 11.15 23.2 59.9 39.6 59.5 511.8 52.2
14.75 33.11 86.3 57.0 85 .7 93.5 89 .1
5 .10 38 .3 99.0 63 .9 96.1 101 96.2
5.35 38.7 100 .0 66.5 100 .0 105 100.0
5.95 3805 9905 65.8 98.9 1018 99.0
6.115 38.0 98.7 65.0 97.7 102 97.1
6.90 36.0 93.0 62.3 93.7 96.8 91.8

TABLE XXV III

FROM THE DEGRADATION OF HBJICELLULOSE Y
BY THREE DIFFERENT EI‘IZYME PREPARATIONS

THE EFFECT OF pH ON THE ACCUMULATION OF END PROWCTS

1249

 

 

 

buns pl! Reducing substances (as glucose)
prepsrstdon 2 hours 8. hours 211 hours
Ig./ng. 1 of ng./mg. 5 of ng./mg. Z of
prot. 71. max. prot. N. m. prob. N. m.
I 8.15 21.1; 7h.8 293 61.3 311. 61.2
11.70 323 99.1 1160 96.2 506 98.6
5030 ”I 9352 11113 9207 1882 93.9
5 .91: 326 100 .0 h? 8 100.0 513 100.0
6.130 297 91.1 1185 93.1 1177 93.0
6.70 270 82.8 386 80.7 1120 81.9
II b .015 50.06 79 .6 m .0 73.01‘ 99 Co 58.09
5 .10 62.0 97.5 109 1(1) .0 166 98.8
5.35 61.8 96.5 108 99.1 16!. 97.6
5 .83 63 .6 100.0 109 100 .0 168 100 .0
6.110 56.8 89 .3 101 92.7 160 95.2
6.61 55.2 “.8 96.9 88.9 m6 %.9
8.75 83.8 98.2 62.5 88.1 102 93 .6
5 .10 1.5.2. 97 .6 69.1 97.5 107 98.2
5.35 116.5 100.0 70.9 100.0 109 100.0
5.90 115 .0 96.8 66.0 93.1 105' 96.3
6.210 112.6 91.6 61.5 86.7 99.2 91.0
6.90 39.0 83.9 59.7 811.2 91.0 83.5

 

‘-
‘_-s

TABLE XXIX

THE EFFECT OF pH ON THE ACCUMULATION OF END PRODUCTS
FROM THE DEGRADATION OF SALICIN

BY THREE DIFFERENT ENZYME PREPARATIONS

150

 

 

 

Fnzy-e pH Reducing substances (as glucose)

preparation 20 minutes fiw_r 2 hoursw 28 hour;*
ng./hg. 1 of ng./ng. S of mg./ng. iyof

prot. N. max. prot. N. max. prot. N. max.

1 8.15 28.3 62.9 153 67.1 1120 75.2

8.75 83.0 95.6 218 95.6 1870 98.6

5.35 85.0 100.0 228 100.0 1890 100.0

5.95 80.0 88.9 195 85.5 1360 91.3

6.85 30.0 66.7 155 68.0 988 63.1

II 8.15 2.8 63 12.6 68.6 102 67.5

5.35 3.8 100 19.5 100.0 151 100.0

5.95 3.6 95 17.0 87.2 115 76.2

III 8.15 1.8 69 8.5 65 88.6 50.6
8.73 2.0 77 9.5 73 78.8 89.0

5.35 2.6 100 13.0 100 88.1 100.0

5.98 1.9 73 9.7 75 82.9 98.1

6.85’ 2.0 77 9.6 78 62.2 70.6

6.85 1.2 86 7.5 58 88.9 51.0

 

151

were calculated. These relative values, also presented in Tables XXV-
XXIX, were used to construct the pH—activity curves shown in Figures 25-29
The curves for three incubation times were compared.

The curves in Figure 25 for Preparation I acting on cellulose sodium
sulfate, show two distinct maxima at 20 minutes and 2 hours. The larger
peak occurred at pH 5.1. The Smaller one at pH 6.2—6.3 was no longer
definitely in evidence after 28 hours. Preparation 11 also had two activ-
ity peaks, the major one occurring at pH 5.1 at 20 minutes, the secondary
one at pH 5.8. After 2 hours, the major peak had shifted to pH 8.9. For
Preparation 11], there was only one distinct peak (pH 5.1) during the
early stages of the reaction. Later, the activity optimum shifted to
pH 8.75. On the alkaline side of the optimum pH, the curve was at first
rather flat, with some indication of a peak at pH 6.2-6.5.

‘When cellulose was used as the substrate (Figure 26) only one distinct
maximum was evident for each enzyme preparation at 2 hours. The maxima
occurred at pH 8.95, 5.25, and 5.10 for Preparations I, II, and 111,
respectively. A second peak at pH 6.8 was noted for Preparation I at
8 hours; it became more distinct at 28 hours. At the same time the pH
of the major maximum had changed to 5.1. Preparation 11 showed only one
peak at 8 hours, but at 28 hours there was some indication of a peak at
about pH 5.9. The pH of the major maximum changed from 5.25 at 2 hours
to 5.1 at 28 hours. Preparation III showed a second peak at pH 5.9 at
8 hours, but it did not persist at 28 hours.

Two almost equal maxima may be seen in the pH-activity curves for
Preparation I and hemicellulose X. The first shifted from pH 5.2 at 2 hours

to 8.9 at 28 hours. The second Optimum was stationary at pH 5.9.

 

PERCENT OF MAXIMUM ACTIVITY

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Prep. No. I
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I l I .J
4 5 6 7

pH

Figure 25. The effect of pH on the hydrolysis
of ce llul ose sodium sulfate by three
enzyme. preparations. The relative activ-
ities at other pH levels were calculated
by assigning a value of 100 percent to the
maximum activity of each enzyme preparation.

  

 

 

PERCENT OF MAXIMUM ACTIVITY

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8 hours
\
r' ——Prap. No.11 _
---- Prep. No.111
J i J l l
4 5 6 7

pH

Figure 26. The effect of pH on the hydrolysis

of wheat straw cellulose by three enzyme
preparations. The relative activities at
other pH levels were calculated by assign-
ing a value of 100 percent to the maximum
activity of each enzyme preparation.

PERCENT OF MAXIMUM ACTIVITY

 

 

/ f 2 hours

 

IOO

90

80

7O

60

 

 

 

.. l 24 hours _

 

 

 

50

 

1 .
,' Prep. No.1
1' — — Prep. No.11:
l
r- ” ------ Prop. No. III a
I ' I 1 L
4 5 6 7
pH

Figure 27. The effect of pH on the hydrolysis
of hemicellulose X by three enzyme prepar-
ations. The relative activities at other
pH levels were calculated by assigning a
value of 100 percent to the maximum activ-
ity of each enzyme preparation.

PERCENT OF MAXIMUM ACTIVITY

 

I00—
90—

80— [I -

 

I 2 hours

 

HDCIF

90-

80

70

 

. 8 hours

 

IOO

90

80

7O

  

24'hours

  
 
 

 

Prep. No.1
— — Prep. No.11
----- Prep. No.IlI _

 

 

50

 

prI

Figure 28. The effect of pH on the hydrolysis
of hemicellulose Y by three enzyme prepar-
ations. The relative activities at other
pH levels were calculated by assigning a
value of 100 percent to the maximum
activity of each enzyme preparation.

