THE UPASHS) AND ”Pubs OF CGAGQ’LASE
POSITIVE STAPHYLGCOCCUS AUREUS

 

Thasés f1»? t‘l’w Dagma- ai‘ Fla. D.
MICHIGAN STATE GE‘t‘EVEREITY
Dharam Vi? Vackhm
19653

 

LIBRARY
IW‘

Michigan State
University

 

This is to certify that the

thesis entitled

The lipase(s) and lipids
of coagulase positive
Staphylococcus aureus

presented by

Dharam.Vir Vadehra

has been accepted towards fulfillment
of the requirements for

__P_h°_D;_degree in_F00d Science and
Mierdbiology and Public
Health

I ,"\/(ici U-vvgg, mm " F5 c‘

. ’ nix/#99353 I” M W
Major profeié)

rat. .1 y, r q 6 4,

Date

 

0.169

THE LIPASE(S) AND LIPIDS 0F

COAGULASE POSITIVE STAPHYLOCOCCUS AUREUS

 

BY

Dharam Vir Vadehra

AN ABSTRACT OF A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Departments of
Food Science and
Microbiology and Public Health

l966

ABSTRACT

THE LIPASE(S) AND LIPIDS 0F
COAGULASE POSITIVE STAPHYLOCOCCUS AUREUS

By Dharam Vir Vadehra

The activity of the lipase(s) of two strains of coagulase

positive Staphylococcus aureus was determined in milk fat incubated

 

at IS, 22 and 30 C for 8 days. Hydrolysis of fat was measured by
acid degree values. Neutral lipids were separated into component
groups on a florisil column. Free fatty acids were determined by
temperature programmed gas chromatography. The acid degree values
were 25-50% greater at 22 than at IS C and 4-7 times greater at
30 than at 22 C. The lipases liberated as much as 0.48 g of fatty
acids per g of fat during 8 days incubation at 30 C. The enzyme
showed a predilection for the palmitic acid-glycerol bond. Addition
of fatty acids C4 to C18 inclusive to inoculated sterile milk caused
inhibition of §. agrggg as follows: (a) complete at 0.05 and 0.10%
concentration of Clo,and (b) partial at 0.05 and complete at 0.l0%

concentration of C The samples showing inhibition were negative

8'
for peptonization, coagulase,-and change in pH. Addition of oleic
and stearic acid to sterile milk inoculated WIth.§°.22£EE§ resulted
in an increase in nonprotein nitrogen, and the C4 to C12 acids caused
a decrease in protease activity.

A lipase—rich fraction was isolated from the cell-free super-
natant of a 24 hr broth culture of §. aureus B-l20. The organisms

were grown in trypticase soy broth at 37 C with continuous agitation.

Initial concentration of lipase was achieved by precipitation of the

Dharam Vir Vadehra

cell-free supernatant with equal volumes of absolute ethanol. This
fraction was further purified by differential precipitation at pH
8.6 and 4.3. Subsequent purification using Sephadex G-200 and
BioGel 300 yielded a preparation which showed a 350—450 fold increase
in Specific activity when compared to the original cell-free super—
natant. Lipase production by the cells appears to be affected by
air supply, agitation, age and pH of the cultures during growth.
The optimum pH for lipase production at 37 C was about 8.0; however,
another optimum was found at pH 9.0 suggesting the possibility of
more than one lipase. The presence of air was essential for lipase
production and maximum lipase activity was produced by 5 day old
cells. The purified lipase had an optimum pH of 8.5 at 37 C. The
electrophoretic mobility was £7.78 x 10—5 cmZ/volt/sec. The sedi—
mentation coefficient for the two peaks was 2.85 and 8.5, reSpectively,
and the molecular weight was 100,000. The purified lipase was
capable of hydrolyzing a variety of natural oils and fats. The
amount of free fatty acids liberated from these fats and oils was
similar except in the case of hydrogenated soybean oil (iodine value
< 3) in which the activity was reduced to one-third as compared to
natural oils and fats. Gas chromatographic analysis of a hydrolyzed
synthetic triglyceride with palmitic, stearic and oleic acid at the
<1, B and a' positions, respectively, indicated that the enzyme is
capable of hydrolyzing the glycerol-fatty acid bonds at all three
positions. The yield was 40% palmitic, 20% stearic and 39% oleic
acids.

Formaldehyde, mercaptoethanol, cysteine, glutathione, and

terramycin had an inhibitory effect on the lipase activity while

Dharam Vir Vadehra

hydrogen peroxide,streptomycin and sodium taurocholate had a
stimulatory effect on the activity.

During storage the enzyme lost less than 10% of the activity
over a period of 2i days at 4 and -23 C. Storage at 4 C or freezing
was the preferred method of preserving the lipase activity of the
purified preparation, as compared to lyophilization where as much
as 40% of the activity was lost.

The enzyme lost 7% of the activity when heated for 30 min at
50 C and was completely destroyed after 30 min at 70 C.

The cells of S. aureus contained l2.4% nitrogen, 4.5% phosphorus,
approximately 4% lipids, 78% moisture, and on ignition yielded 13%
ash. Fractionation of the total lipids yielded 77% phospho and 23%
neutral lipids. Diglyceride was the major component of the neutral
lipids, constituting approximately 45% of the total. Lecithin was
the predominant phospholipid, constituting approximately 46-52% of
that fraction. Analysis of phospho and neutral lipids for constituent
fatty acids by gas chromatography revealed similar fatty acid com-

position in both of these components.

THE LIPASE(S) AND LIPIDS OF

COAGULASE POSITIVE STAPHYLOCOCCUS AUREUS

 

BY

Dharam Vir Vadehra

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Departments of
Food Science and
Microbiology and Public Health

l966

Dedicated
to

Mr. and Mrs. Sri Ram Vadehra

ACKNOWLEDGEMENTS

The author wishes to thank all with whom he has been
associated during the course of his study in this country and who
were instrumental in making his stay a pleasant and memorable one.
Special thanks to to the following persons:
Dr. L. G. Harmon, Professor of Food Science,for his guidance,
encouragement and counsel in academic and personal matters. His
philosophy of life will be a constant afflatus throughout the time
to come.
Dr. Harold L. Sadoff, Professor of Microbiology, for acting as joint
major professor and giving his valuable time for discussions and
preparation of the manuscript.
Dr. J. Robert Brunner, Professor of Food Science, for allowing use
of his laboratory facilities and for his enriching philosophical and
scientific discussions.
Dr. Richard V. Lechowich, Department of Food Science, for his help
in the preparation of the manuscript and willingness to share his
laboratory facilities.
Dr. Hans A. Lillevik of the Department of Biochemistry for serving
on the guidance committee.
Dr. Bernard S. Schweigert, Chairman, Department of Food Science, for
providing financial assistance.
Miss Delores M. Fang for her secretarial help.
Last but not least the author thanks his parents Mr. and Mrs.
Sri Ram Vadehra for their sacrifices and constant inspiration for a

higher education, and to whom this thesis is reSpectfully dedicated.

TABLE OF CONTENTS

Objective of Research . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . .
Literature Review . . . . . . . . . . . . . .
Lipases . . . . . . . . . . . . . . . .
Microbial lipases . . . . . . . . . .
Lipid composition of microbial cells
Experimental Procedure and Results .
Growth and action of_§._au£gg§ on milk
Growth of_§._ag£eu§ . . . . . . . . . .
pH changes during growth . . . . . . .
Determination of acid degree value . .
Analysis of the milk lipids .

Esterification of fatty acids . . .

Measurement of volatile fatty acids . .

Effect of free fatty acids on growth of S. aureus .

Measurement of nonprotein nitrogen

Effect of fatty acids on coagulase activity of

S. aureus 0 I O O O O I O O O O O 0

Effect of fatty acids on the pH production by

'S. aureus in skimmilk . . . . . . .

Effect of fatty acids on coagulability of

.inoculated with §. aureus . . . . .
Isolation and purification of lipase . .
Preparation of the cell-free supernatant
Assay for lipase . . . . . . . . . . .

Measurement of protein concentration .

skimmilk

Page

l0
l0
l0

ll

2]

23

23

3O

30
33
33
33

34

Lipase production .

Effect of agitation and air .

Effect of age . . . . . . .

Effect of pH on lipase activity .

Effect of added chemicals on lipase production

Presence of intracellular lipase

Concentration of staphylococcal

Ammonium sulfate precipitation

Precipitation with acid . .

Precipitation with ethyl alcohol and acetone

Purification of staphylococcal

lipase

lipase .

Characterization of the enzyme preparation . .

Optimum pH for lipase action

Optimum temperature for lipase activity .

Storage stability of purified lipase

Thermal inactivation of the lipase

Action on various natural fats and oils .

Action of lipase on a synthetic triglyceride

Effect of chemical agents

Free boundary electrophoresis

Sedimentation coefficient and molecular

Chemical composition of S. aureus cells

Determination of moisture

Determination of nitrogen

Determination of phOSphorus .

Determination of ash . . .

iv

Page
34

36
36
37
4O
43
43
45
45
47
47
52
52
56
56
56
6O
64
65
66
69
73
73
73
73

73

Determination of total lipids .

Separation of neutral and phospholipids . . . . . . .

Thin layer chromatography of neutral and phOSpholipids.

Analysis of neutral lipids

Analysis of phOSpholipids .

Analysis of fatty acids . .
Discussion . . . . . . . . . . .
Summary . . . . . . . . . . . . .

Literature Cited . . . . . . . .

Page
74
74
76
78
78
78
84
94

98

LIST OF TABLES
Page

Table I. Number of organisms per ml in cereal milk
inoculated with S. aureus and incubated
at 15, 22, and 30 C for 8 days . . . . . . . . . l2

Table 2. Changes in the pH of cereal milk inoculated
with S. aureus and incubated at 15, 22,

and 30 C for 8 days . . . . . . . . . . . . . . 13

Table 3. Acid degree values of fat extracted from
milk inoculated with S. aureus and
incubated as indicated . . . . . . . . . . . . 15

Table 4. Components of lipids extracted from cereal
milk inoculated with S. aureus strain S-1
and incubated as indicated . . . . . . . . . . . 18

Table 5. Amount of free fatty acids liberated by
S. aureus strain S-l when grown in cereal
milk under the conditions indicated . . . . . . 20

Table 6. Volatile fatty acids recovered from 10 g of
cereal milk (10% milk fat) inoculated with
S; aureus strain S-l and incubated as
indicated . . . . . . . . . . . . . . . . . . . 22

Table 7. Population per ml when S. aureus strain S-l
was grown at 30 C in skimmilk containing
0.01% free fatty acids . . . . . . . . . . . . . 25

Table 8. Population per ml when S. aureus strain S-l
was grown at 30 C in skimmilk containing
0.05% free fatty acids . . . . . . . . . . . . 26

Table 9. POpulation per ml when S. aureus strain S-l
was grown at 30 C in skimmilk containing
0.1% free fatty acids . . . . . . . . . . . . . 27

Table 10. Amount of tyrosine liberated when skimmilk
containing added fatty acids was inoculated
with S. aureus strain S-1 and incubated
8 days at 30 C . . . . . . . . . . . . . . . . . 28

Table 11. Coagulase activity of skimmilk containing fatty
acids, inoculated with S. aureus strain S-1
and incubated at 30 C for 8 days . . . . . . . . 29

Table 12. Changes in pH when skimmilk containing added
fatty acids was inoculated with S. aureus
strain S-1 and incubated at 30 C . . . . . . . 31

vi

Table 13.

Table 14.

Table 15.

Table 16.

Table 17.

Table 18.

Table 19.

Table 20.

Table 21.

Page

Coagulability of skimmilk containing added
fatty acids when inoculated with S. aureus
strain S-1 and incubated at 30 C for
8 days . . . . . . . . . . . . . . . . . . . . 32

Effect of age and agitation on the activity of
lipase produced PY.§' aureus strain B-120 when
grown in trypticase soy broth at 37 C.
(Lipase activity measured in olive oil emulsion
for 10 min at 37 C and pH 8.0.) . . . . . . . . 38

Effect of air and agitation on the activity of
lipase produced by S. aureus strain B-lZO
grown in trypticase soy broth at 37 C.
(Lipase activity measured in olive oil emulsion
for 10 min at 37 C and pH 8.0.) . . . . . . . . 39

Effect of pH on activity of lipase produced by
'§. aureus strain B-120 grown in trypticase soy
broth for 24 hr at 37 C. (Lipase activity
measured in olive oil emulsion for 10 min
at 37 C and pH 8.0.) . . . . . . . . . . . . . . 41

Effect of chemical agents on activity of lipase
produced by S. aureus strain B-120 grown as
indicated at 37 C for 24 hr. (Lipase activity
measured in olive oil emulsion for 10 min at
37 C and pH 8.0.) . . . . . . . . . . . . . . . 44

 

The lipase activity of various fractions obtained
by precipitation of the cell-free supernatant
obtained from S. aureus strain B-120 with
different concentrations of ammonium sulfate.
(Lipase activity measured in olive oil emulsion
for 10 min at 37 C and pH 8.0.) . . . . . . . . 46

Lipase activity of the fractions resulting
from treating the cell-free supernatant obtained
from S. aureus with absolute ethyl alcohol.
(Lipase activity measured in olive oil emulsion
for 10 min at 37 C and pH 8.0.) . . . . . . . . 48

A comparison of the various methods for the
initial concentration of staphylococcal lipase.
(Lipase activity measured in olive oil emulsion
for 10 min at 37 C and pH 8.0.) . . . . . . . . 49

Activity of the staphylococcal lipase (strain B-120)
obtained from the cell-free supernatant at
each state of fractionation. (Lipase activity
measured in olive oil emulsion for 10 min at
37 C and pH 8.0.) . . . . . . . . . . . . . . . 55
VII

Table

Table

Table

Table

Table

Table

Table

Table

27.

28.

Amount of free fatty acids liberated from
various natural lipids by purified
staphylococcal lipase (strain B-120).
(Lipase activity measured in olive oil

emulsion for 10 min at 37 C and pH 8.0.) . . .

Effect of hydrogen peroxide, formaldehyde and
2-mercaptoethanol on lipase activity
measured in olive oil emulsion for 10 min
at 37 C and pH 8.0 . . . . . . . . . . . .

Effect of various chemicals on lipase activity

of S. aureus measured in olive oil emulsion
for 10 min at 37 C and pH 8.0

Physical characteristics of the purified
staphylococcal lipase preparation . . . .

Nitrogen, phosphorus, ash, total lipids and

moisture content of cells of S. aureus strain

B-120 grown as indicated for 24 hr at 37 C .

Phospho and neutral lipid composition of the
lipids of S. aureus strain B-IZO using the

silicic ach and countercurrent distribution

methods of separation . . . . . . . . . .

Composition of the neutral lipid fraction
extracted from S. aureus strain B-120 when
separated into various components as indi-
cated using thin layer chromatography

Composition of the phospholipid fraction
extracted from S. aureus strain B-120 when
separated into various components as indi-
cated using thin layer chromatography . .

viii

Page

63

67

68

72

75

77

79

8O

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

2.

