3 I. r- 1?. .3”: E. ._,.., '.“ “.4... ,. . , ‘ , “v ‘ ‘fl'ifz ! f '1 ' " . ‘ - l 'r ' ' “3' - _ . . » "g . ' ' x 7 ' or Twfllfi ‘ 3 ~"‘ 543‘!" ‘ r “fad-era , . 1&3 mi?“ 8‘- .- .- ZKii’fia ‘ ‘4 ”‘11:. Fifi“:- \ ‘2: 'Mu“ It .g '3 ‘ A? 21%,. .\ 49-; no": u- - I-«wrc‘u Ty. car-r? ~31?” t“ ..,..... p», _ .. -.. '1" 3": $1ij J .5 ”2.111.. -0 14:!" rug 1 o . ~35: ..... .3133 £311. ‘2 .J J n . .L .. -<\O‘-v'._. v1-I-. r w, r; 333 A: . X?! Wu : w! ’ -l:. r I) ‘ - any: ,. ,'va 1": d n ”u ‘uu ’ '. > ,1 .r:.._. w... a. ‘L . .. - *7. . H’WFF'. . '. "g“ «m 5-»5~_::,re3? “5:”:‘rl' ‘MTJo .S‘r."‘.:" ‘J'ul .1 I". J ’ u. - ‘. . - KKK" . - a! r' . . :’—. a 1 3%.,3 7 , '.’j",’;‘ , ~3 ‘ .17! 9: 1'! h) Lu -.I Unto. . V ‘nv ‘LHH 3 1293 00908 563 4 This is to certify that the thesis entitled PURIFICATION AND CHARACTERIZATION OF XYLANASE FROM THE FUNGAL MAIZE PATHOGEN , COCHLIOBOLUS CARBONUM presented by FRANK ROBERTSON HOLDEN has been accepted towards fulfillment of the requirements for M.S. degree in Botany and Plant Pathology; WM NEW J Major professor Date AugMJr (O; [I (N 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution v' \ :Efiugfi‘l Michigan State University \ I PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. _ DATE DUE DATE DUE DATE DUE 4 fifi MSU Is An Affirmative Action/Equal Opportunity Institution chS-q PURIFICATION AND CHARACTERIZATION OF XYLANASE FROM THE FUNGAL MAIZE PATHOGEN, COCHLIOBOLUS CARBONUM by Frank Robertson Holden A THESIS Submitted to Michi an State Universi in partial ful illment of the requ1rements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1991 ABSTRACT PURIFICATION AND CHARACTERIZATION OF XYLANASE FROM THE FUNGAL MAIZE PATHOGEN, C0 CHLIOB 0L US CARB ONUM by Frank Robertson Holden Xylans are the most abundant hemicellulose and are found in all land plants. They are a major component of plant cell walls, making up to 40% by mass of the primary cell wall of monocots and a significant portion of the secondary cell walls of dicots. There is a considerable amount of indirect evidence that enzymes that degrade xylan (xylanases) are important in pathogenesis. Three xylanases and a B-xylosidase were purified from cultures of the maize pathogen, Cochliobolus carbonum grown on xylan or corn cell walls. The major xylanase was found to have a molecular weight of 24,000 by SDS-PAGE and 8,500 by gel filtration chromatography and a high isoelectric point (pI greater than 9.5). The other xylanases had similar properties and the B-xylosidase was shown to have a molecular weight of 41,000 by SDS-PAGE and 31,500 by gel filtration chromatography. The major xylanase was purified to homogeneity and was analyzed for amino acid composition. It was also digested with trypsin and the resulting peptides were purified and sequenced. Using the tryptic peptide sequences, two degenerate oligonucleotides were synthesized and used as probes against a genomic DNA library made in lambda. Approximately 50 lambda clones were picked from the library. ACKNOWLEDGEMENTS I wish to thank Jonathan Walton for all his help in preparing this thesis and too many other things to mention. I would also like to thank Joseph Leykam for the help in preparing the peptides and the amino acid sequencing. I thank Ray Hammerschmidt for his help by being on my committee and his support. Finally, I wish to thank Laura for just being herself. iii TABLE OF CONTENTS Page LIST OF TABLES vii LIST OF FIGURES viii LIST OF ABBREVIATIONS ix INTRODUCTION 1 MATERIALS AND METHODS 6 Fungal Growth Conditions 6 Enzyme Assays 8 Purification by Conventional Liquid Chromatography 10 Purification by High Performance Liquid Chromatography (HPLC) 11 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS—PAGE) 12 Isoelectric Focusing (IEF) 14 Measurement of Xylanase Activity versus pH and Temperature 15 Thin Layer Chromatography of Xylanase Reaction Products 15 Enzymatic Digestion of Xylanase I 16 Separation of Tryptic Peptides by Reverse Phase HPLC 17 Preparation of Samples for N-Terminal Sequencing 19 iv Page Amino Acid Sequencing 20 Amino Acid Composition 20 Oligonucleotide Synthesis 20 32F Labeling of the Oligonucleotides 22 Probing Southern Blots of Genomic DNA from C carbonum 23 Screening a C. carbonum Genomic DNA Library Made in Lambda EMBL3 24 RESULTS 27 Effects of Different Media on the Production of Xylanase 27 Inhibition of Xylanase Production by Sucrose 27 Comparison of Xylanase Production in Toxin Producing and