PERCENT OF MAXIMUM ACTIVITY

 

90-

80-

70-

60-

 

IOO-
90r-
so-

p70~

60-

 

 

I00—

90c

80-

7O

60

 

   

24 hours \

 

Pffip. No.1 \
----- Prep. No.1]! \ ..

 

I
I
I
I
I
I
I
I
I
I
I
I
I

 

50

“—
0|
0)

pH

Figure 29. The effect of pH on the hydrolysis
of salicin by two enzyme preparations.
The relative activities at other pH
levels were calculated by assigning a
value of 100 percent to the maximum
activity of each enzyme preparation.

152

Preparation II also had two activity peaks, but in this case one of the
peaks was not very pronounced. The pH of the major maximum was 5.8,
coinciding nicely with the secondary optimum of Preparation 1. The minor
peak of Preparation II came at about pH 8.7, somewhat lower than the
mayor peak of Preparation I. The activity of Preparation Ill showed
only one Optimum pH range at 5.8-5.8. Above pH 5.8, the activity showed
little tendency to decline until pH 6.5. The result was a broad, flat
curve.

The results with hemicellulose I were similar to those with hemi-
cellulase X. Both Preparations I and II had two distinct maxim-a. The
peaks at the higher pH coincide very well at pH 5 .95. The smaller peak
of Preparation I, at pH 8.7, held constant throughout the 28-hour reaction

period. The secondary peak of activity of Preparation II was at pH 5.0-5.1.
The activity of Preparation 11 I was highest at pH 5.8; no other peak was
evident.
The single optimum for the action of Preparation I on salicin was
at pH 5.1-5.2. The main optimum for Preparation III was at pH 5.35; a
smaller peak at pH 6.8 was quite distinct at 20 minutes, less so at 2
hours, and had disappeared entirely at 28 hours. Only three pH levels
were used with Preparation II, so pH-activity curves could not be constructed.
The results described above are summarized in Table XXX and are in
'eneral agreement with the interpretation given to the comparison of
aecific activities of the enzyme preparations at a constant pH. The
idence suggests that at least two of the protein components were capable
catalyzing the hydrolysis of each of the substrates used in this study.

the case of wheat straw cellulose, most of the activity appeared to be

153

TABLE XXX

pH OF ACTIVITY MAXINA OF THREE
ENZYME PREPARATIONS

(TheMpH of the main peak is given first.)

anyme preparation

 

 

Substrate I II III
Cellulose sodium sulfate 5.], 6.3 h.9-5.1, 518 5.1, --
Wheat straw cellulose 5.1, 6.b 5.1-5.3, 5.9? 5.1, 5.9?
Hemicellulose X b.9-5.2, 5.85-5.95 5.8, h.7 5.h-5.8, ~-
Hemicellulose Y 5.9;, 14.7 5.95, 5.04.1 5.11, -
Salicin ‘S.1~5.2, - 5.35, 6ob

 

associated with a single component, but at least one other probably con-
tributed a small part of the activity.

The results with Preparation III, eSpecially'with reapect to its
action on cellulose and cellulose sodium sulfate, deserve further comment.
The evidence suggests that the maximum which occurred at pH 5.1 with
Preparations I and II was due to component 2. Preparation III supposedly
contained no component 2, yet the only clear-cut maximum obtained with
this preparation was at pH 5.1. Two different enzymes could, of course,
have the same optimum pH. However, a second possibility which should
not be neglected is that, in Spite of the electrOphoretic evidence to the
contrary, Preparation III may have contained a small amount of component
2. On the other hand, the PH Optima for Preparation III with hemicelluloses

X and Y, and salicin do not coincide with any of the optima of the other

two preparations.

194

It should also be emphasized that the occurrence of two peaks in
pH-activity curves does not necessarily constitute proof that two enzymes

are involved, especially with substrates such as the hemicellulose prepar-

ations. With these substrates, the occurrence of two peaks could be

interpreted on the basis of different pH Optima for the hydrolysis of
two types of linkage.

End Products of Enzymatic Hydrolysis

Chromatograms of the cleared, concentrated hydrolysates of cellulose

sodium sulfate and wheat straw cellulose are shown in Figure 30. The R?

value of each of the spots is given in Table m1, in which the spots are

numbered downward from the spot corresponding to glucose (the largest,

densest spots).

The major end product in each case was D-glucose. 'Ifim unidentified

spots can be seen in the chromatogram of cellulose sulfate hydrolysates

obtained with Preparation I (columns A and B, Figure 30). The R? values

of these Spots, 0.110 and 0.114, do not correSpond to the RE value of any

of the known sugars used in later chromatograms. With Preparation II a

prominent spot at R8 0.52 was found. The same Spot occurred with the hy-

drolysate from Preparation Ill (Rg 0.51;) as well as a Spot (kg 0.69) corres-

ponding to the position of cellobiose (58 0.71).

With wheat straw cellulose and enzyme Preparation I (column F, Figure

30), the Spot correSponding to glucose appears to be elongated. The tap

portion of this spot was a bright lemon-yellow color in contrast to the

brown color characteristic of glucose. Its R8 of 1.08 corresponded fairly

well to that of fructose in a later chromatogram. There was also an indi-

cation of the presence of fructose in the hydrolysates of cellulose sodium

 

 

3
{F 2
2 2
3 3
A B E [3 E E-

Figure 30. Products of hydrolysis of cellulose sodium sulfate and
cellulose by three enzyme preparations at pH 5.1.

A and B. 0%, Preparation I, 21: and 96 hr., reap. D. cs5,
Preparation II, 96 hr. 8. CSS, Papa-ation III, 96 hr. F. 'heat
straw cellulose, Preparation 1, 96 hr.

C. Glucose (g) and cello-
biose (c).

155

TABLE XXXI

Hg VALUES 0? SPOTS C'N CHROWTOGRMIS 0F ENZYMATIC HYDROLYSATES
0F CELLULOSE SODIUM SULFATE AND WHEAT STRAW CELLULOSE

(See Figure 30)

 

 

 

Enzyme Time of Spot
Column Substrate prep. hydrolysis No. - ' hg
hours

A 085 I 21; 1 0.99
2 0.39

3 0.13

8 083 I 96 l 0.99
2 0.h0

3 0.31;

D 068 II 96 l 1.00
2 0.52

B 083 III 96 1 1.00
2 0.69

0.511

F cellulose I 96 f 1.08
l 0.99

C Known glucose g 1.00
Known cellobiose c 0.71

sulfate produced by Preparation I. To be more certain, an.e1uate of the
correSponding area of a streak chromatogram of the solution used for
column B was concentrated and rechromatographed by the two-dimensional
technique previously described. Some glucose was also eluted, but the
identification of fructose was quite definite due to its greater concen-
tration and more intense spot, and because its Rg values with the butanol-
pyridinedwater solvent (1.11) and phenol-water (1.25) matched those of an

authentic sample of fructose (1.11 and 1.28).