LIST OF FIGURES
Page

Changes in population Of.§' aureus strain 5-]
when grown in skimmilk containing 0.01,
0.05 and 0.10% of the fatty acids indicated . . . 24

Standard curve indicating protein concentration
(lysozyme) vs optical density at 660 mp using
the method of Lowry_gt_al. (1951) . . . . . . . . 35

Milliliters of 0.0lN NaOH required to neutralize
the free fatty acids formed by the lipolytic
enzymes produced by S. aureus strain B-120
grown in trypticase soy broth at 37 C at the
pH indicated. (Lipase activity measured in
olive oil emulsion for 10 min at 37 C and
pH 8.0.) . . . . . . . . . . . . . . . . . . . . 42

An elution diagram of a lipase preparation on
Sephadex G-200 using tris buffer pH 8.1,
F72 = 0.1. Lipase concentration is repre-
sented as micrograms of protein and the
lipase activity as ml of 0.01N NaOH required
to titrate free fatty acids liberated by the
lipase in olive oil emulsion for 10 min at 37 C
and pH 8.0 . . . . . . . . . . . . . . . . . . . 51

An elution diagram of a lipase preparation on
BioGel 300 using tris buffer pH 8.], [‘72 = 0.1.
Lipase concentration is represented as micro-
grams of protein and the lipase activity as ml
of 0.01N NaOH required to titrate free fatty
acids liberated by the lipase in olive oil
emulsion for 10 min at 37 C and pH 8.0 . . . . . 53

Flow sheet for the isolation and purification of
staphylococcal lipase . . . . . . . . . . . . . . 54

Milliliters of 0.01N NaOH used to titrate the
free fatty acids liberated by the action of
staphylococcal lipase in olive oil emulsion
for 10 min at 37 C at various pH values . . . . . 57

Milliliters of 0.01N NaOH required to titrate the
free fatty acids liberated from olive oil by
staphylococcal lipase after 10 min at pH 8.0 at
various incubation temperatures . . . . . . . . . 58

Percent loss of activity of staphylococcal
lipase when stored at various temperatures
for the times indicated . . . . . . . . . . . . . 59

ix

 

Page

Fig. 10. Percent loss in the activity of staphylococcal
lipase heated in tris buffer at pH 8.1 at

the various times and temperatures indicated. . . 61
Fig. 11. Percent loss of staphylococcal lipase activity

when heated in tris buffer at pH 8.1 at the

various temperatures for the time indicated . . . 62

Fig. 12. Free boundary electrophoretic pattern of

purified staphylococcal lipase in veronal
buffer at pH 8.6 F—/2 = 0.1 after 3,000
seconds of electrophoresis . . . . . . . . . . . 70

Fig. 13. Sedimentation velocity pattern of 1% lyOphilized
purified staphylococcal lipase solution in
veronal buffer at pH 8.6 F_/2 = O.l centrifuged
at 50,790 rpm at 20 C . . . . . . . . . . . . . . 71

Fig. 14. The gas chromatographic pattern of methyl
esters of fatty acids of phospholipids
extracted from cells of_§. aureus B-120 . . . . . 82

Fig. 15. The gas chromatographic pattern of the methyl
esters of fatty acids of neutral lipids
extracted from cells of_§. aureus B-120 . . . . . 83

OBJECTIVES OF THE RESEARCH
Whole milk is inferior to skimmilk as a substrate for the

growth of Staphylococcus aureus. Since the only difference between

 

the two substrates is fat, then either fat.EEI.§E or its derivatives
are deleterious to the growth of the organism. The first part of
this dissertation therefore is concerned with the elucidation of

the nature of the changes produced in milk fat during growth of

-§. aureus. The effect of added fatty acids on the growth and enzyme
activity of S. aureus was studied using skimmilk as a substrate.

After studying the gross changes produced PY.§° aureus lipase,
the isolation and purification of the enzyme was deemed desirable.
The factors affecting the production and activity of this enzyme
were also investigated.

During the investigation of changes in the lipid portion of
milk caused by growth of S. aureus, the lipids of this organism
were also extracted along with the milk fat. The S. aureus lipids
were unimportant in the compositional changes of milk fat as they
represented only 0.002% of the total lipid extracted, but the
qualitative and quantitative analyses Of.§° aureus lipids were con-

sidered worthy of investigation.

INTRODUCTION

The lipases are ubiquitous in nature being present in all
biological systems. Several microorganisms possess strong lipolytic
enzymes. The lipases exist both extracellularly and intracellularly
but exolipases have been investigated more thoroughly because they
are easier to obtain. The lipases are biologically important enzymes
in the degradation of fats and oils to make available the fatty acids
for energy and nutrition in cellular metabolism. The beneficial
effect of lipases during the ripening of various varieties of cheese
has been amply demonstrated by research workers. Excessive amounts
of liberated fatty acids can also have a detrimental effect on the
growth and fermentation ability of the organisms.

Lipases have many commercial uses. In foods, butter oil is
being lipolysed under controlled conditions to deveIOp a Specific
type of aroma used extensively in certain bakery products. Lipases
could also be used to alter the composition of the natural fats and
oils to meet changing consumer demands. A combination of several
lipases with different specificities might be necessary to achieve
this goal. In human medicine, lipases are being tried to lower the
blood lipid level. The great concern and confusion of professional
and nonprofessional people about dietary fats and their possible
relationship to coronary diseaseshave opened new avenues for the use
of lipases. It is possible that lipases in association with
cholesteraseserw phOSpholipases could be used to modify lipid compo-
sition. Lipases could also have a possible use in the mobilization
of depot fats and fat around the cardiac muscles.

A possible relationship between pathogenicity and lipase

2

production by Staphylococcus aureus has been suggested since_§. aureus
is the only pathogenic organism known to produce a detectable
exolipase (Davies, 1954).

‘Recent studies on microbial lipases indicate that some of these
lipases have a significant difference in their Specificity as com-
pared to classical pancreatic lipase. With the improved instrumen-
tation during the past decade, the interest in fundamental and
applied research on lipolytic enzymes has been renewed.

The purpose of the investigation reported herein was therefore
to: a) study the effect of growth of_§. aureus on milk fat and the
effect of fatty acids on the growth of_§. aureus; b) isolate, purify
and characterize the exolipase of S. aureus; c) determine the
chemical composition of §. aureus with special emphasis on lipid

composition.

LITERATURE REVIEW

During the past several years there has been an extensive in-
vestigation of the physiology of staphylococci. This renewed inter-
est has been brought about by both the medical profession and dairy
and food bacteriologists interested in the resistance of the
staphylococci to antibiotics and heat. Several theses have been
written on the thermal resistance of staphylococci in milk including
those by Busta (1963), Walker (1964), and Zottola (1965). The
subject of thermal resistance of the staphylococci in certain foods
has been thoroughly reviewed by Angelotti _£ _1. (1960). Ecology
is important in the growth, death and fermentation ability of most
organisms including staphylococci. During 1962-1964 Peterson _£ ad.
published a series of papers entitled llStaphylococci in Competition”.
They studied naturally occurring mixed populations in preconked
frozen food and the total number and proportion of staphylococci
in mixed cultures capable of growth in artificial media. The
effect of pH, salt, sugar, eggs and lipids on pure and mixed cul-
tures of staphylococci has also been investigated by Peterson
_£‘_l. (l962a,b and l964a,b,c). Walker (1964) observed that skim-
milk was superior to whole milk as a medium for the growth of
staphylococci. Free fatty acids liberated from the whole milk were
suggested as the possible cause of inhibition Of.§° aureus. Some
earlier work by Costilow and Speck (1951) on streptococci showed
that some of the fatty acids are inhibitory to the growth of the
streptococci. The liberation of these fatty acids in biological

systems results from the action of lipolytic enzymes on tri-

glycerides.

Lipases. There is some confusion about the definition of
“lipase” and Hesterase”. Bier (1955) suggests that this confusion
arises from the fact that both groups are usually nonspecific in
their action and the nature of either the fatty acids or the alcohol
residue has a secondary effect, influencing only their rate of
hydrolysis. Several workers have used the term lipase synonymously
with esterase. According to Desnuelle (1961a), a true lipase is
one which splits emulsified esters of glycerin and fatty acids with
long chains, such as triolein or tripalmitin. This definition is
arbitrary except that it has a physiological basis. Marchis-Mouren
_£ ad. (1959) suggested that a lipase hydrolyses di and monoglycerides
as well as triglycerides although the rates are in decreasing order
of the saturation of the glyceride molecule. On the other hand,
Overbreek (1957) and Desnuelle (1961a) prefer to use the term
aliesterase or simply esterase for the “so called lipases'l which act
on solutions of triglycerides with short chain fatty acids. “Wheat
germ lipase” for example, in the true sense of the word, is an
esterase, while pancreatic lipase is a true lipase.

The importance of lipases in digestion and metabolism has been
amply demonstrated. Their role in the food industry is two fold
involving both beneficial and detrimental effects. In Spite of
their fundamental importance, our understanding of these enzymes
is rather meager.

The lipase subjected to the most extensive study is the one
produced by the pancreas. This enzyme has been purified by Willstatter

and Waldschmidt-Leitz (1923), Meyer t l. (1947), Boissonnas (1948),

and more recently by Sarda t l. (1957), Marchis-Mouren (1959) and

Coleman (1963). The interest in pancreatic lipase has been from
the physiological and pathological standpoint and there is an
excellent review by Desnuelle (l961a).

Microbial lipases. Our knowledge on microbial lipases is

 

limited and scattered. Goldman and Rayman (1952) reported that
fungi and some bacteria produce lipases of importance in the food
industry. Most of the work on bacterial lipases has been done on
crude preparations and concerns the comparison of relative rates of
hydrolysis of various natural and synthetic triglycerides. Shipe

(1951) studied the relative Specificity of lipases from Penicillium

 

rogueforti and Aspergillus niger. The enzymes studied were intra-

 

cellular lipases extracted from the mycelia. The optimum tempera-
ture, pH, effect of calcium and effect of several inhibitors were
also reported. Fukumoto §t_al. (1963) and |wai__t__l. (l964a,b)
crystallized lipase from 5°.fli325 and have studied the enzyme
physically and chemically. The comparisons of the lipolytic ac-
tivity of the various microorganisms have limited validity since
the assay conditions, time and temperature of incubation, differ-
ence in solubility, ease and stability of emulsion, and the size of
1 fat globules have an effect on the rate of hydrolysis and the assay
method. At least five or six different methods have been exten-
sively used for assaying lipase activity. These methods involve one
of the several basic techniques such as using a) chromogenic Sub-
strates, Foster__t__l. (1955) and Greenbank (1950); b) manometric
techniques, Willart and Sjbsstrom (1959); c) reduction of surface

tension, Dunkley and Smith (1951); d) spectrophotometric determina-

tion, Bbrgstrom (1957) and e) the pH stat method of Marchis-Mouren

_£._l' (l959) and Parry sf 21' (1965). Recently (1962) the Inter-
national Commission on Enzyme Nomenclature has defined a lipase
unit and recommended standardized techniques for measuring activity.

Bacterial lipases have not been studied extensively. Members

of the genera Pseudomonas, Achromobacter are prolific producers of

 

lipases. Nashif and Nelson (l953a,b,c) published on the lipase of

Pseudomonas fragi. Factors affecting the lipase production and

 

the action of this lipase on cream and butter as well as some of

the characteristics of the enzyme were described. Mencher and

Alford (1965) isolated a lipase-rich fraction from f. fragi. Shahani
t al. (1964) isolated an intracellular lipase-rich fraction from

Achromobacter lipolyticum while Khan t 1. (1964) reported the

 

existence of an extracellular lipase in the same organism. Earlier
work of Richards and ElSadek (1949) and Wilcox _t-_l. (1955) on the
action of microbial lipase was done on milk fat. They applied the
techniques of differential volatility and paper chromatography to
steam distillates and suggested that differences in Specificities
existed among the lipases of various organisms.

Alford et_al. (1964) in an extensive survey tested over 73
microorganisms for their ability to produce lipase. Using lyophilized
cell-free supernatants they reported that the Specificity of micro-
bial lipases falls into three main categories: a) attacks primarily
the<1 position, b) attacks the a, B or a' positions, and c) attacks

the oleic acid linkage irrespective of position. The third type of

activity is shown by Geotrichum candidum. They claim this type of

 

activity is unusual because the enzyme Specificity is primarily for

the acid and the position is of secondary importance. Jensen _t _l.
(1965) have confirmed this report.

The interest in the study of staphylococcal lipase dates back
to the work of Gillepsie and Alder (1952) and Alder__t__l. (1953)
who associated the ”egg yolk factor'l with virulence. They also
suggested that this egg yolk factor has lipase activity. However,
Rosendal (1962) presented evidence to the contrary showing no
relationship between the egg yolk factor and virulence. Burns and
Holtman (1960) suggested a possible relationship of the egg yolk
factor to pathogenicity. Blobel _t_al. (1961) isolated the egg yolk
factor and later Shah and Wilson (1963, 1965) studied some of its
characteristics. This factor has been reported to have lipase
activity but it is not clear if it is lipase BEE 23'

Renshaw and SanClemente (1964) reported on partial purification
of staphylococcal lipase but the characteristics have not yet been

determined.

Lipid composition of microbial cells. One of the earliest

 

references to bacterial lipids was that of Nencki (1886). However,
interest in the study of bacterial lipids was dormant until Anderson

(1943) began his studies on the lipids of Mycobacterium tuberculosis

 

to determine if any correlation existed between lipid composition
and pathogenicity. Woodbine (1959) in a review of microbial lipids
emphasized the importance of microorganisms as a potential source of
human food. Most of the research on bacterial lipids has been per-
formed to find an answer to intrinsic scientific inquiry and mode of
synthesis of lipids.

The amount of lipids present in bacteria varies from 8-40%

(Larson and Larson, 1922). The quantity depends on the method
employed for extraction of lipids and the growth medium. Salton
(1953) reported that ether extraction before or after methanol
treatment removes only one-third of the lipids isolated from the

cell walls of Escherichia coli. For complete release and extrac-

 

tion of lipids, hydrolysis with 6N HCl at 100 C for several hr is
recommended by Luria (1960). This method is less applicable when
the individual composition of various labile components is desired.
Larson and Larson (1922) also reported that the presence of

glycerol in the growth medium increased the lipid content of most

organisms by 3-5 fold. Cook and Woodbine (1958) using Aspergillus

 

_g§£u§ Showed that agitation during growth had a beneficial effect
on lipid production. Kates (1964) has reviewed the literature on
bacterial lipids but not much information is available on the
lipids of S. aureus. Macfarlane (1962) and Polonovski 3; El.
(1962) have recently studied the lipids of S. aureus but report

somewhat conflicting data.

EXPERIMENTAL PROCEDURES AND RESULTS
Growth and Action of S. aureus on Milk Fat

The work reported describes the effect of a) growth of S.
aureus on milk fat and b) the presence of fatty acids on the growth
and enzyme activity of S. aureus in skimmilk.

Two strains of S. aureus were used in this study. The strain
B-120, responsible for a food posioning outbreak, was obtained from
the Northern Regional Research and Development Division, Agricultural
Research Service, U.S. Department of Agriculture, Peoria, Illinois.
Strain S-l was isolated from milk from a cow in the Michigan State
University dairy herd,which had clinical staphylococcal mastitis.
These strains had the typical characteristics of S. aureus as des-
cribed by Breed t l. (1957).

Growth of S. aureus. Both the B-120 and S-l strains of S. aureus

 

were used to study the gross changes produced in the lipid fraction
of milk. However, during the latter part of the study only strain
B-lZO was used.