Nonproducing Strains 28 Purification of Xylanase 28 Determination of the Number of Xylanases 32 B-xylosidase and B-glucosidase 32 Determination of Molecular Weights by SDS-PAGE and Gel Filtration 35 Isoelectric Focusing 39 pH and Temperature Profiles of Xylanase Activities 39 TLC of Xylanase Products 39 Enzymatic Digestion of Xylanase I and Peptide Sequences 42 Peptide Homology Search 44 Amino Acid Composition of Xylanase I 44 Oligonucleotide Synthesis 47 Page Establishing Hybridization Conditions for Probing with Labeled Oligonucleotides 47 Probing the Genomic DNA Library Made in Lambda 48 DISCUSSION 50 BIBLIOGRAPHY 56 vi LIST OF TABLES ' Page Table 1. Mineral salts medium. 7 Table 2. Recipe used for SDS-PAGE. 13 Table 3. Peptide 13 and 22 sequences and the oligonucleotides made from them. 21 Table 4. Recipes used for molecular biology. 25 Table 5. Purification table for xylanase. 30 Table 6. Effect of temperature on xylanase activity. 41 Table 7. Peptide sequences. 43 Table 8. Homology search of xylanase sequences. 45 Table 9. Amino acid composition of xylanase I. 46 Table 10. Physical properties of some xylanases. 53 vii Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. LIST OF FIGURES Effect of sucrose on xylanase activity and fungal dry weight. 29 Hydrophobic interaction HPLC. 31 Cation exchange HPLC showing three xylanases and a B-xylosidase. 33 Cation exchange HPLC showing a single xylanase. 34 SDS-PAGE of xylanase I prior to digestion with trypsin. 36 SDS-PAGE showing three xylanases and a B-xylosidase. 37 Gel filtration HPLC showing the molecular weight of xylanase I. 38 Gel filtration HPLC showing the molecular weight of xylanase II and B-xylosidase. 38 Effect of pH on activity of xylanase I, II and III. 40 Figure 10. Southern blot of C. carbonum genomic DNA probed with Oligonucleotide 1 and 2. 49 viii ATP CM DEAE DTT EDTA HF BA HPLC z; mm mM mg uCi ul um LIST OF ABBREVIATIONS adenosine triphosphate beta Celsius carboxy methyl diethylaminoethyl dithiothreitol (ethylenedinitrilo)tetraacetic acid gram hexafluorobutyric acid high performance liquid chromatography isoelectric focusing Luria broth molar millimeter(s) millimolar milligram(s) microCurie(s) microliter(s) micromolar m1 mw N “8 nm OD % P pI PTH RVU SDS SDS-PAGE TFA TLC Tris UV v/v w/v milliliter(s) molecular weight normal nanogram(s) nanometer(s) optical density percent para isoelectric point phenylthiohydantoin relative viscometric units sodium dodecyl sulfate sodium dodecyl sulfate polyacrylamide gel electrophoresis trifluoroacetic acid thin layer chromotagraphy tris(hydroxymethyl)aminomethane ultraviolet volume to volume weight to volume INTRODUCTION Many plant pathogens need the ability to penetrate the plant cell wall before successful infection can occur. Penetration of the plant cell wall can occur by mechanical force or by enzymatic degradation. Research on the role of plant cell wall degrading enzymes in infection is considerable and most bacterial and fungal plant pathogens are known to produce a battery of plant cell wall degrading enzymes (Cooper, 1984). However, few of these enzymes have been studied for a role in pathogenesis. Nearly all plant cell wall degrading enzymes directly implicated in pathogenesis are from bacterial pathogens. A few cell wall degrading enzymes which when selectively mutated in bacterial pathogens result in reduced virulence, directly implicating the enzymes in pathogenesis. The best evidence showing involvement of a cell wall degrading enzyme in pathogenesis comes from transposon .mutagenesis of the pectin methyl esterase gene of Erwinia Chrysanthemi 3937. The mutant pathogen became noninvasive in Saintpaulia plants (Boccara et a1., 1989). Other evidence suggests that certain cell wall degrading enzymes are, in some cases, important but not essential in bacterial pathogenesis. The 2 endoglucanase gene in Pseudomonas solanacearum was disrupted by transposon mutagenesis causing a ZOO-fold decrease in endoglucanase activity and significantly less virulence on tomato (Roberts et 31., 1988). The major endoglucanase activity in Xanthomonas campestris pv. campestris was disrupted by transposon insertion and pathogenicity tests of these mutants demonstrated that this enzyme plays only a minor role in of pathogenesis on turnip and radish (Glough et 31., 1988). All four pectate lyase genes in E. chrysanthemi were deleted causing a severely reduced ability to degrade