156

Eluates of the unknown Spots of columns B, D, and E were also obtained

from streak chromatograms. Samples were analyzed for reducing sugars (as

glucose) and tested qualitatively for free sulfate before and after acid
hydrolysis. The eluate correSponding to R? 0.36 (enzyme Preparation I)

gave a doubtful test for free sulfate (barium chloride) before, and a

positive test after hydrolysis. Hydrolysis increased the reducing sugar

content by 60 percent. The material eluted from the Hg 0.1h area (Prepar-
ation I) gave a positive test for sulfate only after hydrolysis, and a
three—fold increase in reducing sugar content accompanied the liberation
of sulfate. The eluate from the area suSpected of containing cellobiose

(enzyme Preparation III, column B) contained no free sulfate either before

or after acid hydrolysis. Its reducing power increased 2.1 times after

hydrolysis. The material causing the spot at Rs 0.5% (Preparations II

and III) increased in reducing power by 60 percent after acid hydrolysis

and contained sulfate ion only after hydrolysis. Thus, aside from cello-

biose, the unknown Spots all contained bound sulfate. Whether their re—

ducing power after acid hydrolysis was a consequence of the loss of the
sulfate radical or was due to the liberation of new reducing groups by

hydrolysis of63-1, b glucosidic linkages is not known.
The most important finding was that, except for glucose, the end

products of hydrolysis of cellulose sulfate with Preparation I were different

from those obtained with Preparation II and III. This suggests the exist-

ence of more than one enzyme acting either on the original substrate or on

the intermediate products.

Fructose appeared only in the hydrolysates prepared with enzyme Prepar-

atdxni I. ILts appearance, therefore, can be correlated only with the presence

157

of component 1. If the reasonable assumption is made that fructose was
not an original constituent of cellulose sodium sulfate and wheat straw
cellulose, than it must also be assumed that component 1 in some way
brings about the formation of fructose, probably from glucose. The con-
centration and clearing treatments were the same for all the hydrolysates,
so it is unlikely that fructose was formed after the enzymatic reaction
was stopped.

A very faint spot above the glucose Spot in column F (wheat straw
cellulose) indicated the presence of a small amount of xylose in this
substrate.

ChromatOgrams of the 80 percent ethanol soluble end products of the
enzymatic hydrolysis of hemicellulose fractions X and Y are presented in
Figures 31 and 32, respectively. The areas in which spots occurred were
numbered on the completed chromatogram, starting with the spot having the
highest Hg. The HR values of the unknown and known Spots are given in
Tables XXXII and XXXIII.

The solutions used for a and B in Figure 31 were fractions obtained
from the same hB-hour enzymatic hydrolysate of hemicellulose X, and those
used for C and D were obtained from a 96-hour hydrolysate produced by
enzyme preparation I. The fractions represented by.A and C were from the
80 percent ethanolic extracts of the residues obtained by evaporation of
the alcoholic filtrates of the original hydrolysates to dryness. Fractions
C and D were obtained by dissolving in water the residues remaining after

extraction with 80 percent ethanol. All the fractions were deionized as

previously described. It can be seen that the same constituents were

present in the two fractions of both hydrolysates. However, undesirable

c.

l

 

at
U.
9
W
M
a...

1.1.
S.)
)

Ca

Figure 31. Praducts of hydrolysis of hemicellulase X by three enzyme

preparations at pH 5.35.

H.
E.
F.
G.

A and B. Preparation I, [:8 hr. C and D. Preparation I, 96 hr.

Preparation II, 96 hr. I. Preparation III, 96 hr.
Xylose (X), lucose (G), and glucuronic acid (01A).
Arabinose (A , galactose (Ga), and galacturonic acid (GaA).

Mannose (M) and fructose (F).

158

TABLE XXXII

Rg VALUES 0!" SPOTS ON CHROMATOGiAMS OF ENZYMATIC
HYDROLYSATES OF HMICRLULOSE X

(See Figure :1)

Spot number

 

 

 

Colmmi l 2 ‘7? HO 5 6
1.20 1.03 0.8h 0.69 0.50 0.21
1.19 1.02 0.8b 0.65 0.L9 0.0-0.23
1.20 1.0h 0.87 0.71 0.51 0.17
1.20 1.02 0.8h 0.67 0.51 0.0-0.23
1.19 1.06 0.83 0.h7 0.20
0.99
1.20 1.08 0.86 0.50 0.21
1.01
E X: xylose G: glucose GIA: glucuronic acid
1.20 1.00 0.20-0.3h
F A: arabinose Ga: galactose Gaa: galacturonic acid
1.08 0.88 0.17-0.38
G F: fructose M: mannose
1.08 1.11

trailing of the Spots was noted with the aqueous extracts. No qualitative

differences between the end products of the b8—hour and 96-hour hydrolysates

were found .

The Spot in area 1 was probably xylose. The Spots in area 2 of A-D
were elongated; the leading edge had the dark brown color of the aldopentoses
and the trailing edge had the light brown color characteristic of the aldo-
hexoses. The R8, calculated from the estimated center of the spot, was

slightly higher than 1.00 in each case. It seemed probable that Spot 2

TABLE XXXIII

Hg VALUES OF SPOTS ON CHROMATOGRAMS OF ENZYMATIC
HYDROLYSATES OF HEMICELLULOSE Y

(See Figure 32)

Spot number

 

 

Column 1 2 3 ’3 5* 45 “

A 1.15 0.98 0.83 0.h8 0.03-0.16

B 1.17 1.01 0.87 0.71 0.53 0.18

G 1.13 1.37 0.88 0.18
1.01

H 1.18 1.07 0.87 0.18
1.00

C x: xylose 1.18

D g: glucose 1.00

E a: arabinose 1.07

’31

ea: galactose 0.91

was made up of two incompletely resolved sugars.

The Spot in area 3 had an Rg slightly lower than that of galactose,
but its color was considerably darker. It was found later that this Spot
was not galactose.

Spots b, S, and 6 could not be identified with any of the known
sugars used.

The hydrolysates of hemicellulase X obtained with enzyme Preparations
II and III gave Spots in areas 1, 3, S, and 6, probably identical with
those obtained with enzyme Preparation I. The amount of the substance re-
sponsible for spot h was relativelv smaller than in the hydrolysates pro-

duced with enzyme Preparation 1, and area 2 was resolved into two distinct

160

Spots with the Hg.valJPS and colors of arabinose and glucose.

The results with hemicellulose Y hydrolysates were qualitatively much
the same as those described for hemicellulose X. ‘With this substrate also,
area 2 was resolved into 2 Spots corresponding to arabinose and glucose
only in the hydrolysates produced by enzyme Preparations II and III
(Figure 32 and Table XXXIII). Spots b and S were not detected‘gith cer-
tainty in the hydrolysates from enzyme Preparations II and III.

The unknown Spots were investigated further by isolating the sub-
stances from the appropriate areas of streak chromatograms as described
in the section on methods. Portions of the eluates were analyzed for
reducing power before and after acid hydrolysis, and other portions were
rechromatographed before and after acid hydrolysis. The results with the
end products of hemicellulose X hydrolysis are shown in the chromatograms
in Figures 33 and 38. The Hg values and color of the spots, and the per-
centage change in the reducing power brought about by hydrolysis are
presented in Table XXIIV.