The organisms were grown in sterile cereal milk (milk con-
taining 10% milk fat). Five hundred milliliter quantities of milk
were dispensed in one liter Erlenmeyer flasks and sterilized at
121 C for 20 min. The inoculum was grown either in sterilized
cereal milk or trypticase soy broth (Baltimore Biological Labora-
tories, Baltimore, Maryland) at 32 C for approximately 18 hr.
Enough inoculum was added to give a final population of approximately
1,000,000 cells per ml in the substrate. The cultures were incu-

bated for a period of 8 days at 15, 22 and 30 C. One flask of

10

11

medium was inoculated with a culture grown for 18 hr in trypticase
soy broth. This flask was incubated at 30 C and treated the same

as the other samples. Uninoculated samples were incubated along
with the inoculated samples and served as controls. Another flask
with added inoculum was frozen and stored at -23 C during the period
of Study.

Fifty milliliter samples were withdrawn aseptically from each
flask immediately after inoculation and mixing, and at 24 hr inter-
vals thereafter up to 8 days. The number of organisms per ml of
sample was determined according to Standard Methods (1960) using
plate count agar (Difco Laboratories, Detroit, Michigan).

The data obtained are summarized in Table l. The temperature
had a profound effect on the rate of growth. At 30 C the maximum
population occurred within 48 hr and then declined during the re-
mainder of the incubation period. However, at 22 and 15 C there
was a continuous gradual increase in population through the 8 day
incubation period. The variation in the number of viable cells at
the end of the incubation period is noteworthy.

pH changes during growth. The pH of each sample was deter-

 

mined using a Beckman glass electrode pH meter Model H~2. The changes
observed in pH during the 8 day incubation period of cereal milk
inoculated with_§. aureus B-120 and S-l are recorded in Table 2.

Determination of acid degree value. The milk fat was extracted

 

with a Separatory funnel from the lyophilized samples using 50 ml of
absolute ethyl alcohol, followed by the same amount of ethyl ether
and finally taken up in 50 ml of hexane. The solvents were removed

from the lipid extract under vacuum using a Rinco evaporator with an

12

pmc.Ecmomp “o: i

 

 

 

 

_ v - i .. . v - .. _ v .. on 90:88 802
N. mm mm owe 00. one 00.. 0mm N.w om
ooN. com. com o.m omq 0mm 0mm oq. N.w mm
owm 0.0 o.n ooq 0mm 0mm o.N cm 0.0. m. .im
we we co. 0mm owm com comm 005. ..o On
1 com. 000. 0mm 0mm oqm mm 0.. N.o mm
com com. com com omq oom 0mm .w q.o m. Om.im
Amco....E c.%l.E Loo ucnoQ
w m o m q m N . o mcsumcmaemu Em.cmmco
co.omn:oc. mo mxmo co_omn:oc_ mo c.mcum

 

.m. up pmumnsoc. pcm msmcsm .W.Lu.3 omum.300c. x..E .mmcmo

m>mp w co. u on can «mm

c. .E coo mEm_cmmco mo meEJZ .. o.nmh

l3

pmc_Ecmump go: i

 

 

 

 

 

 

mm.a - mm.a - - o~.a - - om.a om ._occcoUv acoz
00.0 0N.0 0..0 0N.0 00.0 00.0 00.0 00.0 0N.0 00
00.0 00.0 00.0 0..0 00.0 00.0 00.0 0N.0 00.0 NN
00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 0. .im
00.0 0..0 00.0 00.0 0..0 0N0 00.0 00.0 0N.0 00
00.0 0..0 0..0 00.0 0..0 00.0 00.0 00.0 0N.0 NN
00.0 0N.0 00.0 00.0 00.0 00.0 00.0 00.0 0..0 0. 0N.im
:0
w m 0 0 0 m N _ 0 mcsumcmmEmu Em.c©mco
co_um0:oc. mo mxmo co_um0:oc_ mo c.mcom
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.NN «0. pm pmomnzoc_ 0cm mzocnm .w.zo.3 poum.:o0c. x..E .mmcoo mo In 000 c. mmmcmcu .N p.0mk

14

air driven motor and a laboratory aspirator. The evaporation was
performed at 60 C. The extracted lipids were usually cloudy at this
point and were centrifuged at 10,000 X g for 15 min. The final dry-
ing of the extracted lipids was done on a steam bath under a constant
stream of nitrogen.

The extracted and dried milk fat was tested for rancidity using
the method proposed by Thomas__t_al. (1955). The rancidity was
expressed as acid degree value which iS defined as ml of 1N methanolic
potassium hydroxide required to titrate 100 g of fat.

The method consisted of taking 1 m1 of melted milk fat in a
syringe and dissolving in 5 ml of fat solvent consisting of hexane
and N-prOpanol. The titrant was 0.025 N methanolic potassium
hydroxide and the indicator was 1% alcoholic phenolpthalein. The
fat solvent alone was titrated to obtain the blank reading. The
acid degree value (ADV) was calculated using the following equation.

m1 KOH used for sample - ml KOH used for blawzx normality x 100
weight (g) of fat in the sample

 

ADV =

In these calculations 1 ml of milk fat was considered as 0.90 g.

The degree of hydrolysis of milk fat during the growth Of.§°
aureus in cereal milk at various incubation temperatures was measured
by acid degree value. The data are shown in Table 3.

The acid degree value increased as the incubation time pro-
gressed at all temperatures and there was a positive correlation
between the acid degree value and temperature. The acid degree
value was only 25~50% greater at 22 C than at 15 C but at 30 C the
acid degree value was 4-7 times greater than at 22 C. These results

indicate a definite preference for the production and/or action of

15

poc.Ecmump yo: i

 

 

 

 

 

N0.0m i i i N0.00 a i 0N.0. 00.N 00
0N.0. i i i 0N.0. i i 0..0 .0.N NN x..E c.
00.0. - - - Ea - - 8:0 0.; 2 E63 Tm
0..0.. 00.0.. NN.N.. 0N.N0. “mo. 00.0w 00..0 00.0N 00.0 00
00.0. 00.0. 00.0. 00.0. 0N.N. Nn.0 N0.m .0.0 m0.m NN x._E c_
00.N. .0... N0.0. 00.0 .0.“ .0.N .0.0 00.0 0N.0 0. c30c0 0N.i0
£0000 >00
ommo.oq>cu c.
00.00 m0.00 .0.0N 0N.00 m..00 00.00 .0.00 0N... 00.0 00 czocm 0N.i0
m:.m> mmcmov 0.o<
0 m 0 0 0 m N . 0 ocsomcmmEou E:.:oo:.
co.um0:oc. mo m>m0 co.0m030c_
po~mo.0:. mm
pagansoc. 0cm mzocsm .w.;p.3 pm~m.:oOc. x..E Eocw pmpomcuxo um» 00 mm:.m> mocmmp p.o< .0 p.0mF

l6
lipase at 30 C by the enzyme(s). The data also indicate that
lipolytic activity varies considerably between strains, with strain
B-120 being almost twice as active as strain S-l. The cells acti—
vated in cereal milk were more lipolytic than those activated in
broth.

Analysis of the milk lipids. Cells of S. aureus S-l inere

 

grown in milk as described previously. The samples removed initially
and thereafter every 24 hr were not lyophilized; however, the lipids
were extracted in the usual manner. The extracted lipids were

dried and l g was dissolved in 20 ml of N-pentane. The free fatty
acids and the remaining portion of the neutral lipids were separated
using Amberlite (Mallinckrodt Chemical Works, St. Louis, Missouri)
IRA-400 resin which had been prepared in the following manner. Ten
grams of resin were stirred with 25 ml of 1N NaOH for 5 min. The
base was removed by washing several times with water. This was
followed by dehydration of the resin using absolute ethanol. The
alcohol was finally replaced by N-pentane.

The dissolved fat was applied to the resin and stirred with a
magnetic stirrer for 6 min. The supernatant was removed and the
resin was washed two additional times with 25 ml of N-pentane. The
washings were collected, filtered and concentrated under vacuum.

The final drying was done on a hot plate under a constant stream

of nitrogen. The residue of neutral lipids was weighed. The free
fatty acids which were adsorbed by the resin were calculated by
difference. The neutral lipids were further separated into various
fractions using Florisil column chromatography as recommended by

Carroll (1961). Florisil (activated magnesium silicate manufactured

17

by Floridin Co., Tallahassee, Florida) as supplied was deactivated
by mixing 7 g of water with 100 g of florisil in a tightly stoppered
Erlenmeyer flask and allowed to set overnight. A 20 x 1.6 cm column
with a water cooled jacket was used. The preparation of the column,
application of the sample, and the solvents for elution were exactly
the same as suggested by Carroll (1961). The solvents were
evaporated and the quantities of various fractions determined gravi-
metrically. The separation of cholesterol esters and triglycerides
was not very sharp, so they are reported as a single fraction.

The result of fractionation of the hydrolyzed fat on a flori-
sil column is Shown in Table 4. The data Show an increase in the
lipolytic activity, evidenced by an increase in the amount of free
fatty acids as the time increased from 0-8 days and the tempera-
ture increased from 15-30 C. Also, the amount of tri and diglycerides
decreased in approximate proportion to the increase in free fatty
acids as the temperature increased and the time progressed. The
quantities of monoglycerides and cholesterol remained uniform or
showed only slight fluctuations during incubation.

Esterification of fatty acids. The free fatty acids adsorbed

 

to the resin were converted into their methyl esters by the method
'of Hornstein _t 21' (1960). The methanolic hydrochloric acid used
for esterification was prepared by adding concentrated sulfuric

acid into concentrated hydrochloric acid and bubbling the liberated
gas into absolute methyl alcohol. After an increase of 5-10% in
weight, the bubbling was stopped and the mixture stored at a temper-

ature of 4 C. The methanolic hydrochloric acid was generally used

within a week and was discarded after 10 days. Twenty five milli-

18

mcmumm .OLmumo.oLo mopz.oc. e

 

 

 

 

 

 

 

 

0.0a 0.0m 0.0a o.ma 0.0a 0.0m m.aa 0.0m m.ma oo. emcm>oUmt
mmmucmocoa
ca 00 om 00 cm on co ca 00 00 .otmcmm_o£u
000 ems 0.0 000 0.0 00k 000 00k 0km o.a amme_tmo>_m_ce
o. o. cm 0 m an 0. ON 00 o www.cmu>_m_o
m o. o. m m m o o m o www.cau>.m0coz
owe owm mam omm 0mm ck. mmm ma. om. om ma_um >cum. amen
0mm 00 0 comimE
a 0 _ w 0 . m 0 _ o ecsomeoU
mxmv c. we.» .0cucoo
Om mm m._ Umum—DUOC_COZ

 

Auv mczumcanmh

pmumo.pc. mm poumnsoc. 0cm
.im c.mcum msocnm .w.£u.3 poum.300c. x..E .mocoo Eoc0 pmuomcpxo mp.a_. mo mucoCOQEOU .0 p.0mh

19

liters of methanolic hydrochloric acid mereadded to the resin and
stirred for 25 min. The supernatant was removed and the process
repeated twice with 15 ml of fresh methanolic hydrochloric acid,
and Stirred for 5 min. Fifteen milliliters of deionized water were
added to the supernatant and the esters were extracted with 50 m1 of
N-pentane. The extraction was repeated twice with 20 ml of N-
pentane. The combined N-pentane extracts were washed with water

to remove any acid present and then dried over anhydrous sodium
sulfate. The solvent was evaporated and the samples analysed on a
temperature programmed F and M gas chromatograph (Model 500, F and
M Corporation, Avondale, Pennsylvania). The temperature was pro-
grammed at the rate of 7.9 C/min over a range of 80-200 C. Twenty
microliters of the evaporated samples were injected into the
column. The final volume of the evaporated material and the amount
of the material injected for analysis by gas-liquid chromatography
was the same in all cases for ease of comparison. Known quantities
of pure fatty acids were mixed and esterified on the resin in
exactly the same way aS the lipolyzed fat. The quantitation of

the unknown samples was done by comparison to the standard and
known mixture of fatty acids. The data obtained from the chromato-
graphic analysis are shown in Table 5. These data include only the
fatty acids with 10 or more carbon atoms. L0wer chain fatty acids
were determined in a separate experiment.

Measurement of volatile fatty acids. During the preparation

 

of the samples for gas chromatographic analysis,extensive losses of
volatile acids made it apparent that another method of measuring

these volatile acids was needed. The rapid distillation method of

20

 

 

 

 

 

 

 

 

 

Rm.mq mm.mm w_.m_ NN.NN mm.o om.¢ qm.m ow.o qm.q mm.m o_m_o
om.om m¢.m_ o.w ¢_.m_ wm.¢ mm._ mm.m om.m mo.m om._ o_cmoum
mw.mm no.0q m_._m ow.qq oq.c_ mm.m _m.m_ ow.m o¢.q oo.m o_u_E_mm
mm.qm om.a_ aa.~ w©.q_ aq.a o_.m _o.m mm.m qN.N mm.o u_bm_t>z
NN.w mo.© mm.m mm.m om.m No._ oo.m m... _w.o mm.o o_c3m4
~_.o mo.q ow._ q_.q mm._ mm.o mm.m “woo «0.0 qm.o o_camu
pm; #0 m com mp_om >uumw mo mE
w q _ w q _ w a _ o p_o<
mme c_ mE_H _0cucoo
om mm mm amum_suoc_c02
AQV mcsumLanoH
Umumompc_ mco_u_pcoo mzu coca:
x__E _mmcmo c_ czocm cog; _iw c_mcum msmcsm .w.>n poumconm_ mp_om >ubmm omcv mo uc:OE< .m o_nmh

2l

Kosikowski and Dahlberg (l946) was adapted for this work. The
method involved digesting and hydrolyzing 10 g of inoculated cereal
milk with magnesium sulfate and sulfuric acid. The volatile
materials were distilled over with precaution being taken not to
exceed a temperature of llO C and to collect the same quantity each
time. Since alcohol was used to wash the condenser, both the water
soluble and water insoluble acids were measured by this method.

The volatile acidities were measured as the ml of base re-
quired to neutralize the acids. The data are presented in Table 6.
These results indicate a progressive increase in lipolysis as the
temperature increased from 35-30 C and the incubation period in-
creased from 0-8 days.

Effect of free fatty acids on growth of_§. éELSflE' The effect

 

of free fatty acids on growth of §.'§u£§u§ strain S~l was deter-
mined using a skimmilk substrate. Tne skimmi F was reconstituted
from non-fat dry milk solids to give a final concentration of lOZ

solids. The saturated fatty acids CA through C} and oleic acid,

8’
were added in 0.0l, 0.05 and O.lZ concentrations to reconstituted
skimmilk. The mixtures were homogenized at 500 psi. Tne mixture

was recycled once again to achieve greater homogeneity. The milk

was sterilized, inoculated with §. aureys and incubated at 30 C

 

for 8 days. During the incubation period samples were drawn
aseptically every 24 hr and were analysed for changes in pH and
population. The coagulase test was performed an the samples by the
tube method described by Chapman _£ _1. (l94l). The cogulation was

measured by Standard Methods (I960) using tlate count agar.

The changes in the number of organisms per ml of the cereal

22

Table 6. Volatile fatty acids recovered from l0 9 of cereal milk
(lOZ milk fat) inoculated with §. aureus strain S-l and
incubated as indicated

 

Days of incubation

 

 

 

0 l 4 8
Incubation ml of O.lN KOH required to titrate
temperature (C) volatile fatty acids
l5 0.5 1.33 l.92 2.l5
22 0.5 l.74 2.l8 2.97

30 0.3 3.50 5.&7 7.28

 

23

milk inoculated with é. aureus and containing various concentrations
of fatty acids are shown in Fig. l. The log of the count is plotted
against time. None of the fatty acids tested were inhibitory at
0.0l% concentration. Caprylic acid was the most effective in
suppressing the growth of the organisms causing complete permanent
inhibition at O.l% concentration and temporary inhibition at 0.05%
concentration. The other saturated fatty acids (C4 to C18) and
oleic acid added in concentrations of 0.0l, 0.05 and O.lZ had no
effect on the growth of §. aureus strain S-l in skimmilk (Tables

7, 8, 9).

Measurement of nonprotein nitrogen. The effect of fatty acids

 

on the proteolytic activity of §. aureus was determined. At the
end of 8 days of incubation, l ml aliquot of milk sample was with-
drawn and added to 5 ml of lOZ trichloracetic acid. The trichlor-
acetic acid soluble nonprotein nitrogen was determined in the super-
natant using the method of Lowry _t_al. (l95l). The results were
expressed as micrograms of tyrosine liberated. The quantitation
was done using a standard curve prepared with L-tyrosine.

The data are presented in Table l0. The short chain fatty
acids C6 to C12 inclusive caused partial inhibition of proteolysis
of skimmilk when added to the skimmilk in concentrations of 0.05

and O.l0%. Butyric acid caused some inhibition at O.lOZ.

Effect of fatty acids on coagulase activity of §. aureus. The

 

skimmilk with added fatty acids and inoculated with §. aureus was
tested for coagulase activity. The results were recorded as posi-
tive (+), negative (-), or doubtful Qt). The data obtained at the

end of the 8th day of incubation at 30 C are summarized in Table ll.

LOG OF COUNT PER ML.

24

   

 

 

 

 

   
 

' BUTYRIC
. CONTROL ’
I z ,
"- =0.0I% FATTY ACID
— =0.05%FATTY ACID
-~- =O.l0% FATTY ACID
‘ 'o‘~-~- ’-------
r I” ----
.- l' r
' I
. I '
I I
, I
' ,’ CAPRIC

CAPRYLIC

 

 

I l A I A I l I l l l l l l I l I
o I 2 J ‘ 5 C 7 . O I 2 J ‘ 5 O 7 0
TIME IN DAYS

Fig. l. Changes in population of E. aureus strain S-l when grown
in skimmilk containing 0.0l, 0.05 and O.l0Z of the fatty
acids indicated.

 

Table 7.

25

Population per ml when S. aureus strain S-l was grown

at 30 C in skimmilk containing 0.0l% free fatty acids

 

Days of incubation

 

 

 

Fatty acids added 0 l 3 5 8
S. aureus per ml

. 7 7 7 7
Butyric 5500 4.5xl0 9.2xl0 20xl0 l0xl0
Caproic 8l00 22 xl07 48 xl07 l7xl07 l6xlO7
. 7 7 7 7
Caprylic 6000 27 xlO 52 xl0 3lxlO 20x10
Capric 6500 5.5xl07 Al xlO7 22xl07 l6xl07
Lauric 6600 3.5xl07 32 xlO7 30xl07 27xl07
. . 7 7 7 7
Myrlstlc 9400 4.0xl0 22 xl0 2lxl0 l6xl0
. . 7 7 7 7
Palmltic l2000 3.0xl0 40 xl0 28xl0 l6xl0
. 7 7 7 7
Stearic l4000 4.0xl0 23 xlO 37xl0 l4xl0
Oleic 14000 3.1x107 48 xl07 10x107 l0xl07
7 7 7 7

None (control) 6000 5.0xl0 4l xlO 38xl0 l6xl0

 

Table 8.

26

Population per ml when S. aureus strain S-l was grown at
30 C in skimmilk containing 0.05% free fatty acids

 

Days of incubation

 

 

 

Fatty acids added 0 1 3 5 8
§. aureus per ml
. 7 7 7 7
Butyr1c 5,700 5.0xl0 43xl0 44x10 30xl0
. 5 7 7 7
Capr01c 9,300 6.0xlO 29xl0 49xl0 38xl0
Caprylic 6,000 - - - -
Capric 6,000 - - - -

. 6 6 6
Laurlc 6,lOO 10 xlO 3.0xl0 35xl0 7.0xl0
Myristic 6,500 5.0xl07 12x107 14x107 13x107
Palmitic 18,000 37 x107 32x107 28x107 18x107

. 7 7 7 7
Stear1c 14,000 55 xl0 20x10 3lxl0 24xl0
Oleic 9,000 5.1x107 23xl07 39x107 36x107

7 7 7 7
None (Control) 6,500 5.4xl0 4lxl0 39xl0 l6xl0

 

- no growth (inhibition)

27

 

 

 

 

 

Table 9. POpulation per ml when §. aureus strain S-l was grown at
30 C in skimmilk containing O.l% free fatty acids
Fatty acids Days of incubation
added 0 l 3 5 8
§. aureus per ml

Butyric 5,000 2x107 25x107 24xl07 23x107
Caproic 4,200 28x107 6lxl07 6Oxl07 2lxl07

. 6 7 7
Capryl1c 6,300 - l0xlO 50xl0 36xl0
Capric 6,000 - - - -
Lauric 6,000 1x107 12x107 11x107 2x107
Myristic 6,400 4xl07 l50xl07 38xl07 contaminated
Palmitic 5,000 3x107 25x107 24xl07, 22x107

. 7 7 7 7
Stearic l3,000 l07xl0 26xl0 4lxl0 l8xl0
Oleic 9,000 200xl07 30x107 42xl07 15x107

7 7 7 7

None (control) 9,000 4xl0 40xl0 3lxlO l0xlO
- no growth inhibition

28

Table 10. Amount of tyrosine liberated when skimmilk containing
added fatty acids was inoculated with_§. aureus strain
S-l and incubated 8 days at 30 C*

 

Concentration of fatty acid (Z)
Fatty acid added 0.0] 0.05 0.]

mg of tyrosine per ml of milk

Butyric l.00 l.08 0.75
Caproic l.l7 0.96 0.36
Caprylic l.25 0.38 0.29
Capric l.05 0.27 0.27
Lauric l.55 0.75 0.80
Myristic 0.86 lost 0.89
Palmitic l.30 l.30 l.34
Stearic 0.92 l.l9 l.l9
Oleic 0.96 l.07 l.l7

 

*Two samples which did not contain fatty acids were used
as controls. Noninoculated skimmilk and skimmilk
inoculated with S. aureus and incubated 8 days yielded
0.27 and 0.9l mg of tyrosine per ml of milk, reSpectively.

29

Table ll. Coagulase activity of skimmilk containing fatty acids,
inoculated with S. aureus strain S-l and incubated at
30 C for 8 days*

 

Concentration of fatty acid (Z)

 

 

Fatty acid added 0.0l 0.05 0.l0
Butyric

Caproic

Caprylic + + -
Capric + - _
Lauric + - i
Myristic + + +
Palmitic +
Stearic

Oleic + + +

 

*
Samples of skimmilk containing no added fatty acids
were coagulase positive when inoculated with S. aureus
and coagulase negative when not inoculated.

30

All of the samples were coagulase positive, indicating substantial

growth of S. aureus, except; a) those which contained 0.l% of C8,

acids, and b) those which contained 0.05% of C and

and C l0

C

l0 l2

C12 ac1ds.

Effect of fatty acids on the pH production by §. aureus in

 

skimmilk. The skimmilk with added fatty acids was inoculated with
_§. aureus and incubated at 30 C. Samples were withdrawn after l,

3 and 5 days of incubation and the changes in pH observed. The
data are summarized in Table l2. A direct relationship existed
between pH and inhibition of the growth of S. aureus, with the
highest pH prevailing in those samples in which growth of S. aureus
was inhibited.

Effect of fatty acids on coagulability of skimmilk inoculated

 

with S. aureus. The growth of §. aureus in skimmilk produces enough

 

acidity to cause the precipitation of casein and hence the coagula-
tion of milk. However, when S. aureus was grown in skimmilk with
added fatty acids at 30 C it was observed that all samples did not
show coagulation. The inoculated samples were examined at 0, l, 3,
5 and 8 days of incubation for visual signs of coagulation. The
data are shown in Table l3 with coagulation indicated by (+) and

noncoagulation as (-).

3]

Table l2. Changes in pH when skimmilk containing added fatty
acids was inoculated with §. aureus strain S-l and
incubated at 30 C

 

 

 

 

Concentration % Hr of incubation
Fatty acid added of fatty acid 24 72 l20
pH

None 6.3 5.8 5.l
Butyric 0.0l 6.3 5.9 5.4
" 0.05 6.2 5.6 5.0

" O.lO 6.6 5.8 5.3
Caproic 0.0l 6.3 6.l 5.2
" 0.05 6.3 5.7 5.2

" 0.l0 5.8 5.5 5.]
Caprylic 0.0] 6.3 5.6 5.l
" 0.05 6.l 6.2 .5.9

" O.lO 6.0 6.0 5.9
Caproic 0.0] 6.3 5.9 5.8
” 0.05 6.2 6.1 6.l

" O.l() 6.] 6.] 6.l
Lauric 0.0] 6.3 6.0 5.5
" 0.05 6.2 6.3 6.0

" O.l0 6.2 6.2 5.8
Myristic 0.0l 6.2 5.8 5.3
" 0.05 6.2 5.9 5.l

" O.l0 6.3 5.9 5.3
Palmitic 0.0l 6.2 5.9 5.2
" 0.05 6.2 5.8 5.2

" 0.l0 6.] 5.9 5.l
Stearic 0.0] 6.2 5.8 5.2
" 0.05 6.] 5.7 5.2

" O.lO 6.0 5.7 5.0
Oleic 0.0l 6.2 5.8 5.]
" 0.05 6.l 5.8 5.3

" 0.l0 6.0 5.7 5.2

 

32

 

 

co_um_:mmoo .amunaou u H.

co_um_:mmoococ u 1

co_um_:mmoo n +

+ + + + + + + + + 1 1 1 1 1 1 20.0
+ + + + + + + + + 1 1 1 1 1 1 0200;
+ + + + + + + + + 1 1 1 1 1 1 03:50..
+ + + + + + 1 + + 1 1 1 1 1 1 032;:
- h + - - + - - - - - - - - - 2.33
1 1 + 1 1 1 1 1 1 1 1 1 1 1 1 o_camu
1 1 + 1 1 + 1 1 + 1 1 1 1 1 1 o__xLam0
+ + + 1 + + 1 + 1 1 1 1 1 1 1 £0..de
+ + + + + + + + 1 1 1 1 1 1 1 07:33
0_. m0. _0. 0_. m0. _0. 0.. m0. .0. 0_. m0. _0. 0.. m0. _0. poppm U_om xuumm

 

Amu p_om >uumm mo co_umcucooc00

 

 

w m

 

m

 

 

_ 0

co_umn:oc_ mo mx00

 

madcam .w.Lu_3 toga—zooc_ c053 mp_om >uumm popom mc_:_m~coo x__EE_xm mo >u___nm_:mm00

m>mv w cod 0 om um voumnnoc_ 0cm .1m :_mcum

.m. m_nmh

Isolation and Purification of Lipase

Ԥ. aureus strain B-l20 was used as a source of lipase since
preliminary work showed that this strain produced a very active
lipase. A culture grown for l8 hr at 37 C in IQ ml of trypticase
soy broth was used to inoculate approximately l,250 ml quantities
of trypticase soy broth in two liter Erlenmeyer flasks. The cells
were grown at 37 C for 24 hr with continuous agitation using a
rotary shaker at approximately l50 rev/min.

Preparation of the cell-free supernatant. At the end of in-

 

cubation, the cells were removed from the medium in a refrigerated
Servall centrifuge operated at 8,000 X g for 20-30 min. The super-
natant was collected and assayed for lipase activity. The cells
were washed at least twice with normal saline (0.9% sodium chloride),
centrifuged and then resu5pended in distilled water and lyophilized.
The lyophilized cells were stored at -23 C.

Assay for lipase. In preliminary work, cream containing 35%

 

fat was used as the substrate and the reaction mixture was in-
cubated at 37 C for l hr. The liberated fatty acids were titrated
with alcoholic potassium hydroxide to the phenolpthalein end point.
However, this method was not reproducible and was deemed unsatis-
factory.

The method adopted was reported in the Worthington catalog
(l964) and used olive oil as the substrate. An emulsion was pre-
pared by blending a mixture of 20 ml of olive oil with l65 ml of
5% gum acacia and l5 9 of ice in a Waring blender for a total of
IO min (5 min at low speed and 5 min at high speed). Then the

following were pipetted into a l50 ml beaker to form a substrate
33

34

for the enzyme:

Olive oil emulsion 5.0 ml
Sodium taurocholate l5 mg/ml 2.0 ml
Sodium chloride 3 M 2.0 ml
Calcium chloride 0.075 M l.0 ml
Deionized water 5.0 ml

One milliliter of the enzyme solution was added to this substrate
mixture and the pH adjusted to 8.0. The reaction mixture was in-
cubated in a reciprocating shaker bath at 37 C for l0 min. At the
end of incubation the mixture was electrometrically titrated to pH
8.0 with 0.0lN KOH. The ID min incubation period included the
titration time. The lipase activity was defined in terms of l'units",
with a unit being equal to one micromole of acid produced per mg

of the protein in l min at 37 C under the Specified conditions.

Measurement of protein concentration. The protein concentra-

 

tion was determined by the method of Lowry_§t_al. (l95l) with

certain modifications. The solution of CuSO '5H20 and potassium

4
sodium tartrate were added directly to the sodium carbonate solu-
tion and the phenol reagent was diluted l:l with water. Lysozyme
3X crystallized was used to prepare a standard curve for quantita-
tion. Protein concentration was plotted against optical density

measured at 660 mp and the data are shown in Fig. 2.

Lipase production. During earlier investigations it was found

 

that cultures which were not shaken during incubation did not pro-
duce lipase. The effect of several physical and chemical factors

influencing lipase production by S. aureus is described below;

35

«.0

n.0

v.0

0.0

0.0

0.0

Cu.

0:

00.

00

w2>Nom>4 m0 mz<¢00¢0_2

00

.23: 01m mm 330.. .6 aofide 9: 9:3 as 80
um >u_mcmn _mo_uao m> AoE>~0m>_V co_umcucoucou c_000ca mc_umo_tc_ o>cao vcmvcmum

0...

00

00

0e

0n

ON

0.

 

 

q

l1

W

n

q

‘1

J

A

-

 

 

«.0

n.0

0.0

0.0

Ho

0.0

0.0

.N

A .USNBO 7V9I1d0

.m.m

36

a) rate of agitation and atmOSphere, b) age of culture, c) pH, and
d) added chemicals.

Effect of agitation and air. Four two liter Erlenmeyer flasks
containing l,000 ml of sterile trypticase soy broth were inoculated
with S. aureus and incubated at 37 C under the conditions indicated
below: a) without agitation, b) with agitation, c) with agitation
and under vacuum, and d) without agitation but with sterile air
Supplied through filter tubes. The vacuum in flask in method (c)
was created by using a laboratory aSpirator operated at maximum
water flow for 30 min. At the end of this period the exit tube
was sealed. After I, 2 and 3 days of incubation a portion of the
medium was removed aseptically and the cells were removed by cen-
trifugation at 8,000 X g for 20-30 min. The supernatant was assayed
for lipase activity.