The only eluate unaffected by acid hydrolysis was that from area 1.
The Spot appeared at the same location before and after treatment with
O.h N hydrochloric acid at 120° C. All of the other acid-hydrolyzed
eluates gave one or more new Spots. The hydrolyzed eluate from area 2
gave spots corresponding to xylose and glucose, that from area 3 gave
a single spot correSponding to xylose, and the reducing substances in the
eluates from areas h and S were apparently made up of three constituents;
(a) a relatively large proportion of xylose, (b) smaller quantities of
arabinose, and (c) a uronic acid. (The very faint uronic acid Spots,

identified mostly by their distinctive red color, were indicated on the

 

 

Figure 32. Products of hydrolysis of hemicellulose I by three enzyme
preparations at pH 5. 35.
land B. Preparation 1, 1:8 and 96 hr. , respectively.
Go Preparation II’ 96 hr. Ho ”emu” III , 96 me
C. Xylose (x). D. Glucose (g). E. Arabinose (ax F. Galactose (ga).

 

Figure 33. Effect of acid ludrolysis on the isolated end products in the
enzymatic hydrolysates of hemicellulase x.
A, C, and H. Blunts: of areas 1, 2, and 3, respectively,
before hydrolysis (Figure 31). B, D, and I. mates of areas 1,
2, and 3, respectively, after hydrolysis. E. Iylose (x). F.
Glucose (g). G. Arabians- (a).

 

Figure 31;. Effect of acid lmlrolysis on the isolated end products in
the easy-tic hydrolysates of henicsllulose I.
A, c, and H. Eluatea of areas 1;, 5, and 6, respectively
before hydrolysis (Figure 31). B, D, and I. Eluatss of areas ,
, and 6, respectively, after tudrolysia. E. Xylose (x). 1".
Glucose (g). G. Arabinose (a).

161

TABLE XXXIV

EFFECT OF ACID HYDRGLYSIS ON THE END PRODUCTS 0F
ENZYMATIC HYDROLYSIS OF HEMICELLULOSE X

(Data from Figures 33 and 3h. Source of eluates:
streak chromatograms of solution used for C, Figure 31.)

 

Figure No. Description Change in Spot Color** Rg
and column of reducing No.
eluate* power
%
33A spot 1 1 P 1.1h
33B spot l-H -S 1 P 1.1h
330 Spot 2 1 P 1.06
2 H 0.99
33D Spot 2-H -2O 1 P 1.1h
2 H 1.0?
33H spot 3 1 P 0.91
33I Spot B-H 7O 1 P 1.16
BhA spot h 1 P 0.7h
BLB Spot b-H 100 1 P 1.16
2 P 1.0h
3 U 0.39
3hC spot 5 l P 0.52
3bD Spot S—H lbO 1 P 1.16
2 P 1.07
3 U 0.1.0
BhH Spot 6 1 ? 0.0h-o.19
BhI Spot 6-H lhO 1 P 1.17
2 P 1.06
3 U 9.hh
33! x xylose 1.15

BhE x xylose 1.16

TABLE XXXIV concluded

 

 

162

 

Figure No. Des cription Change in Spot Col or“ R0
of reducing No. ’
eluate* power
33F g glucose 1.00
BhF g glucose 1.00
330 a arabinose 1.05
336 a arabinose 1.06

 

*The letter “H" after the number indicates the acid hydrolyzed portion

of the eluate.

**Colors are indicated as follows:

P, the dark brown color charac-

teristic of aldopentoses; H, the lighter brown color given by aldohexoses;
U, the dark red color given by uronic acids.

163

chromatogram, before it was photographed, by a pencil mark.) The hydro-
lyzed eluate from area 6 also contained the Spots correSponding to xylose

and uronic acid, but showed no trace of the arabinose spot, possibly

because the concentration was too low.

The reducing equivalent of all the eluates except the ones from

spots 1 and 2 were increased by acid hydrolysis. Because of the empirical
and non-Specific nature of the method used to estimate reducing substances,

the results can be used only in a qualitative sense. Thus it is probable

that the material responsible for spot 6 was a higher oligosaccharide

than that of spot 3, but the number of residues per molecule cannot be
estimated from the results.

The chromatograms obtained with the hydrolyzed and unm'drolyzed

eluates containing the end products of hydrolysis of hemicellulose Y are

shown in Figures 35 and 36. The Rg values and color of the spots and the
percentage increase of reducing power of the acid hydrolysates are pre-
sented in Table XXIV.

The results with the eluates from areas 1, 2, and 3 were the same.
as before1 (Figure 35). The results with the remaining eluates were dif-

ferent (Figure 36). No uronic acid was detected in the hydrolyzed eluates

of areas 1; and 5. Moreover, xylose and glucose, instead of xylose and

arabinose, were identified in the hydrolyzed eluates. The hydrolyzed

eluate of area 6 yielded spots correSponding to xylose, glucose, and uronic

acid. The amount of glucose in all three cases was small in comparison

to the amount of xylose.

1A small amount of area 2 was evidently eluted with the xylose of
area 1, and a small amount of Spot 3 with area 2.

2'
H

e

’1
m
0

O
n
’11;
O
Ii

Figure 35. Effect of acid hydrolysis on the isolated and products in
the enzymatic hydrolysates of hemicellulase I.
A, C, and H. Eluates of areas 1, 2, and 3, respectively, before
hydrolysis (Figure 32). B, D, and I. Elmtes of areas 1, 2, and 3,
respectively, after hydrolysis. E. Xylose (x). F. Glucose (g).
G. Arabinose (a).

 

Figure 36. Effect of acid hydrolysis on the isolated end products in the
enzymatic hydrolysates of hemicellulase I.
A, c, and H. Eluates of areas )4, S, and 6, respectively
before hydrolysis (Figure 32). B, D, and I. Eluates of arses ,
5, 83d 61 remflimly’ after hydrolysifl. Es Xylose (I). F.

Glucose g). a. arabinose (a).

16b

TABLE XXXV
EFFECT OF ACID HYDROLYSIS ON THE END PRODUCTS OF
ENZYMATIC HYDROL‘ISIS OF HEMICELLLEOSE Y

(Data from Figures 35 and 36. Source of eluates:
estreak chromatogram of solution used for E, Figure 32)

 

 

Figure No. Description Change in Spot Color“ Hg

and column 'of reducing No.
e1uates* power

35A spot 1 l P 1.21

2 H 1.09

35B Spot l-H -S 1 P 1.20

2 P 1.08

35C spot 2 1 H 0.99

2 P 0.87

35D soot 2-H -7 l P 1.20

2 P 1.09

3 H 1.01

35H Spot 3 l P 1.22

2 P 0.90

351 Spot 3-H DO 1 P 1.22

36A spot b 1 P 0.72

368 Spot h-H 86 1 P 1.22

2 H 1.01

360 Spot 5 1 P 0.50

36D Spot S-H 112 1 P 1.21

2 H 0.99

36H Spot 6 1 P 0.15

361 spot 6-H 12b 1 P 1.22

2 H 1 .01

3 U 0.30

39E x xylose 1.20

363 x xylose 1.21

 

165

TABLE XXXV concluded

 

 

 

Figure No. Description Change in Spot Color“? R9
of reducing No. °
eluates* power
35? g glucose 1.00
361'“ g glucose 1 .00
35G a arabinose 1.09
360' a arabinose 1.10

 

*The letter "H" after the number indicates the acid-hydrolyzed portion
of the eluate.

**Colors are indicated as follows: P, the dark brown color character-
istic of aldOpentoses; H, the lighter brown color given by aldohexoses;
U, the dark red color given by uronic acids.