Effect of age. The cells were grown in trypticase soy broth

 

for a maximum period of 7 days at 37 C under the following condi-
tions: a) continuous agitation throughout the incubation period,

b) agitation during the first 24 hr of incubation, and c) no agi-
tation during incubation. Samples were withdrawn at regular
intervals. The cells were removed by centrifugation and the super-
natant was assayed for lipase. The data in Table I4 indicate that
continuous agitation of the.§°.§2£§£§ cells resulted in a progressive
increase in the lipolytic activity of the cell-free supernatant when
the organism was grown in trypticase soy broth. The maximum acti-
vity was achieved with extracts from cells incubated 5 days, followed
by somewhat lower activity from cells incubated 7 days at which time

when the experiment was terminated. However, when the cultures

37

were agitated for the first 24 hr only, followed by quiescent in-
cubation (Table l4, column 2) there was only a slight increase in
the lipolytic activity on the second day and this was followed by a
decrease in activity during the remainder of the incubation period.
There was no evidence of lipase activity in the extracts from cells
grown in a still culture.

Lack of enzyme activity in still cultures Of.§° aureus grown in
trypticase soy broth at 37 C (Table I4) indicated that either air
or agitation was necessary for the production (release) of lipase
by §. aureus. An experiment was conducted to find which of these
two factors was reSponsible for the presence of lipase in the cell-
free supernatant. The data in Table l5 show an increase in lipase
activity with an increase in the age of §. aureus cells in all
cases. As shown in Tables l4 and l5 the extract from the non-
agitated cultures had no lipase activity. There was only slight
lipase activity when the growth medium was agitated under vacuum.
However, when air was bubbled into the growth medium, the cell-free
supernatant showed an amount of lipase activity similar to the
activity of the cell-free supernatant obtained from cells grown
with continuous mechanical agitation.

Effect of pH on lipase activity. Fifty milliliter portions of

 

trypticase soy broth were adjusted to an initial pH of 4.0, 4.5,
5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and l0.0 with O.lN

NaOH or HCl. The medium was autoclaved in l25 ml Erlenmeyer flasks
and inoculated with an l8 hr culture of_§. aureus. The organisms
were grown in the above medium for 24 hr in a reciprocating shaker-

water bath at 37 C. The medium was centrifuged and the supernatant

Table l4.

Effect of age and agitation on the activity of lipase
produced by §. aureus strain B-lZO when grown in

trypticase soy broth at 37 C.
measured in olive oil

pH 8.0.)

(Lipase activity
emulsion for l0 min at 37 C and

 

Days of incubation

Conditions for growth of cells

 

No agitation

Agitation for
first 24 hr

Continuous
agitation

 

ml of 0.0lN NaOH used to titrate free

 

at 37 C fatty acids liberated from olive oil

l 0.0 1.3 l.3

2 0.0 l.8 2.8

3 0.0 l.l 3.8

4 0.0 l.3 4.2

5 .0 l.3 4.5

6 0. l. 4.0

7 .0 l.4 4.l
Nonlnoculated broth_ 0.0 0.0 0.0

(Control)

 

39

Table l5. Effect of air and agitation on the activity of lipase
produced by §. aureus strain B-l20 grown in trypticase
soy broth at 37 C. (Lipase activity measured in olive
oil emulsion for l0 min at 37 C and pH 8.0.)

 

Conditions for growth of cells

 

Mechanical No mechanical
Days of agitation Continuous agitation but
incubation No under mechanical bubbled with
at 37 C agitation vacuum agitation filtered air

 

ml of 0.01N NaOH used to titrate free fatty
acids liberated from olive oil

l 0.0 0.3 l.3 l.0
2 0.0 0.5 2.7 2.7

3 0.0 0.6 3.6 3.2

 

40

assayed for lipase activity. The cells were washed with deionized
water and transferred to preweighed aluminum evaporating dishes.
The cells were dried at llO C for 24 hr and the dry weight deter-
mined.

The lipase activity of the cell-free supernatant obtained from
_§. aureus grown in trypticase soy broth at 37 C at different pH
between 4.0 and l0.0 is shown in Table I6. Also recorded in Table
I6 is the dry weight of the S. aureus cells. Visual observations
were made during the growth of the cells and at the end of the in-
cubation period. There was no turbidity at pH 4.0 and 4.5 and only
slight turbidity at 5.0, 9.5 and l0.0. Interestingly, growth in the
form of clumps was observed between pH 6.5 and 8.5 while clumps were
not observed at other pH values and most of the cells settled to
the bottom. The data in Table I6 are plotted in Fig. 3 which shows
that there were two optima for lipase production, one at pH 8.0 and
the other at pH 9.0. The optimum for cell production ranged from
pH 7.0 to 9.0 (Table 16, column 4).

Effect of added chemicals on lipaseyproduction. Previous work

 

indicated that staphylococcal lipase may be an inducible enzyme
(Table 3). Experiments were conducted to elucidate this phenomenon.
One or two percent of glycerol, Tween 65, sorbitan monooleate poly-
oxyethylene (Tween 80), olive oil,and corn oil were added to
trypticase soy broth and incubated at 37 C without agitation. The
cell-free supernatant obtained at the end of incubation was assayed
for lipase activity. The lipids from the supernatant containing
olive oil and corn oil were extracted three times with N-pentane.

The solvent was evaporated and the hydrolyzed oil titrated to the

4l

Table l6. Effect of pH on activity of lipase produced by
§. aureus strain B-l20 grown in trypticase soy broth
for 24 hr at 37 C. (Lipase activity measured in olive
oil emulsion for l0 min at 37 C and pH 8.0)

 

 

ml of 0.0lN
NaOH required
to titrate mg of_§. aureus
free fatty acid cells recovered
Initial pH of Final pH of liberated from per 50 ml of
the medium the medium olive oil the medium
4.00 4.05 0.20 8.6
4.50 4.65 0.60 7.5
5.00 4.90 0.60 29.0
5.50 4.95 0.65 40.9
6.00 5.50 l.25 53.5
6.50 6.35 4.05 60.0
7.00 6.70 5.90 77.7
7.50 6.95 6.70 87.]
8.00 7.25 7.30 84.l
8.50 7.45 5.00 70.4
9.00 7.75 7.l0 68.0
9.50 7.80 l.70 36.7

l0.00 7.95 0.90 20.2

 

ml OF 0.0] N NaOH

42

 

8.0

7.0 -

5.0 1-

4.01

3.0 1-

l.0r

 

 

 

4.0 5.0 6.0 7.0 8.0 9.0 I0.0

Fig. 3.

Milliliters of 0.0lN NaOH required to neutralize the free
fatty acids formed by the lipolytic enzymes produced by
.§. aureus strain B-l20 grown in trypticase soy broth at
37 C at the pH indicated. (Lipase activity measured in
olive oil emulsion for ID min at 37 C and pH 8.0.)

 

43

phenolpthalein end point with 0.025N NaOH. The acid degree value
was calculated as described previously.

The data obtained are presented in Table I7. Apparently none
of the added chemical substances had a stimulating effect on the
lipase production by_§. aureus under the conditions of this experi-
ment. However, when the lipids were extracted from the cell-free
supernatant and titrated to the phenolpthalein end point with
0.025N KOH to determine acid degree value, they showed extensive
lipolysis.

Presence of intracellular lipase. Five grams of the cells
obtained from still cultures were washed in a Waring blender (l
min at slow Speed and l min at high speed). The cells were
separated by centrifugation and disintegrated by sonification for
5 min at 7-9 amps. The cell debris and intact cells were removed
by centrifugation at 20,000 X g for 20 min. The supernatant from
the washings in the blender and the cell-free supernatant from
sonification were analyzed for lipase activity. The supernatant
obtained after §. aureus cells were washed in a Waring blender and
centrifuged showed no lipase activity when tested on olive oil.
The results were also negative with the cell-free supernatant ob-
tained from sonified cells. These observations suggest that under
the conditions prevailing in this experiment there was no evidence
for the existence of an intracellular lipase in S. aureus B-l20.

Concentration of staphylococcal lipase. The extracellular
nature of the staphylococcal lipase and the presence of this enzyme
in the growth medium makes it mandatory to concentrate the lipase

into smaller volumes of liquid for convenience of handling. Several

44

Table 17. Effect of chemical agents on activity of lipase
produced by S. aureus Strain B-120 grown as indicated
at 37 C for 24 hr. (Lipase activity measured in olive
oil emulsion for 10 min at 37 C and pH 8.0.)

 

Micromoles of acid

 

Basal medium produced per mg of

Trypticase soy broth protein in the Acid degree

(TSB) ,growth medium value
TSB, agitated 0.3-0.5 -
TSB, stationary 0.0 -
TSB + 1% Tween 65, stationary 0.0 -
TSB + 1% Tween 80, stationary 0.0 -
TSB + 2% olive oil, stationary 0.0 15.4
TSB + 2% corn oil, stationary 0.0 12.2
TSB + 1% glycerol, stationary 0.0 -

 

- not determined

45

methods were tried and are described below. All operations were
performed at 4 C or lower, unless otherwise specified and all
fractions were analyzed for protein content and lipase activity, as
described previously.

Ammonium sulfate precipitation. The cell-free supernatant was
precipitated with 40, 50, 60, 70, 80 and 100% ammonium sulfate.

The precipitates were dissolved in deionized water and dialyzed.
The supernatants were first dialyzed against tap water followed by
pervaporization and dialysis against deionized water.

The data in Table 18 indicate the activity of lipase pre-
cipitated with various concentrations of ammonium sulfate. In the
interest of clarity and valid comparison the lipase activity is pre-
sented in I'specific units”. A specific unit is defined as the
micromoles of free fatty acids produced per mg of the protein in
the enzyme solution under the experimental conditions.

The lipase activity in the supernatant showed a progressive
decrease as the percent concentration of ammonium sulfate was in-
creased above 40% saturation. Lipase activity in the precipitate
increased as the concentration of ammonium sulfate increased from
40-80% saturation. At 100% saturation there was a decrease in
activity probably due to denaturation of the enzyme protein.

Precipitation with acid. Zolli and SanClemente (1963) repor-
ted that lipase can be precipitated from the cell-free supernatant
by dialyzing against acetate buffer at pH 4.3 containing 10% v/v
ethyl alcohol. Direct precipitation of the supernatant was tried
in this experiment with some success. The precipitate obtained by

adding 10% v/v of absolute ethyl alcohol to the cell-free supernatant

46

Table 18. The lipase activity of various fractions obtained by
precipitation of the cell-free supernatant obtained from
.§' aureus strain B'l20\Nlth different concentrations of
ammonium sulfate. (Lipase activity measured in olive
oil emulsion for 10 min at 37 C and pH 8.0.)

 

 

 

Percent saturation of Lipase activity (Specific units)
ammonium sulfate Precipitate Supernatant
40 8-10 8-10
50 15-18 4- 7
60 12-17 2- 5
70 15-16 0- 4
80 17-20 1- 4
100 15-18 -

 

- not determined

47

at pH 4.3 had a lipase activity of 13 units which was lower than
the activity of the precipitate obtained with ammonium sulfate
(80% saturation).

Precipitation with ethyl alcohol and acetone. The cell-free

 

supernatant was precipitated with 40, 50, 60 and 70% ethyl alcohol.
The mixture was constantly stirred during addition of alcohol. The
precipitate was dissolved in water while the supernatants were
dialyzed against deionized water. The process was repeated using
acetone as a precipitant. The precipitates and supernatants obtained
were tested for lipase activity and the data obtained are shown in
Table 19. The data indicate that the precipitation with a 50% con-
centration of alcohol gave the most lipase activity per mg of pro-
tein and as the concentration of alcohol increased to 70% the
activity of the precipitate decreased.

The results obtained with acetone precipitation were similar
to those obtained with alcohol, except 50% acetone gave a precipi-
tate with slightly lower activity than the one obtained with 50%
alcohol.

A comparison of the various methods indicates that precipita-
tion with 50% alcohol was the preferable method for initial concen-
tration. The data Showing the optimum concentrations for each of
the various methods are summarized in Table 20.

Purification of staphylococcal lipase. The precipitate re-

 

moved from the cell-free supernatant with 50% ethyl alcohol showed
the most activity and this crude preparation was further purified by

the following procedure;

48

Table 19. Lipase activity of the fractions resulting from treating
the cell-free supernatant obtained from S. aureus
with absolute ethyl alcohol. (Lipase activity measured in
olive oil emulsion for 10 min at 37 C and pH 8.0.)

 

Lipase activity_(5pecific units)

 

 

Percentage of alcohol Precipitate Supernatant
40 10-12 5-6
50 24-28 0-1
60 14-16

70 10-12

 

49

Table 20. A comparison of the various methods for the initial
concentration of staphylococcal lipase. (Lipase
activity measured in olive oil emulsion for 10 min at
37 C and pH 8.0.)

 

Units of lipase activity

 

 

Method of precipitation Precipitate Supernatant
80% ammonium sulfate 17-20 1-4
10% v/v ethanol at pH 4.3 11-16 4-5
50% v/v ethyl alcohol 24-28 0-1

50% v/v acetone 22-24 0-2

 

50

Step 1. The alcohol precipitate was dissolved in
deionized water and the pH adjusted to 8.6. The
mixture was cloudy and was centrifuged for 30 min
at 1000 X 9.
Step 2. The pH of the supernatant obtained from
Step 1 was slowly adjusted to pH 4.3 with O.lN
HCl. The resulting precipitate was removed by
centrifugation at 18,000 X g for 30 min.
Step 3. Use of column chromatography using Sephadex G-200.
The columns were packed with Sephadex or BioGel according to
the directions of Flodin (1962). The height of the packed material
was generally 15 times the internal diameter of the various columns.
The packed columns were checked with blue dextran 2000 for unifor-
mity of packing and void volumes. The buffer solution was passed
through the packed column for at least 12 hr before chromatograph-
ing the protein solution. A 1-2% solution of the protein in tris
(hydroxymethyl) amino methane-hydrochloric acid buffer pH 8.1, F72
= 0.1 was used to charge the column. The buffer was prepared
according to the method of Long (1961). The columns were eluted
with tris buffer pH 8.1, r72 = 0.1 at room temperatures. The
eluant was passed through an ultraviolet monitor and the fractions
collected every 20 min using an automatic fraction collector. The
chromatographic pattern obtained with Sephadex G-200 is shown in
Fig. 4. The tube numbers were plotted against protein concentra-
tion and lipase activity.
Step 4. The fractions showing maximum lipase activity

from Step 3 were pooled and concentrated by

51

I-IODN N [0'0 .40 [W

.0.0 In 0cm 0 mm on c.E 0. L00 co.m.:Eo ..o o>..o c. omma.. may

>3 woumcmn.. mn.um >uumw 00L» mamcu.u Op voc.:uoc Iomz 2.0.0 00 .5 mm >u.>.uum mmma..
0:0 0:0 c.muoLa mo mEmcmoLo.E mm noucomocaoc m. co.umcucmocou omma.4 ...0 u N\1L

...m :0 mewan m.cu m:.m: 00~10 xmnmcaom co co_umcmaoca omma.. m *0 Emme.n co.u:.o c<

mum’s... was...

 

.uN a. AV. 0 _
o . s . . . . -
\ III! 1
\ IIInII
O.N .. \\ II
, \ II
\ I
s I ~
s — 0
961 ~ . ~
. . ~
~ _ -
~ - -
~ -
oar . _ .
u ‘ .
g — -
~ —
. f \ p .
00 s
\
\
.s
I
0:0 I
ZO_P<¢PZUOZOO z_m...0¢a .l.ll.
>._..>_._.0< mmdmj l

 

.a .3...

l

0.

l

00.

 

 

NIJJOUd J0 SNVHQOUONV

52

pervaporation. The concentrated material was then
chromatographed using a 40x2.3 cm tube packed with
BioGel 300. The chromatographic pattern is shown
in Fig. 5. Most of the lipase was eluted in the
first peak, though the second peak showed some lipo-
lytic activity. A direct relationship between protein
concentration and lipase activity was observed in
the fractions obtained under the first peak.
A schematic flow sheet for the isolation procedure is shown in
Fig. 6. The data showing the degree of purification during the
various steps of purification are summarized in Table 21. The
final purified lipase preparation possessed 350-450 times more

lipolytic activity per mg of protein than the cell-free supernatant.

Characterization of the Enzyme Preparation

The lipase-rich fraction isolated by the procedure outlined
above was assayed at different pH values and temperatures to deter-
mine the optimum conditions for lipolytic activity. The storage
stability and thermal inactivation at various temperatures were
determined. The action of this enzyme on various natural and
synthetic substances was also ascertained. In addition, the
inhibitory or stimulatory effect of various chemical agents on
lipase activity was investigated and the electrophoretic mobility,
sedimentation coefficient and molecular weight were determined.

Optimum pH for lipase action. One milliliter of the enzyme
solution was added to the substrate and the pH adjusted to one of

the following values: 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0,

53

NOON NIO'O J0 I”

.0.0 :a new 0 um um c.E 0. Lo; co_m.:Eo ..o o>..o c. omma.. ozu >0

tmumcmn..mu.um >uumm mop. cameo.“ cu nmc.:coc Iomz 2.0.0 00 .8 mm >u.>.uom mmma..

050 new c.muoLa mo mEmLmoLo.E mm voucMmocaoc m. co.umcucoucoo mmma.4 ...0 u NNI.
...0 :0 commas m.cu mc.m: 00m .ooo.m co co.umcmaoca mmma.. 0 mo Emcmm.u co.u:.m c< .m .0.0

cumin: won...
0N ON 0. 0. m

 

Lou

 

 

ms; 084 JO snvaaoaam

o. a 22552328 2.323111 1 8.
>553 “.2531

 

 

 

54

24 hr old culture of S. aureus in trypticase soy broth

1

[—Centrifuged at 10,000 X g for 20 min
7 l
Supernatant Cells discarded
[diluted with equal volume

 

of absolute alcohol

 

 

[Centrifuged at 700 X g for 20 min

 

 

l
Supernatant discarded Precipitate dissolvgd in
‘ water and adjusted to pH 8.6

liCentrifuged at1000 X g for 30 min]

 

 

 

l
Supernatant Precipitate discdrded
adjusted to pH 4 3

 

l c

lCentrifuged at 18,000 x g for 30 min]
1 I 1 l
Supernatant discarded Precipitate resu5pended in
tris buffer pH 8.1 pervapqrated

 

 

l

[ySephadex 0-200]

 

 

lst deak 2nd1peak
dialyzed and pervaporated discarded

 

 

[BioGel 300j

l

lst deak 2ndjpeak
(Final Prep) discarded

 

Fig. 6. Flow sheet for the isolation and purification of staphylo-
coccal lipase.

55

Table 21. Activity of the staphylococcal lipase (strain B-120)
obtained from the cell-free supernatant at each state
of fractionation. (Lipase activity measured in olive
oil emulsion for 10 min at 37 C and pH 8.0.)

 

 

 

Specific activity (units) Degree of
Stage of fractionation Precipitate Supernatant purification
cell-free supernatant 0.3 - 1
.50% alcohol 25.0 2 83
pH 8.6 6.0 32 106
pH 4.3 60.0 — 200
Sephadex G-200 95.0 12 316
BioGel 300 135.0 5 450

 

- not determined

56

9.5, 10.0, 10.5 and 11.0. The reaction mixture was incubated at 37 C
for 10 min. At the end of incubation, the pH was adjusted back to
the original value with 0.01N NaOH.

The milliliters of 0.01N NaOH required to titrate were plotted
against pH and the data are shown in Fig. 7. The enzyme appeared
to have a single pH optimum around 8.5. lbwever, the enzyme was
active over a wide range of pH mostly on the alkaline side of
neutrality.

Optimum temperature for lipase activity. The optimum tempera-

 

ture for the action of the lipase preparation was determined at pH
8.0 using olive oil as the substrate. The enzyme activity was deter-
mined at 5, 15, 25, 30, 35, 37, 40, 45, 50, 55, 60, 65 and 70 C.

The substrate was conditioned at the test temperature before the
enzyme solution was added. The activity represented as ml of

0.01N NaOH required to neutralize the liberated fatty acids was
plotted against the incubation temperature and the graph obtained

is shown in Fig. 8. The optimum temperature under experimental con-
ditions was around 45 C. There was little activity at 70 C.

Storage stability of purified lipase. The lipase preparation

 

was stored at 37, 32, 22, 16, 4 and —23 C for a maximum period of

21 days. One milliliter aliquots were assayed for lipase activity.
The percentage of loss of activity was plotted against the storage
time and the results are indicated in Fig. 9. The high degree of
stability of this enzyme is shown by the fact that only 7% of the
activity was lost after 21 days of storage at 4 C. At 37 C the
enzyme preparation lost 50% of its activity after 21 days of storage.

Thermal inactivation of the lipase. Thermal destruction studies

 

ml OF 0.0! N NaOH

57

 

6.0

5.0 r-

2.0 F

 

 

()1) 1 1 1 1 1 1

 

 

5.0 6.0 7.0 8 .0 9.0 10.0 11.0
p H

Fig. 7. Milliliters of 0.01N NaOH used to titrate the fatty acids
liberated by the action of staphylococcal lipase in olive
oil emulsion for 10 min at 37 C at various pH values.

12.0

7.0

6.0

5.0

4.0

3.0

ml OF 0.0! N NaOH

2.0

1.0

20

Fig. 8.

58

 

 

1 1 1 1 1 1, l l 1 J

 

 

25 30 35 40 45'50 55 60 65 70
TEMPERATUREPC)

Milliliters of 0.01N NaOH required to titrate the free
fatty acids liberated from olive oil by staphylococcal
lipase after 10 min at pH 8.0 at various incubation
temperatures.

PERCENT LOSS OF ACTIVITY

 

59

 

 

 

 

 

 

0
fi 40
10 - fi-23C

160
20 -

220
3° " 32c
40 r-

50 - 320
60 1-
100i 1 1 4AA 1 1 1
0 1 2 3 7 14 21
TIME (DAYS)
Fig. 9. Percent loss of activity of staphylococcal lipase when

stored at various temperatures for the times indicated.

60

were performed using tris buffer pH 8.1,r_ /2 = 0.1 as the heating
menstrum. The design of the apparatus used for thermal resistance
studies was based on the unit devised by Malin (1953) and modified
by Walker (1964). The enzyme solution was heated at 50, 55, 60, 65
and 70 C. The samples were withdrawn every 5 min for 30 min and
were immediately cooled in pre-chilled tubes.

The lipase activity was determined for the heated aliquots and
correSponding unheated controls and the percentage loss of activity
in the heated samples was calculated. Fig. 10 shows a plot of the
percent loss of activity vs time. The percent loss of activity was
also plotted against the heating temperature and the graph is shown
in Fig. 11. The enzyme lost 6% of the activity after exposure to
50 C for 30 min and 95% of the activity when heated at 70 C for 30
min.

Action on various natural fats and oils. The lipase activity
of the enzyme preparation was tested on several oils and fats in-
cluding olive oil, soybean oil, safflower oil, corn oil, cottonseed
oil, butter oil, lard, turkey fat, beef tallow and hydrogenated soy-
bean oil. The hydrogenated soybean oil was flaky and solid at room
temperature and had an iodine value of less than three. Animal fat
from the fresh tissue was extracted with chloroform-methanol (2:1).
The ability of staphylococcal lipase to act on a wide variety of
natural oils and fats is shown by the data in Table 22. The
staphylococcal lipase did not show any preference for animal or
vegetable fats or oils. It is noteworthy that the emulsion of
hydrogenated soybean oil (iodine value < 3) was also hydrolyzed by

this enzyme.

PERCENT LOSS OF LIPASE ACTIVITY

IO
20
30
40
so
so
70
so

90
100

61

 

 

-

1 l I I I

55C

6°C

7°C

 

. 10.

5 10 15 20 25 30

TIME IN MINUTES

Percent loss in the activity of staphylococcal lipase

heated in tris buffer at pH 8.1 at the various times
and temperatures indicated.

 

62

 

 

 

 

O

10 -

20 -
>.
I: 30 -
>
E 0
q 40-
h.
0 50~ 10111111
(I)
(I)
C)
‘1 60 .
1~
E
Q 70 *-
0:
it

80 _ 30min

90 -

100 A 1 1 1

37 50 55 ‘ 60 65 70

TEMPERATURE (°Cl

Fig. 11. Percent loss of staphylococcal lipase activity when
heated in tris buffer at pH 8.1 at the various
temperatures for the time indicated.

63

Table 22. Amount of free fatty acids liberated from various natural
lipids by purified staphylococcal lipase (strain B-120).
(Lipase activity measured in olive oil emulsion for 10
min at 37 C and pH 8.0.)

 

ml of 0.0lN NaOH required to titrate

 

Lipid free fatty acids
Olive oil 4.10
Soybean 011 3.40
Safflower oil 4.25
Corn 011 4.20
Cottonseed 011 3.30
Butter oil 4.00
Lard 3.30
‘Turkey fat 3.80
Beef tallow 4.10

I+ydrogenated soybean oil 1.40

 

64

Action of lipase on a synthetic triglyceride. The only synthe-
tic triglyceride tested had palmitic, stearic and oleic acids at a,
B, and a‘ positions. Since only a small quantity of this glyceride
was available, the following procedure was adopted to prepare an

The following ingredients were added in a sonifier tube.

emulsion.
Synthetic triglyceride 50.0 mg
10% gum acacia solution 0.2 ml
0.075 M calcium chloride 0.2 ml
tris buffer pH 8.1 2.0 ml
Deionized water‘ 5.0 ml

The tube was then placed in a water bath maintained at 65 C
and the mixture sonified at 7-9 amps for 4 min. One milliliter of
the enzyme preparation was added to the above emulsion and the re-
action mixture incubated at 37 C for 1 hr. The hydrolyzed glycerides
vvere extracted with N-pentane and analyzed for constituent fatty
aczids by gas chromatography. Thin layer chromatographic analysis
muss also performed at the end of the incubation period. The tech-
riicque used was essentially as given in the standard texts of Bobbit
(15963), Randerbath (1963) and Stahl (1965). The solvent used for
development was ether, hexane, and acetic acid mixed in the propor-
tl<>r1 of 30:90:l, reSpectively. Concentrated sulfuric acid was used
to develop the Spots. The percentages of the three free fatty
aCids were as follows: palmitic, 40%; stearic, 21%; oleic, 39%.
The=ana1ysis of hydrolyzed fat on thin layer chromatography showed
onl‘Y faint Spots for mono and diglycerides, with triglycerides and

‘Frefii fatty acids predominating.

65

Effect of chemical agents. The effect of several chemical
compounds on the lipase activity was determined using olive oil as
the substrate. In each trial a control titration was performed on
a substrate containing the enzyme but no additive. The percentage
of stimulation or inhibition was calculated from the titration value
obtained on the control. The assay for lipase was performed by
adding 1 ml of the enzyme solution to the substrate mixture des-
cribed previously.

The chemicals used in the experiments were mostly oxidizing
agents (hydrogen peroxide), reducing agents (glutathione, cysteine
and ascorbic acid), or agents which are known to effect -SH or S-S
bonds (2-mercaptoethanol, iodoacetic acid, N_ethyl maleimide and
p-chromomercuribenzoate). Certain antibacterial agents (formaldehyde,
penicillin, streptomycin, aureomycin, terramycin, phenol and
potassium cyanide) were also tested for their effect on lipase
activity. The following chemical agents (control excluded) were
used in the reaction mixture.

Hydrogen peroxide. The enzyme-substrate mixture was
mixed with 0.1, 0.2, 0.3, 0.4, 0.5, 0.8 and 1.0 ml of a 35% hydro-
gen peroxide solution. The pH was adjusted to 8.0.

Formaldehyde. Quantities varying from 0.1 to 1.0 ml of

 

40% formaldehyde solution (formalin) were added to the enzyme-
substrate mixture to study the effect on enzyme activity.

2-Mercaptoethanol. One-tenth, 0.2, 0.3, 0.4, 0.5, 0.8
and 1.0 ml of 2-mercaptoethanol were added to the enzyme-substrate
mixture to determine the effect on lipase activity.

Glutathione, cysteine and ascorbic acid. A 5% solution

66

of each of the three components was prepared. Aliquots of 0.5, 1.0,
2.0, 3.0 and 5.0 ml were added to the enzyme-substrate mixture to
determine the effect of enzyme activity.

lodoacetic acid, N-ethylmaleimide and p-chloromercuri-

 

benzoate. The effect of these compounds on lipase activity was
determined by adding 0.5, 1.0, 2.0, 3.0 and 5.0 ml of a 5% aqueous
solution of each of these compounds to the reaction mixture.

Penicillin, streptomycinLgaureomycin and terramycin. A

 

5% stock solution of each of these antibiotics was prepared in de-
ionized water. One, 2, 3 and 5 ml of the antibiotic solution were
added to the enzyme-substrate mixture to determine the effect of
these antibiotics on enzyme activity.

Phenol and potassium cyanide. The effect of a 5% solu-
tion of these reagents was studied using 0.5, 1.0, 2.0, 3.0 and
5.0 ml of inhibitor in the reaction mixture.

The data in Table 23 indicate that the lipase activity was
stimulated by hydrogen peroxide and inhibited by formaldehyde and
2-mercaptoethanol. The data in Table 24 show that reducing agents
inhibited the lipase activity. Antibiotics had a variable influence
with terramycin inhibiting activity, streptomycin and aureomycin
stimulating activity, and penicillin causing no appreciable change.
Chemical compounds such as iodoacetic acid, p-chloromercuribenzoate,
phenol and potassium cyanide inhibited the lipase activity.

Free boundary electrophoresis. A 1% solution of the lyophilized
enzyme preparation was prepared and dialyzed against veronal buffer
at pH 8.6, F72 = 0.1 for 12 hr with constant stirring at 4 C. The

Tiselius electrophoresis apparatus, Model 38 manufactured by

67

Table 23. Effect of hydrogen peroxide, formaldehyde and
2-mercaptoethanol on lipase activity measured in olive
oil emulsion for 10 min at 37 C and pH 8.0

 

Chemicals used

Quantities of chemical agent (ml)

 

O 0.1 0.2 0.3 0.4- 0.5 0.8 1.0

 

ml of 0.01N NaOH required to titrate
free fatty acids

 

Formaldehyde
Hydrogen peroxide

2-mercaptoethanol

4.4 3.4 2.1 1.8 1.6 1.2 0.8 0.1
4.4 5.3 6.2 7.1 7.0 5.9 4.5
4.5 3.6 2.7 1.8 1.0 0.0 0.0 0.0

 

68

Table 24. Effect of various chemicals on lipase activity of
§. aureus measured in olive oil emulsion for 10 min at
37 C and pH 8.0

 

Quantities of a 5% solution of chemical
agents (ml)

 

 

 

0 0.5 1.0 2.0 3.0 5.0

m1 of 0.01N NaOH required to titrate
Chemicals used free fatty acids
Glutathione 3.0 2.3 0.4 0.0 0.0 0.0
Cysteine 3.0 1.3 0.4 0.0 - -
Ascorbic acid 3.0 - 2.0 1.5 - 0.9
lodoacetic acid 3.3 2.6 1.7 - - 0.0
n-ethylmaleimide 3.1 3.3 3.0 3.1 3.3 3.1
p-chloromercuribenzoate 3 2 2. 1 5 - - 0.0
Penicillin 3.1 - 2.8 2.8 3.0 -
Streptomycin 3.0 - 3.3 4.2 5.7 6.6
Aureomycin 3.0 - 4.2 5. - 7.2
Terramycin 3.3 - 2.7 l 0 0.7 -
Phenol 3 2 2.8 3 - - 1 4
Potassium cyanide 3.1 1.2 0.0 - - -

 

- not determined

69

Perkin-Elmer Corporation was used for analysis employing a 2 ml cell.
The electrophoretic mobility (p) was calculated using the following

equation

where d = the distance traveled in cm; A = the cross sectional area
of electrophoretic cell; K = the conductivity cell constant; t =
time in seconds; I = current in amps; R = resistance in ohms; and

m = the magnification factor of the optical system. The initial
and final pictures of free boundary electrophoretic analysis at the
end of 3,000 seconds are shown in Fig. 12.' The enzyme preparation
showed a single peak at pH 8.6 using a veronal buffer system, and
can be considered electrophoretically homogereous.