166

\v
110

9*.13ence of a significant extent of hydrolysis of the oligo-
saccharides of Spots 3—6 for hemicellulose X was obtained when small

portions were. incubated at 1400 C. with enzyme Preparation I.

The reducing
equivalent of each eluate remained essentially unchanged after 2h hours
of incubation.

Thus it is probable that the Spots represent end products
and not intermediate products.

Chromatograms of hydrolysates of hemicellulose Y and hemicellulose

Y taken at 1.5, 6, 20, and [:8 hours are shown in Figures 37 and 38, re-
apectively. R

P values are presented in Tables XXXVI and XXXVI}.

Enzyme
Preparation I was used in both cases.

The concentrated hydrolysates were
not deionized. The amount of reducing sugars (as glucose) applied at

each starting Spot (except a) was kept constant at about 65 micrograms

in the case of the hemicellulose X hydrolysates, and at about 70 micro-

grams with the hydrolysates of hemicellulose Y.

The volumes of the original
hydrolysate represented in Spots A-E were 0.1, 0.1, 0.011, 0.02 and 0.015

m1., reapectively, in the case of the hemicellulose X hydrolysates, and

0.011, 0.01;, 0.032, 0.022, and 0.016 ml. for the hemicellulase Y hydrolysates.
Care. was taken to treat all samples alike in order that a constant pro-

portion of each component would be carried through the procedure.

The
experiment was undertaken to determine the order of appearance or dis-

appearance of the end products or intermediate products of hydrolysis.

The results with the two substrates were similar.

The major excep-
tion was the occurrence of Spots in areas 3 and S in the solution of hemi—

cellulose x before the enzyme was added (A, Figure 37). These Spots did
not occur with the suSpensions of hemicellulose Y (A, Figure 311).

Another
difference, noted previously, was the relatively small amount of the

 

 

 

 

Figure 37. Effect of time of reaction on the relative proportions of end

products obtained by the enzymatic hydrolysis of hemicellulase X.
Enzyme Preparation I, 16.? pg. of protein nitrogen per milliliter
of reaction mixture, pH 5.3.

A. Before enzyme was added. B. 1.5 hr. C. 6 hr.
D. 20 hr. E. 118 hr. F. Xylose (x) and glucose (3). G. Arab-
inue (a o

 

 

(fl

'9 .3.

 

Figure 38. Effect of time of reaction on the relative proportions of

end products obtained by the enzymatic hydrolysis of hemicellulase Y.
Enzyme Preparation I, 16.7ug.. of protein nitrogen per milliliter
of reaction mixture, pH 5.3.
A. Before enzyme was added. B. 1.5 hr. C. 6 hr.
D. 20 hr. E. h8 hr. F. Xylose (x) and glucose (g).
c. Arabinosa (a) .

 

I
l
w
l

 

TABLE XXXVI

R VALUES OF SPOTS ON CHROMATOGRAMS 0F ENZYMATIC

 

 

 

 

 

g HYDROLYSATES 0F HEMICELLULOSE x
(See Figure 37) ‘4_r‘
Spot number
Column 1 21* 22* 37 (h 5 6—
A 0.8b 0.h5
B 1.15 1.06 0.97 0.82 0.61 0.h6
c 1.18 1.07 0.99 0.86 0.65 0.h9 0.21
D 1.19 1.09 1.00 0.86 0.6L 0.b9 0.15
E 1.19 1.09 0.99 0.85 0.b7 0.1L
F xylose 1.19; glucose 1.00
G arabinose 1.09

AA

*2 indicates the upper spot of area 2, and 22 indicates the lower
spot of the same area.
TABLE XXXVII

a VALUES or SPOTS 0N CHROMATOGRAMS or ENZYMATIC
3 HYDRDLYSATES OF HEMICELLULOSE I

(See Figure 38)

Spot number

 

 

Column 1 21* 22* 3 h 5 6
B 1.20 1.10 1.00 0.86 0.61. 0.1.8 0.17
C 1.19 1.08 0.99 0.85 0.66 0.h8 0.17
D 1.19 1.08 0.99 0.85 o.b7
E 1.19 1J08 0.99 0.86 0.h8
F Xylose 1.20; glucose 1.00

C)

arabinose 1.09

 

*2, indi

An LL- -A--‘-

cates the upper Spot of area 2, and 22 indicates the lower spot

168

substances causing Spots h and 6 in the hydrolysates of hemicellulase Y.

lith both substrates, the relative concentrations of xylose (spot 1),
arabinose (top Spot of area 2), and the substance causing Spot 3 increased
throughout the course of the reaction. The relative intensity of the
glucose spot (lower Spot of area 2) and of Spots h and 6 decreased as the
reaction proceeded, whereas the intensity of Spot 5 increased during the
first part of the reaction and then decreased. It should be emphasized
that the changes in concentration are relative; only positive changes may
be interpreted as evidence that the substance was increasing in absolute
concentration. The concentration of the substances which were represented
by the spots with decreasing intensities may have remained constant or
actually increased. On this basis, it appears that the reactions which
resulted in the appearance of glucose and Spots b, S, and 6 approached
completion more rapidly than those which resulted in the appearance of
xylose, arabinose, and the material of Spot 3. It is probable that the
substance represented by spot 3 was an end product, refractive to further
enzymatic hydrolysis. The results are inconclusive with reSpect to the
status (end product or intermediate) of the oligosaccharides of Spots h,
S, and 6.

Spot 5 in these chromatograms exhibited a peculiar characteristic
not found with the correSponding spots on previous chromatograms. .a
crescent-shaped area can be seen in the photographs, especially in Figure
38. The color of the crescent was the dark red color characteristic of
the uronic acids. The remainder of the Spot was the dark brown color
characteristic of pentoses.

Some of the material of Spot 5 was isolated by elution from the

lim-
m_
_ 'r.

1‘ 4‘ .

 

169

appropriate area of a streak chromatogram. The eluate was rechromato-
graphed by the two dimensional technique, using the usual butanolzpyridine:
water mixture in one direction, and phenol:water in the second direction.
Two Spots, one having the red color of a uronic acid and the other the
color produced by pentoses, were found. The R3 of the red Spot was 0.23
and that of the brown spot was O.b8 in the first solvent.

It will be recalled that the solutions used for the chromatograms in

Figures 37 and 38 were not treated with the ion-exchange resins, whereas

 

those used for the previous chromatograms were treated. On the basis of

 

the results obtained with the unhydrolyzed eluate of Spot 5, it may be
concluded that uncombined uronic acid occurred in the hydrolysates. The
abnormally rapid migration of the free uronic acid into the area occupied
by spot 5 was an artifact, caused, perhaps, by the presence of inorganic
ions. It is also possible that free uronic acid was originally present
in the hydrolysates used for previous chromatograms, but was removed by
the anion-exchange resin.

In summary, evidence was obtained which indicated that D-xylose,
L-arabinose, D-glucose, uronic acid, and an unknown di- or trisaccharide
of D-xylose were end products of the hydrolysis of both hemicellulose
fractions by g. verrucaria enzyme preparations. Inconclusive evidence
was also obtained suggesting that three other oligosaccharides found in
enzymatic hydrolysates of the hemicelluloses were end products rather
than intermediate products. The last-mentioned oligosaccharides appeared

to be different in the hydrolysates of the two hemicellulases.