Sedimentation coefficient and molecular weight. The enzyme

 

preparation was analyzed using a Spinco model E, analytical ultra-
centrifuge. The sedimentation velocity and sedimentation equilibrium
were determined at 50,790 and 12,000 rpm, respectively. The calcula-
tions were made using the standard texts of Svedberg and Pedersen
(1952), Schachman (1959) and William5(l963).

The sedimentation velocity and molecular weightvnye determined
using a 1% enzyme solution at 20 C and the sedimentation pattern is
shown in Fig. 13. The sedimentation velocity determination (Fig. 13)
suggests that the enzyme preparation is heteroger in regard to
the size of the protein molecules. The electrophoretic mobility,
sedimentation coefficient and molecular weight of the highly puri-

fied preparation are shown in Table 25.

.m_moco;aoLuoo~o wo mucooom ooo.m Louwm ..o n N\1L «o.m Ia
um Cowman _mcoLo> c_ omma__ .mooooo_>£amum po_emcsa wo chuuma u_uoco£aocuoo_o >mecson omen

 

71

.u ON um Ea; oomaom um pom:m_cucou _.o u N\1L «o.w Ia um Lommsn _mcoco>
c. co_u:_0m mmma__ _mooooo_>;amum po_w_csa voN___;Qo>_ N_ mo cLouuma >u_oo_o> co_umucoe_pom .m_ .m_u

 

 

 

72

Table 25. Physical characteristics of the purified staphylococcal
lipase preparation in veronal buffer pH 8.6, r-/2 = 0.1.

 

Physical characteristic Value

Electrophoretic mobility

Ascending -7.78 x lO-ScmZ/volt/sec
Descending -8.12 x lO-ScmZ/volt/sec

Sedimentation coefficient
lst peak 2.85 Svedberg
2nd peak 8.50 Svedberg

Molecular weight 100,000

 

Chemical Composition of_§. aureus Cells
é. aureus B-120 was grown in sterile trypticase soy broth with

and without 3% glycerol for 24 hr at 37 C with continuous agitation.

At the end of incubation the cells were removed by centrifugation
and washed as previously described. The washed and packed cells
were analyzed for moisture and then lyophilized for use in other
experiments.

Determination of moisture. One gram of wet packed cells was

 

weighed into an aluminum dish. The sample was then placed in a

vacuum oven maintained at a temperature of 100 C. Dishes were

3

.I b OSLDIA‘.“ M‘- “M”
o

‘p”

weighed every 12 hr until a constant weight was obtained.
Determination of nitrogen. The lyophilized cells were dried

to constant weight in a vacuum oven at 100 C. The dried cells were

analyzed for nitrogen using the microkjeldahl procedure described in

Official Methods of Analysis of the Association of Official

Agricultural Chemists (AOAC),(1960). The percentage of protein was

calculated from the percent nitrogen using a factor of 6.25.

Determination of phosphorus. The total phosphorus present in

 

the dried cells was determined by the method of Sumner (1944). The
cells were digested with lON H2504 for 45 min at approximately 130
C. Complete oxidation was achieved by the addition of hydrogen
peroxide. Excess hydrogen peroxide was avoided since it interferes
\Nlth color development.

Determination of ash. The direct ashing procedure as recommen-
ded by AOAC (1960) was used. The cells were weighed into an ashing

dish that had been previously ignited and cooled. Ignition was

73

performed in a muffle furnace at 550 C for 24 hr.

Determination of total lipids. The IyOphilized cells were

 

analyzed for total lipids by the following procedures. The cells
were hydrolyzed with 6N HCl at 100 C for several hours followed by
extraction with N-pentane. Five grams of cells were heated with
100 m1 of absolute methanol for 15 min at 65 C. The mixture was
brought to room temperature and stirred with 200 ml of chloroform
for 20 min. The mixture was then filtered through a fine sintered
glass disc funnel. The residue was again subjected to the above
extraction procedure with chloroform-methanol. This method of
extraction is essentially similar to one given by Vorbeck and
Marinetti (1965). The combined solvents were evaporated and the
crude lipid extract dissolved in N-pentane. The water soluble
material was removed by the method of Folsh _t._l. (1957). The
lipids were either analyzed immediately or stored at -20 C for not
over 2 weeks.

The data on the nitrogen, phOSphorus, ash, total lipids and
moisture content of §. aureus B-120 are shown in Table 26. The data
presented show no significant difference in the chemical composition
of the cells grown with and without glycerol.

Separation of neutral and phospholipids. The total iipid extract

 

was separated into phOSpho and neutral lipids using siiicic acid as
recommended by Hanhan (1960). The silicic acid slurry was poured
into a sintered glass disc funnel. The sample was poured on to the
silicic acid and eluted with 250 ml of chloroform. Subsequently, the
phOSpholipid fraction was eluted with 250 ml of absolute methanol.

The countercurrent principle of Galanos and Kopoulas (1962) was also

_.——_ {__ _

x' I ".

r

i» ,_')b-M
1

.
fiul; .-.

75

Table 26. Nitrogen, phosphorus, ash, total lipids and moisture
content of cells of S. aureus strain B-120 grown as
indicated for 24 hr at 37 C

 

Cells grown in

 

 

Cells grown in trypticase soy broth
Chemical component trypticase soy broth +3% glycerol
Percent

Nitrogen 12.4 12.6
PhOSphorus 4.5 4.2
Ash 13.1 12.9
Total lipids

a) 6N HCl hydrolysis 4.2-4.8 4.6-5.2

b) Extraction method 3.9-4.5 4.4-5.1

Moisture 78—82 81-82

 

‘D-u'léifl-A.‘ Anna-T11
F 1

Lil; I

1"

A 1.31"“; ,. .

76

used to isolate the polar and nonpolar lipids from crude extract.

The crude lipid fraction obtained by the solvent extraction
method of Vorbeck and Marinetti(1965) was separated into phospho
and neutral lipids. The results obtained with the silicic acid
method and countercurrent distribution method of separation are
shown in Table 27. The results obtained with the two methods were
similar.

Thin layer chromatograph of neutral and phOSpholipids. Thirty

 

grams of silica gel G were suspended in 60 ml of water and the
slurry was applied to precleaned 20x20 cm glass plates in 0.5 mm
thickness using the Research Specialties Co. (RSCo) applicator. The
plates were allowed to dry overnight at room temperature and acti-
vated at 100 C for at least 2 hr. Five to 10 mg of the sample dis-
solved in benzene were applied by means of a capillary tube about
2.5-4.0 cm from the edge of the plate. The weight of the sample
applied was calculated by the difference between the initial and
final weight of the total sample. Prior to weighing, the solvent
was dried in a vacuum oven at 40 C for 2 hr. Initially the split
plate technique was tried for quantitation in which one-fourth of
the plate was Sprayed for spotting and the other three-fourths of
the plate was used for quantitation. Later, the Split plate techni-
que was discarded in preference to monitoring the plates by ultra-
violet (UV) lamp. The results obtained by UV diagnosis were con-
firmed by Spraying the plates with sulfuric acid in case of neutral
lipids, and the color reagent of Ditmer and Lester (1964) in case of
phospholipids. After the efficacy of UV monitoring was established,

this method was used exclusively for identification of both phOSpho

 

 

Table 27. PhOSpho and neutral

77

lipid composition of the lipids of

Ԥ. aureus strain B-120 using the silicic acid and
counter-current distribution methods of separation

 

Separation method

Silicic acid

Counter-current distribution

PhosPholipids

Percent

Neutral lipids

 

74-81
72-78

21-27
24-29

 

1-422“ ' (tum-1. 1161”
ll

 

1'

78

and neutral lipids. Once the identification was established, the
silica gel strips containing the sample were stripped from the plate
and washed with chloroform when determining phOSpholipids,and with
diethyl ether when determining neutral lipids. The silica gel and
the solvent were passed through an ultrafine porosity sintered glass
disc funnel and the solvent evaporated under vacuum at a temperature
somewhat below the boiling point of the solvent.

Analysis of neutral lipids. The neutral lipids were separated

 

into six component fractions by thin layer chromatography as des-
cribed above. The solvent mixture used for development contained
hexane, diethyl ether and acetic acid in the ratio of 79:21zl,
respectively. The quantities of the various fractions were deter-
mined using a gravimetric procedure. The various fractions were dis-
solved in a definite volume of the solvent. An aliquot or the whole
amount was evaporated in a teflon cup at 40 C until completely dry.
The cups were then put in a vacuum oven at 40 C for 1 hr. At the
end of this period the samples were weighed using a Cahn electro-
balance and the percentage of each fraction calculated. The data
obtained are shown in Table 28.

Analysis of phOSpholipids. The procedure used was similar to

 

that described under neutral lipids, except that the percentage of

each fraction was calculated on the basis of the amount of phosPhorus.
The phosphorus analysis was performed using the method of Sumner (1944).
Table 29 summarizes the data obtained.

Analysis of fatty acids. The fatty acid composition of the

 

phospho and neutral lipids was determined using the method of

McGinnis and Dugan (1965). An F and M gas chromatograph was used for

Table 28.

79

Composition of the neutral

lipid fraction extracted from

é. aureus strain B-120 when separated into various
components as indicated using thin layer chromatography

 

Component

Percent

 

Free fatty acids
Monoglycerides

Diglycerides

Triglycerides
Cholesterol

Cholesterol esters

3-7
15-20
42-48

12-18
16-20
8—12

 

h-..- -

 

 

Table 29.

80

Composition of the phospholipid fraction extracted from
§. aureus strain B-120 when separated into various
components as indicated using thin layer chromatography

 

 

Component Percent
Lecithins 46-52
Cephalins 28-32
Sphingomyelins 18-20

 

‘:
. .7: "9;;

h. . ~.~ ~11 awn: .mn.n¢.-”
l .

81

separation of fatty acids. The temperature used for analysis was
200 C. The gas chromatograms of the two fractions are shown in
Figs. 14 and 15, respectively. The two components appear to have

similar fatty acid composition.

tn'éu

82

 

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pouomcuxo mn_a__o;amo;a mo mn_om >uumm mo mcoumo _>cuoe mo ccouuma o_;amcm05meocnu mmm 054 .q. .m_u

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mm ow cm 8 o. m. m c o

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GEE . mi; ZO_._.zw._.mm
mN ¢N ON @— N. m ¢ 0

 

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DISCUSSION

The two strains of §. aureus used in this study were similar
in their growth and pH pattern when inoculated into cereal milk.
At 30 C a population of approximately 2.3 billion was reached in
about 2 days while 7 days were required to reach similar popula-
tions at 15 and 22 C. The pH of the inoculated cereal milk did not
change appreciably during incubation at 15 C for 8 days. The
samples incubated at 22 C showed a slight decrease in pH while at
30 C the pH decreased approximately one unit during 8 days of in-

cubation.

.7} 'MzmHEQI'fl 15“ n: h—p-“'L-M-fi

The lipolytic enzyme systen of S. aureus was .active on milk
fat as indicated by the increase in free fatty acids during incuba-
tion of inoculated samples. This enzyme system appears to be extra-
cellular since appreciable lipolysis occurred during the first day
of incubation at 30 C. At 15 and 22 C the lipolysis was slower but
showed a progressive increase during incubation. Presumably
autolysis is minimal during the first 24 hr and lipolysis during
that period is due to an extracellular enzyme. However, the intra-
cellular lipases, if present, could contribute to the lipolytic
activity. The autolysis and release of lipase might be a factor
after the cells go into the stationary or death phase and previous
work done by Walker (1964) suggests that the death phase starts
after about 3-4 days of incubation at 30 C with the strains used
in this study.

There was greater lipolysis by the cells grown in cereal milk,

than by those grown in broth, when inoculated into cereal milk. The

84

85

potency of the lipase(s) of_§. aureus is further established from
the calculations based on the total amount of fatty acid liberated.
The maximum amount of free fatty acid liberated was 0.48 g per g of
fat in the inoculated cereal milk when incubated at 30 C for 8 days.
The lipid portion of milk contains approximately 85% fatty acids
which are bonded to glycerol. Calculations based on the total amount
of fatty acids present indicate that about 56.4% of the milk fat in
cereal milk was hydrolyzed by E. aureus during 8 days of incubation
at 30 C. The gas chromatographic analysis of the lipolyzed milk fat
showed that the fatty acid composition was similar to the hydrolyzed
milk fat. Milk fat contains an average of about 32% oleic acid and
20% palmitic acid as reported by Jenness and Patton (1959). The
data summarized in Table 5, however, show a higher percentage of
palmitic acid than any other fatty acids which suggests that the
lipase from_§. aureus has a preference for the palmitic acid-
glycerol bond. These data, however, did not include results of
analysis involving short chain fatty acids. The volatile fatty
acids were determined (Table 6) but the method used for their deter-
mination did not differentiate the various individual acids. No
correlation can be made between volatile acidity and fatty acids
determined by gas chromatography. In fact, the volatile acidity
values include acetic, propionic, butyric, caproic, caprylic and a
substantial portion of C10’ C12 and C14 fatty acids. Kosikowski

and Dahlberg (1946) have estimated that approximately 79, 46 and

16% of the C C

and C acids, reSpectively, are included in

10’ 12 14

the determination used for volatile acids.

Gas chromatographic analysis was performed on the total lipids

86

extracted from cereal milk inoculated with §. aureus. The possi-
bility exists that lipids from autolyzed cells of S. aureus may
have contributed minor amounts of fatty acids to the values re-
ported in Table 5, particularly in samples withdrawn toward the end
of the incubation period and at the higher temperatures. Analyses
showed that é. aureus cells contain about 4% lipid on a dry weight
basis; therefore, the amount of free fatty acids contributed by

the autolyzed cells of §. aureus is very small, if any.

The inhibitory effect of the C C10 and C acids on the

8’ 12

growth of §. aureus is apparently bacteriostatic rather than
bactericidal. Costilow and Speck (1951) also reported that these
fatty acids in concentrations of 0.05-0.10% were inhibitory to

Streptococcus lactis. It would appear that these fatty acids are

 

general inhibitory agents and their exact mode of action may be of
great interest. The conclusion that these acids are bacteriostatic
is supported by the fact that increases in population frequently
followed periods of inhibition. The exact mode of action of fatty
acids in functioning as inhibitors is Speculative. The fatty acids
could accomplish inhibition by reducing the surface tension, or
coating the bacterial surfaces. Also, they could selectively
attack the permease, thus blocking the flow of nutrients and/or
preventing the release of extracellular enzymes. The inhibition of
.§' aureus or inactivity of the extracellular enzyme is evidenced by
a decrease in proteolysis, negative coagulase test, and stability
of pH.

The decrease in proteolysis in the presence of C4 to CIO fatty

acids and the increase in proteolysis caused by stearic and oleic

87

acid suggests that the influence of these acids is related to the

activity or amount of the enzyme systems. This decrease or increase

in proteolysis was independent of population.

The lack of lipase activity in the cell-free Supernatant from

still cultures of S. aureus B-120 and the presence of lipase in the

agitated cultures suggests that agitation increases lipase produc-
tion. The presence of lipase in the cell-free supernatant from
agitated culture is probably due to the effect of incorporated air
on the cells. The cell-free supernatant from cultures grown under
vacuum and agitation did not show any lipase activity indicating
that the lipase production (release) is not due to a mechanical
effect alone. However, the cultures supplied with a constant
stream of air but not mechanically agitated showed considerable
lipase activity. The cells showed no evidence of an ectocellular
lipase, since lipolytic activity was not detectable in the wash-
ings obtained from the packed cells when subjected to agitation in
the Waring blender with normal saline for 2 min. Also, the intra-

cellular milieu 1acked lipase activity. Thus it appears that the

lipase is an extracellular enzyme, the production of which seems to

be related to the air supply in the growth medium. The lipase
activity of the cell-free supernatant was greater when agitation
was achieved with a reciprocating shaker as compared to a rotary
shaker. This difference is probably due to the greater amount of
air incorporated into the medium by the former method. The amount

of lipase activity in the cell-free supernatant was influenced by

the age of the cells. At 37 C the activity increased as the incuba-

tion progressed during the first 5 days. A slight decrease occurred

('3; "A
In ‘1

-v - . .‘II‘VQ!’

 

88

on the 6th and 7th days. Possibly during the later stages of in-
cubation the autolysis of the cells and the release of intracellular
enzymes contributed to the total lipase activity. However, this
does not seem plausible since no intracellular lipase activity was
detected at the end of 24 hr of incubation. It is still possible
that the intracellular enzymes may have been produced and released
during the later Stages of incubation.

The lipase from the cell-free supernatant was easily precipi-
tated by several methods such as ammonium sulfate, alcohol, acetone
and pH adjustment. Most of the reagents had an optimum concentra-
tion for maximum lipase activity. Any further increase in the con-
centration of the salt or the solvent decreased the Specific acti-
vity of the enzyme, presumably due to protein denaturation or to
precipitation of nonenzymic protein. Precipitation of the cell-
free supernatant with equal volumes of absolute ethanol was the
preferred method in this work since it completely removed the
lipase from the cell-free supernatant and also yielded maximum
Specific activity. The precipitate so obtained was flocculent and.
settled out after several hours. The precipitate obtained at pH
4.3 was highly active (200 units/mg of protein),whi1e the super-
natant was almost devoid of activity. This enzyme could be preci-
pitated repeatedly at pH 4.3 indicating that this pH may be its
isoelectric point. Desnuelle (l96lb) reported that the isoelectric
point of pancreatic lipase was pH 5.2. The precipitate at pH 4.3
was further purified using molecular sieving techniques. The two
fractions obtained using Sephadex G-200 were distinctly different.

Most of the activity was concentrated in the first fraction which

 

 

 

89

suggests that this enzyme is a high molecular weight protein. A
plot of protein concentration and lipase activity against tube
numbers (fraction numbers) Shows that there is a tailing of the first
peak, indicating a heterogenels material. However, this tailing
was minimized when the fractions from the first peak were concen-
trated and rechromatographed using BioGel 300. The plot Shows a
straight line relationship between protein concentration and enzyme
activity which is indicative of a homogenean5preparation. No fur-
ther purification was attempted. This purified lipase was tested
for its homogeneity by several other methods. Free boundary
electrophoresis analysis of this preparation in veronal buffer,

pH 8.6,r_72 = 0.1 indicates this preparation is electrophoretically
homogereous. An analysis for sedimentation velocity of the prepar-
ation showed two components, a major component and a rapidly sedi-
menting minor component which were approximately in 95 and 5% con-
centrations, reSpectively. The identity of either of these
components as a lipase has not been established unequivocally. It
is possible that the minor component may be some of the acrylamide
particles which might have passed through the sintered glass disc
during column chromatography.

The purified lipase had an optimum pH of 8.6; however, it
Showed activity over a pH range 6-12. The optimum pH is Similar to
the optimum for pancreatic lipase (pH 8-9) as reported by Bbrgstrom
(1957). An Optimum of pH 9.0-9.2 has been reported for milk lipase
by Chandan and Shahani (1964). Shah and Wilson (1965) reported an
Optimum pH of 8.2 for the egg yolk factor which they claim was a

lipase. The optimum temperature for the action of the staphylococcal

9O

lipase was determined to be 45 C and the enzyme was active over a
wide temperature range. The Optimum temperature for the activity
of milk lipase as reported by Chandan and Shahani (19638) is
approximately 37 C deSpite the fact that a considerable amount of
enzyme is inactivated at this temperature.

The crude staphylococcal lipase preparation and the purified
lipase appear to be very stable at various storage temperatures
used in these experiments. The cell-free supernatant lost less
than 10% of the original activity when kept frozen for more than 6
months. The purified enzyme lost 7% of the activity after 21 days
of storage at 4 C. When stored at 37 C the enzyme lost only 12%
of the activity during the first 24 hr and about 50% of the acti-
vity at the end of 21 days. These results are in contrast to
results reported by Chandan and Shahani (1963b) who observed a
complete loss of activity of milk lipase within 24 hr at 37 C.
Besides showing a high degree of Stability during storage, the
staphylococcal lipase is very heat resistant. For example, heating
at 50 C for 30 min decreased the activity of the enzyme only 6%
and it lost 46, 65 and 80% of its activity when heated for 30 min
at 55, 60 and 65 C, respectively. The high degree of thermal
stability raises the question whether some of the rancidity ob-
served in pasteurized dairy products could be caused by residual
enzyme activity.

The enzyme did not Show any particular preference for differ-
ent substrates. All the natural oils and fats tested were hydro-
lyzed to a similar extent with the exception of hydrogenated soy-

bean oil (iodine value < 3) in which case the activity was reduced

91

by two-thirds. This enzyme when tested for activity on a palmitic,
stearic and oleic acid synthetic triglyceride was found to hydrolyze
the fatty acids in all the three positions a, B and a'. This is in
contrast to pancreatic lipase which attacks a and a' positions only.
These observations on staphylococcal lipase were similar to the
ones reported by Alford__£__l. (1964). Even though staphylococcal
lipase attacks all three positions there appears to be slight pre-
ference for the a and a' positions as compared to the B position,
the ratios being 40:21:39 for a, 8 and a' positions, respectively.
Since the enzyme substrate was incubated for a period of 1 hr there
could be some acyl migration. Another important fact about the
enzyme appears to be the ”all or none'l phenomenon; that is if the
enzyme attacks a triglyceride then that triglyceride will be com-
pletely digested into its constituent fatty acids.

The enzyme activity was inhibited by reducing agents while
oxidizing agents stimulated the activity. Mercaptoethanol,
cysteine and glutathione inhibited the activity while hydrogen
peroxide stimulated the activity. Perhaps -SH groups are not re-
quired by the enzyme or possibly the disulfide bonds are necessary
for enzyme action. Similar results were reported by Wills (1960)
on pancreatic lipase. However, Chandan_g£‘§l. (1963 ) believe that
milk lipase is dependent on the sulfhydral group for the activity.
Whether the disulfide bonds are involved or present in staphyl-
ococcal lipase has not been determined.

The action of antibiotics on staphylococcal lipase was varied.
Penicillin and terramycin inhibited the activity while streptomycin

and aureomycin had a stimulatory effect. The exact mode of action

“it! .

92

or the nature of inhibition or Stimulation is not known. These
observations on the effect of the antibiotics on staphylococcal
lipase are in contrast with the work of Shahani and Chandan (1962)
who reported that all of these antibiotics had an inhibitory effect
on milk lipase.

Under the experimental conditions employed for sedimentation
equilibrium analysis, staphylococcal lipase had a molecular weight

of 100,000. The S2 of the enzyme greparation was 2.85 and 8.5 for

0
the two components. Assuming a globular protein and using the

-1
142/3 (3.29 x 10 ‘6), a sedimentation coefficient of

equation S =
approximately 7.0 is required for a molecule with molecular weight

of 100,000. The molecular weights corresponding to S2O of 2.85 and
8.5 are 28,000 and 130,000, respectively, which suggests that the
enzyme may not be a globular trotein. The elution progerties of

the enzyme with molecular sieving technique using Seghadex G-ZOO

and BioGel 300 definitely suggest that the enzyme is a high mole-
cular weight entity.

S. aureus cells have approximately the same chemical composi-
tion as other bacterial cells. Addition of 3% giycerol to the growth
medium did not change the chemical composition of the cells, except
for a slight increase in the total lipid content. However, this
is in contrast to the results reported by Larson and Larson (1922)
in which they found five-fold increase in the lipid content of S.
.ELEEE when grown in the presence of glycerol.

Macfarlane (1962) reported that g. aureus cells contain 25 mg

of lipids per g of dry weight and this value is Slightly lower than

93

the value of 40 mg/g obtained in this study. The lower value Ob-
tained by Macfarlane may be due to the solvent system used for
extraction. The method of extraction has a definite influence on
the amount of lipid extracted from the cells. The data reported
here showed that hydrolyzing the cells with 6N HCl gave a higher
amount of lipids than heating the cells with methanol followed by
extraction with chloroform. The extent of heating of the cells in
methanol also had a definite influence on the amount of extractable
lipids. As the length of heating time increased up to 30 min the
amount of lipid recovered increased. Macfarlane (1962) reported a
ratio of 1 part neutral lipid to 5 parts of phOSpholipids in §.
_gp£gg§ cells. The lipid fraction of the §._§g£gg§ cells examined in
the work reported herein contained 74-81% phOSpholipids and 21-27%
neutral lipids. A high concentration (42-48%)of diglycerides was
present in the neutral lipid fraction. This confirms the data of
Macfarlane (1962) who reported 51% of diglycerides in the neutral
lipids extracted from §..§g£§g§.

The phOSpholipid fraction contained 46-52% lecithin, 28-32%
cephalin and 18-20% Sphingomyelin. The fatty acid composition of

the phOSpho and neutral lipids was similar.

SUMMARY

Two coagulase positive strains of Staphylococcus aureus,B-120

 

and S-1,were used to determine the effect of a) their growth on

the milk fat, b) added fatty acids on the growth of the cells, and
c) the effect of fatty acids on the activity of enzymes produced by
the cells. The lipase liberated from S. aureus strain B-120 was
isolated, purified and characterized. The lipid composition of this
strain was also determined.

Approximately 106 cells per ml grown in either a) cereal milk
or b) trypticase soy broth were inoculated into sterile cereal
milk. The inoculated medium was incubated at 15, 22 and 30 C for a
maximum of 8 days.

The effect of added fatty acids on growth was determined using
sterile reconstituted skimmilk as the substrate. Saturated fatty
and oleic acid were added at 0.01, 0.05 and

acids C through C

4 18

0.1% concentrations.

The two strains of S. aureus Showed similar results regarding_
pH, pOpulation and acid degree value. The cells grown in trypticase
soy broth showed lower lipolytic activity than those grown in cereal
milk. Maximum lipolytic activity was observed at 30 C and in-
creased with time, with 0.48 g of free fatty acids being liberated
per g of fat at the end of 8 days of incubation at 30 C. Palmitic
acid was the most predominant fatty acid liberated by the lipase(s)
of §. aureus. There was a progressive decrease in triglycerides
with only minor changes in mono and diglycerides as well as choles-

terol. The fatty acids which caused partial or complete inhibition

94

95

of the growth of §. aureus were C and C at 0.05 and 0.10% con-

8 10
centrations. The samples showing inhibition were also negative for
coagulase activity, peptonization and change in pH. The fatty acids
varied in their effect on liberation of nonprotein nitrogen. The
addition of oleic and stearic acid to sterile skimmilk inoculated
with §. aureus caused an increase in the amount of nonprotein
nitrogen while the C4 to C12 acids caused a decrease in protease
activity.

The isolation, purification and characterization of the lipase
of S. aureus was studied using strain B-120. Several factors in-
fluenced the production of lipase by §. aureus cells. Agitation of
the cultures during incubation increased the lipolytic activity of
the cell-free supernatant as compared to unagitated cultures which
did not Show any activity. The addition of Tweens, glycerol and
oils to trypticase soy broth did not increase the lipase activity
of unagitated cultures but the incorporated lipids were hydrolyzed.
Maximum lipase activity was observed when the cells were 5 days old
and the pH of the medium was 8.0 or 9.0. Precipitation of lipase
from the cell-free supernatant was best achieved with equal volumes
of absolute ethanol. Further purification was achieved using differ-
ential pH precipitation at 8.6 and 4.3. The final purification was
achieved with Sephadex G-200 and BioGel 300. The final purified
preparation showed a 350-450 fold increase in Specific activity as
compared to the cell-free supernatant. The preparation Showed a
single peak on a Tiselius free boundary electrophoretic pattern with
a mobility of-7.78 x 10-5cm/volt/sec. The ultracentrifugal analysis

for sedimentation velocity indicated that the purified preparation

96

had two components; a major component with $20 = 2.85 and a minor,
fast moving component with a $20 = 8.5. The molecular weight of
the preparation was determined using sedimentation equilibrium data
and an approximate value of 100,000 was obtained. The enzyme pre-
paration was active over a wide range of pH and temperature, with
optima being 8.5 and 45 C, respectively. A storage temperature of
4 and -23 C was found to be superior for preserving the enzyme acti-
vity of the purified preparation. In general there was a decrease
in activity as the storage temperature increased. The enzyme was
relatively heat Stable. Less than 10% of the activity was lost
when exposed to 50 C for 30 min; however, a 30 min exposure at 70 C
destroyed 95% of the activity. The enzymes were active on all the
lipid substrates tested and the activity was Similar in all cases
except the hydrogenated soybean oil where activity was reduced to
one-third. The gas chromatographic analysis of enzyme degraded
synthetic triglyceride indicated that the enzyme was capable of
attacking the a, B, and a' positions. The effect of several chemi-
cals tested on lipase activity was varied. Formaldehyde, mercapto-
ethanol, cysteine, glutathione and terramycin were inhibitory while
hydrogen peroxide, streptomycin and sodium taurocholate had a
stimulatory effect.

The chemical composition of_§. aureus B-120 was determined on
24 hr old cells which were grown in trypticase soy broth with con-
tinuous agitation at 37 C. The cells were washed and lyophilized
prior to analysis. The cells contained 12.4% nitrogen, 4.5%
phOSphorus, approximately 4% lipids, 78% moisture and 13% ash. The

total lipids were separated into phOSpho and neutral lipids using

97

silicic acid. Both phOSpho and neutral lipids were further
fractionated into the constituent parts using thin layer chroma-
tography and the total fatty acid composition determined by gas
chromatography. Analysis of neutral lipids Showed diglycerides to
be the predominant fraction approximating 45% while free fatty
acids represented only 5%. Lecithins, cephalins, and Sphingo-
nweHns were approximately 50, 30 and 20%, reSpectively, of the
total phOSpholipids. Both phOSpho and neutral lipids had Similar

fatty acid composition.

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