DISCUSSION OF RBULTS

 

 

171

DISCUSSION OF RESULTS

The primary objectives of this study were (1) to determine whether

the cellulase activity of £3 verrucaria culture filtrates is the property

 

of a single or of several of the protein components present, and (2) to

study the relationships among the cellulase, hemicellulase, andP-glucosidase

activities of the culture filtrates. The results will be discussed in

relation to these objectives. Certain observations, perhaps interesting

 

and important in themselves, but not contributing directly to the settle-

test

\

ment of the inquiry, were discussed in the previous section.

EVidence for the multiplicity of enzymes having cellulolytic activity
was manifold. Some of the observations listed below cannot, by themselves,
be considered conclusive, and others are merely suggestive, but considered

together, they present a strong argument for the multiple enzyme hypothesis.

 

(l). The biphasic nature of the activity (CSS) accumulation curve
during the growth of the organism could be the result of an increased
production of a second enzyme at the time of the upward inflection. The
possibility was not specifically investigated, and other explanations are
admittedly possible. Jermyn (1953) found indications of a variable pro-

duction of Cx activity by Stachybotrys atra, and Reese and Gilligan (1953)

 

found it possible, by changing the conditions of culture, to influence

 

the production of one of the Cx components by g. verrucaria.

(2). Although other factors were involved, the failure_to obtain
fractions enriched in cellulase activity (088) by salt and solvent pre-
cipitation may have been due, in part, to the existence of two or more

enzymes with different physical properties.

 

172

(3). It was evident during fractionation by electrophoresis con-
vection that protein component 2 was the most active cellulase when
cellulose sodium sulfate was the substrate, but statistical correlation

of the specific activities of some of the fractions with their electro-

phoretic composition strongly suggested that more than one of the components

was involved. Comparison of the specific activities of the three final
enzyme preparations with cellulose sodium sulfate and wheat straw cell—
ulose led to the same conclusion, but indicated further that the propor-
tion of the total activity attributable to component 2 was greater when
cellulose was used as the substrate than when cellulose sodium sulfate
was used.

The results with respect to the soluble cellulose derivative are in
agreement with the findings of Reese and Gilligan (1953), who presented
evidence indicating the presence of at least two separate enzymes in

E, verrucaria culture filtrates capable of hydrolyzing carboxymethyl-

 

cellulose. It will be recalled that these workers separated the protein
components by paper cgrcratography and incubated transverse sections of
the chromatograms directly with the substrate. Whitaker's (1953) results,
on the other hand, indicated that practically all the cellulase activity

of E, verrucaria culture filtrates can be attributed to a single com-

 

ponent. Whitaker used only insoluble cellulose substrates. The results
obtained in the present investigation, showing that the ratio of activ-
ities of the constituent enzymes apparently depends on the particular
substrate employed, may offer a partial eXplanation for the seemingly
contradictory results reported from different laboratories. Since no

pure fractions were obtained, the results offer no evidence for or against

:7

 

 

 

173

the hypothesis advanced by Reese :t_al. (1950) that two enzymes, C1
(which in some as yet uneXplained manner frees linear polyanhydroglucose
chains from the crystalline matrix of native cellulose) and CK (which
hydrolyzes thel3-l, h-glucosidic linkages), are necessary for the hy-
drolysis of native cellulose.

(b). The differences displayed by the three final enzyme preparations

3
I 1'

J

in reSponse to changes in pH constitute supporting evidence for the

- wa.

multiple enzyme hypothesis.

i
__ .1"— .5“

(S). The difference in end products obtained from the hydrolysates

 

View“ in"

of the three enzyme preparations acting on cellulose sodium sulfate can
be interpreted on the basis of the presence of more than one enzyme.

The occurrence of multiple components in the system studied in this
investigation is not unique. The work of Jermyn (1952b) with the cell-
ulase andl3-glucosidase systems of £3 95123: has already been mentioned.
Giri :2 El! (1952) found indications of multiple components in amylase
systems, and Reed (1950) obtained similar results with a polypalacturonase
system.

The most convincing evidence for the ability of at least one enzyme

to catalyze the hydrolysis of at least portions of the hemicelluloses,

 

as well as cellulose, was obtained by comparing the ratio of Specific
activity of the three final enzyme preparations on both types of substrate.
The comparison indicates that component 2 apparently is the most active
hemicellulase as well as cellulase. Moreover, the relationship between
Specific activity ratios of the three enzyme preparations with hemi-
cellulose X (soluble) and hemicellulose Y (insoluble) were qualitatively

similar to the ratios obtained with cellulose sodium sulfate and.wheat

17h

straw cellulose, indicating that the physical state of the substrate
may have the same effect on hemicellulase as on cellulase activity.

The pH-activity curves for the hydrolysis of the hemicellulose
preparations by the three enzyme solutions give further evidence for
the multiplicity of enzymes actine on the hemicelluloses. Although the
occurrence of two peaks for a single enzyme preparation, with substrates
such as the hemicelliloses, cannot be interpreted as evidence of two
enzymes, the marked difference between the curves for the three enzyme
solutions makes this interpretation more sound.

The similarity of the end products of hydrolysis of the hemicelluloses
by the three enzyme preparations might appear to be contradictory to the
multiple enzyme hypothesis as applied to the hemicellulases. aside from
this, however, the similarity in the relative proportions of end pro-
ducts does serve to validate the comparison of specific activities. If
the end products were different or were present in widely different pro-
portions in the hydrolysates produced by the three enzyme preparations,
then comparisons of Specific activity would be less useful due to the
nonspecificity of the copper reduction method of estimating reducing
supars.

The glucose found with the end products of hydrolysis of the hemi-
celluloses may have been due to partial deeradation of cellulose during
the extraction of the hemicelluloses with strong alkali, or it might
actually be a part of a hemicellulose molecule. In any case, the rela-
tively small amount of glucose present gives assurance that the activity
measured by the copper reduction method was due mostly to cleavage of

bonds other than thefi-l, b-glucosidic bonds.

 

175'

Since a large share of the total activity with both hemicelluloses
and cellulose was apparently contributed by component 2, it appears

probable that the cellulase of E, verrucaria is not absolutely Specific.

 

These results are contrary to those of Grassman $2.31“ (1933) who found
that the xylanase activity of if ggygag preparations could be separated
from the cellulase activity. However, the difference could be due to the
use of preparations from different sources.

The results with salicin as the substrate indicate clearly that

nearly all of thefg-zlucosidase activity was confined to component 1.

 

 

Since component 1 apparently had some action on cellulose sodium sul-
fate, and component 2 may have had some action on salicin, it appears
that, although their specificities are not absolute, a complete separ-
ation of the two enzymes for purposes of classification is justified

in the case of this system. These results do not agree entirely with
those of Jermyn (1952b) who obtained evidence with 2, oryzae preparations
for the existence of several enzymes capable of hydrolyzing both carboxy-

methylcellulose and several P-glucosides.

 

17?

SUMMARY

The present investigation was carried out to study (1) the possible
multiplicity of cellulolytic enzymes and (2) the relationships among
the cellulase, hemicellulase, andz3-elucosidase activities in culture

filtrates of y, verrucaria.

 

The culture filtrates from thirteen different lZ-liter cultures of

the organism were concentrated five to ten times by slow partial freezing.

 

During this process an uneXplained increase in the total activity of
most of the filtrates was observed. Some loss of activity occurred in
the water removed as ice.

Attempts to obtain fractions enriched in cellulase activity by
means of fractional ammonium sulfate, acetone, or ethanol precipitation
of the proteins, and by chromatography on powdered cellulose columns,
ended in failure.

After the material was concentrated further by precipitation of the
proteins with 75 percent acetone or saturated ammonium sulfate, frac-
tionation by electrOphoresis convection was found to be successful.

Fractionations were controlled by the determination of protein

nitrogen, cellulase activity (using cellulose sodium sulfate as the

 

substrate), and by electrophoretic analysis.

The unfractionated enzyme preparations had six electrophoretic com-
ponents. Determinations of specific activity, in conjunction with elec-
trophoretic analysis of fractions obtained during the course of the

fractionation, showed that most of the activity with cellulose sodium

L

 

178

sulfate accompanied the three protein components having the lowest mobil-
ities (components 1, 2, and 3). Three preparations with different ratios
of the protein components were obtained by means of electrophoresis con-
vection. The first preparation contained only components 1, 2, and 3, but
the relative concentration of component 3 was low. The second preparation
contained all the proteins except component 1, and the third had all the
proteins except components 1 and 2.

The specific activities of the three enzyme preparations with cellulose
sodium sulfate, two hemicellulose preparations from wheat straw, cellulose
from wheat straw, and salicin were determined and compared. pH-activity
curves for all of the enzyme preparations with each substrate were con-
structed and compared. The end products of enzymatic hydrolysis of
cellulose sodium sulfate and the hemicellulose preparations were investigated
by means of paper chromatography.

Considered in 2223, the results indicate that the ability to catalyze
the hydrolysis of each of the polysaccharide substrates is shared by at
least two enzymes. Component 2 appeared to be the most active enzyme with
all four polysaccharides, but the proportion of the total activity attrib-
utable to component 2 varied with the substrate. The evidence suggests
that component 1 has some (variable) activity with all the substrates with
the possible exception of wheat straw cellulose. The distribution of the
remaining activity was not clearly indicated by the results.

Component 1 was found to be the most effective enzyme for the hy-

drolysis of salicin.

 

 

 

 

LITERATURE CITED

 

180

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F?-

 

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APPENDIX A

 

186

TABLE XXXVIII

THE EFFECT CF pH ON THE ACCUMULATION OF END PRODUCTS FROM THE DEGRADATION
OF CELLUIOSE SODIUM SULFATE BY THREE DIFFERENT ENZYME PREPARATIONS

(Results are expressed as the mg. of reducing substances (as glucose)
per mg. of protein nitrogen)

 

 

’—

pH Enzyme Preparation I

 

 

 

 

 

 

 

 

 

 

 

Time in hours
__ 173 ‘ l _ 2 # J: .11 8 20 25 __
3.78 110 31.8 610 1090 1800 2920 3180 F‘]
11.21. 261. 61.0 1120 1880 2850 1.250 1.1.80 1
1. .80 283 863 11.80 2370 3570 5070 5320 “ a
5 .10 360 890 161.0 25110 3730 521.0 51.70 ' .
5 .31. 355 887 1610 21.50 3670 5160 51.20 7
5.95 297 820 11.00 2280 3360 1.650 1.870 .
6.85 297 71.3 1360 221.0 3260 1.210 1.1.10 3 .
6.98 156 535 920 1560 2290 3130 3290 1: J
7.37 129 31.8 1630 21.50 s-..
7.55 101 228 1330 2230
p8 Mayne Preparation II
Time in hours

1/1. 1/2 2 1. 8
8.15 52.2 89.7 262 392 523
1..75 75.3 122 331. 1.73 631
5.05 82.6 11.1. 338 1.83 629
5.35 83.7 11.1. 325 1.53 598
5 .90 82.5 135 322 1.1.6 581.
6.1.5 77.2 118 285 1.09 530
6.95 65.0 100 239 328 1.21.
pH Enzyme Preparation III

Tine in hours

1/3 2/3 2 A 1. 10 21. #_ _
14.15 28.3 113.0 101. 157 227 280
11.75 112.3 72.6 167 226 3211 1102
5.08 1.6.1 78.6 160 226 311. 388
5.37 15.5 81.0 158 222 313 378
5.65 1.5.5 80.1 158 203 300 358
6.00 1.1.? 78.0 152 187 283 318
6.51 1.2.6 76.2 11.9 180 262 ' 295
6.90 39.9 62.5 1114 151. 198 231

 

 

187

-TABLE xxxxx

THE EFFECT OF pH ON THE ACCUMULATION OF END PRODUCTS FROM THE DEGRADATION
OF WHEAT STRAW CELLULOSE BY THREE DIFFERENT ENZYME PREPARATIONS

(Results are expressed as the mg. of reducing substances (as glucose)
per mg. of protein nitrogen)

 

 

 

 

 

 

 

 

pH Enzyme Preparation I
Time in hours
1 1 1/2 3 6 20 28
‘80)»; 2005 25.2 318.1 163.2 '48.? 188.6
b.75 27.0 32.6 h6.5 60.h 88.8 90.6
5.10 27.9 33.0 h5.3 60.8 93.2 96.6
5.33 25.9 310’; 165.8 58.0 91.8 98"?
5.95 17.0 21.6 3h.1 86.5 53.7 55.9
6.h5 1h.2 19.9 30.7 h9.2 59.0 61.9
6.81 10.5 13.7 27.3 39.6 50.h 51.1
pH Enzyme Preparation II
Time in hours
1/3 1 3 lo 28
h.1h 3.8 9.0 15.0 26.6 39.2
b.72 5.2 9.2 18.9 37.1 57.0
5.10 5011 11.8 2007 110.1 6100
5.35 5.2 11.2 20.8 80.8 59.2
5.92 8.6 9.9 18.9 35.9 56.1
6.83 3.5 7.2 15.0 28.8 h6.5
6.80 2.5 6.0 13.1 25.0 38.3
pH ‘lnzyme Preparation III
Time in hours
1/2 1 3 11 at
18.20 0.9 1.8 3.9 8.0 1109
h.80 1.2 2.7 5.1 10.h 15.8
5.10 1.7 2.7 h.8 10.7 16.1
5.80 1.8 2.h 8.8 10.8 15.5
6.00 1.2 2.1 8.8 9.8 13.7
6.50 0.9 2.1 3.6 8.3 11.6
6.85 0.6 1.5 3.6 7.1 10.h

 

 

 

188

TABLE XL

THE EFFECT OF pH ON THE ACCUMULATION OF END PRODUCTS FROM THE DEGRADATION
OF HEMICELLULOSE X BY THREE DIFFERENT ENZYME PREPARATIONS

(Results are expressed as the mg. of reducing substances (as glucose)
per mg. of protein nitrOgsn)

 

 

 

 

 

 

 

 

pH Mayne Preparation I
__ Time in hours __ __A
1/3 1 2 - ‘ 1. 11 1/2 20 21. -
11.05 58.0 138 2011 285 1129 1158 1180
11.611 100 210 283 1115 683 905 9811
11.911 102 2111 276 1122 699 913 1010
5.25 103 2111 288 1.22 698 871. 918
5.55 102 203 281 1117 667 856 9011
5 .86 103 212 280 1112 676 893 961
6.22 100 208 261. 1105 671 883 9118
6.117 90 190 2611 393 636 778 816
pH msyme Prepare tion II
Time in hours
_ 1/3 1 _ 2 3 10 _ 21.
11.09 15.9 26.1 314.3 110 .1 66.6 93.2
hOés 20.6 35.2 h6OS 55.5 . 9,403 159
11.95 22 .2 37 .0 118.6 57 . 8 102 166
5 .25 22 .7 39 .7 119 .8 61 .6 109 173
5.3) 23.7 38.8 55 .11 62 .1 132 137
6.20 22.7 37 .0 118.8 57 . 8 110 179
6.110 22 .0 38 .11 116.6 58.2 1011 168
pa Mayne Preparation III
# Time in hours
1/3 1 2 1. 11 20 21.
11.15 8.5 17.8 21.8 30.3 11.6 50.8 511.8
11.75 111.0 27.2 33.11 1111.0 63.1 811.0 93.6
5.10 17 .3 30 .1 38.3 118 .8 72 .6 93 .0 101
5.35 18.5 ”0" 39.2 .8909 7,601 96.8 105
5 e 95 17 0’6 x 01 39 so ‘69 J! 75 so 96 .5 1a}
6J5 17 .9 30.11 37.2 119.0 714 .1 95.0 102
6.90 17 .5 27 .8 35.0 117 .6 69.9 91.8 96 .11

189

TABLE XLI

THE EFFECT OF pH ON THE ACCUMULATION OF END PRODUCTS FROM THE DEGRADATION
OF HEMICELLULOSE Y BY THREE DIFFERENT ENZYME PREPARATIONS

(Results are expressed as the mg. of reducing substances (as glucose)
per mg. of protein nitrogen)

W

pH

Mayne Preparation I

 

 

 

 

 

 

 

 

 

r—I-hr.
Tine in hours 5
1/3 1 2 8 12 2o 28 g
“ E
8.15 61.0 178 288 277 309 312 312 i
8.70 103 250 318 395 887 502 506 .
5.30 85.3 216 308 375 870 878 882 I:
5.98 107 257 326 809 506 510 513 .~‘
6.80 81.2 198 297 377 870 878 876 ‘ "
6.70 72.0 185 268 330 808 815 820
pH mzyme Preparation II
Time in hours _
1/3 1 3 12 1/2 28
11.15 2602 3802 O 56.6 91.1 99.0
8.70 30.6 88.0 71.5 128 162
5.10 32.8 89.7 72.2 133 166
5.35 32.1 88.0 69.7 133 168
5.83 33.9 50.8 69.3 133 168
6.80 31.2 87. 65.6 125 160
6.61 29.1 82.7 65.6 118 186
p8 Mayne Preparation III
‘ #J Time in hours
1/3 ' 1 2 3 11 20 28
8.15 16.? 27.1 33.2 37.5 58.8 61.0 62.2
8.75 25.3 37.2 88.7 88.8 71.8 93.8 102
5.10 26.5 37.5 85.8 50.0 79.2 98.8 107
5.35 25.9 39.0 87.1 53.0 79.2 101 109
So” 23 e6 37 02 ' hSeo 119 e" 7h 07 9608 105
6.80 28.1 36.0 82.6 87.0 69.0 90.1 99.8
6.90 19.6 30.0 39.0 88.3 66.7 88.8 91.0

190

TABLE XLII

THE EFFECT OF pH ON THE ACCUI-RJLATION OF END PRODJCTS FROM THE DEGRADATION
OF SALICIN BY THREE DIFFERENT ENZYME PREPARATIONS

(Results are eXpressed as the mg. of reducing substances (as glucose)
per mg. of protein nitrOgen)

W‘

11

 

 

pH szle Preparation I
Time in hours_
1/3 1 2 3 7 20 28
8.15 28.3 78.7 153 218 526 1050 1120
8.75 85.6 121 218 307 639 1350 1870
5.35 88.8 126 228 312 710 1370 1890
5.95 85.6 110 193 278 552 1230 1360

6.86 37.8 97.8 178 280 510 1080 1180
6.85 31.7 80.6 189 208 866 895 988

 

 

 

 

 

 

pH szme Preparation II
4_ Time in hours
1/3 1 2 8 28
11.15 2.11 7.5 12.6 110.9 102
5.35 3.8 11.2 19.5 60.7 151
5.95 3.6 9.3 17.0 51.8 115
pH Mayne Preparation III
4 A Tine in hours
1/2 1 2 7 20 211
8.15 2.7 5.1 8.0 21.1 39.6 88.6
he73 308 has 1000 2908 6908 7806
5.35 11.5 7.0 12.9 39.6 83.0 88.1
5092‘ 207 501 1000 3002 7300 82.1
6.85 3.0 8.5 8.7 26.7 55.7 62.2
6.85 1.5 8.5 8.0 20.8 80.2 88.9

 

APPENDIX B

192

APPENDIX B
Preparation of Buffers and Reagents

Phosphate buffer, pH 6.9 ionic strength 0.185, used for electrophoretic
analysis and electrophoresis convection.

Nix 13.0 ml. of 8 M monobasic sodium phosphate solution with 160 m1.

of 0.5 M dibasic sodium phosphate solution and dilute to 2 liters

with distilled water.

 

 

Phosphate buffer, pH 5.9 ionic strength 0.185, used for electrophoresis
convection.

Mix 50 ml. of h M monobasic sodium phosphate solution with 60 m1. of

0.5 l dibasic sodium phosphate solution and dilute to 2 liters with

distilled water.

Acetate buffer, pH 5.0 ionic strength 0.15, used for electrophoresis

convection.

Mix 150 ll. of 2 N sodium acetate solution with 38.3 ml. of 3.5 N

 

acetic acid solution and dilute to 2 liters with distilled water.

Citrate buffers from pH 3.5 to pH 7.5, 0.5 M, used for determination
of enzymatic activity.

Dissolve 10.5 g. of citric acid (monohydrate) in a small volume of

water and, using a glass electrode pH—meter, titrate with 10 M sodium

hydroxide to a point a little below the desired pH. Cooling is

necessary during the titration. After adjusting the volume of

193

solution to about 90 ml., add 100 mg. of 'Ierthiolate' and titrate
to the final pH with 1 or 2 N sodium hydroxide. Adjust the final

volume to 100 ml.

Somogyi's (1985) alkaline copper reagent for micro determination of
reducing sugars .

Dissolve 28 g. of anhydrous dibasic sodium phOSphate and 80 g. of

potassium sodium tartrate in about 700 ml. of water, add 100 ml. of

N sodium hydroxide, followed by 80 ml. of 10 percent cupric sulfate

(pentahydrate) with stirring. Add 180 g. of anhydrous sodium sul-

 

 

fate, dissolve, and allow to stand a day or two. Filter into a one-
liter volumetric flask already containing 10 m1. of exactly 1 N

potassium iodate solution. Dilute to 1 liter and mix thoroughly. f

Color reagent used for paper chromatographic detection of reducing

sugars (lukherjee and Srivastava, 1952).

 

Dissolve 0.5 g. of p-anisidine in 2 m1. of phosphoric acid (sp. gr.
1.75), dilute to so ml. with absolute ethanol, and filter. Save the
filtrate (A). Dissolve the precipitate in a minimum of water,
dilute with an equal volume of ethanol. Add phosphoric acid to a

concentration of 2 percent by volume (B). Mix A and B.