pectin, but the bacterium could still survive on pectin as a carbon source and caused significant maceration of potato, carrot and pepper tissue (Payne et 31., 1987; Reid and Collmer, 1988) In fungi, no polysaccharide-degrading enzymes have been directly shown to be important in pathogenesis. It was shown by targeted gene disruption that endopolygalacturonase is not essential for infection of maize by Cochliobolus carbonum (Scott-Craig et al., 1990). Although for the majority of diseases direct evidence is lacking, there is much indirect evidence that cell wall degrading enzymes are involved in pathogenesis: a) Polysaccharide degrading enzymes are produced by plant pathogens when grown on plant cell walls or purified components of plant cell walls as a carbon source (Bateman et a1., 1973; Mullen and Bateman, 1975; Bateman et a1., 1969; Cooper et a1., 1988; Anderson, 1978). b) The timing and extent of production of a particular 3 cell wall degrading enzyme in culture often reflects the importance of that enzyme’s substrate in the host cell wall (Cooper et al., 1988; Cooper, 1984; Anderson, 1978; English et a1., 1971). c) Polysaccharide degrading enzymes are frequently present in infected plant tissue (Mullen and Bateman, 1975; Cooper et a1., 1988; Bateman et a1., 1969). d) Certain plant cell wall degrading enzymes are capable of eliciting host defense responses. EndO-polygalacturonase (EC 3.2.1.15) produces short oligogalacturonides that act as elicitors of plant defense responses such as phytoalexin accumulation in soybean (Davis et a1., 1984) or necrosis in Vrgna unguiculata (Cervone et al.,1987). Pectin lyase can cause the formation of superoxide radical in rice protoplasts (Ishii, 1988). Most if not all plant pathogens produce xylanase (IS-1,4- endoxylanase, EC 2.3.1.8). Although xylanase has not been studied directly for a role in any plant disease, either by traditional mutagenesis or by recombinant DNA methods, indirect evidence suggests that they might be important. The same indirect evidence used to implicate cell wall degrading enzymes in general can be used for xylanase: a) Xylans are present in all land plants. B-1,4-xylan is the major constituent of monocot primary cell walls, making up to 40% or more by mass (Burke et a1., 1974; Darvill et a1., 1980a; Cooper, 1984). Since xylan is the most abundant component of the primary cell wall of monocots, xylanases may play a significant role in the pathogenesis of monocots. Xylans 4 account for approximately 5% of the primary cell wall and a more significant percentage of the secondary cell wall of dicots (Darvill et a1., 1980b) b) Xylanase is produced by plant pathogens when grown on plant cell walls. c) Xylanase is sometimes produced earlier than other enzymes when monocot pathogens are grown on plant cell walls (Mullen and Bateman, 1975; Bateman et a1., 1969). d) Xylanase is present in infected plant tissue (Cooper et al., 1988; Mullen and Bateman, 1975; Bateman et a1., 1969). e) Xylanase from Trichoderma viride can either directly or indirectly elicit plant defense responses (Dean and Anderson, 1991). Xylanase from Trichoderma viride can elicit pathogenesis-related proteins (Lotan and Fluhr, 1990), electrolyte leakage (Bailey, et 31., 1990) and ethylene biosynthesis (Fuchs, et a1., 1989) in tobacco. Xylanase can cause the formation of superoxide radical in rice protoplasts (Ishii, 1988). A great deal of research has focused on the bacterial pathogens of dicots and this has demonstrated the importance of pectin degrading enzymes in these systems. However, due to the different composition of monocot and dicot cell walls, pectin degrading enzymes may not play such a significant role in the pathogenesis of monocots. Instead, enzymes such as xylanase, glucanase and arabanase may be more important. Some researchers believe that the importance of xylanase in pathogenesis, especially in monocots, has been underestimated (Cooper, 1984) 5 The purpose of this research was to prepare the groundwork to test the hypothesis that xylanase is important in plant pathogenesis. Targeted gene disruption of xylanase in C. carbonum and other pathogenic fungi is be possible as a result of this research. This will directly establish whether xylanase is important in these systems. MATERIALS éfig METHODS a owt ndit'ons Conidia of Cocbliobolus carbonum race 1, isolate SB111 (HC-toxin producing), or race 2, isolate 1309 (HC-toxin nonproducing), from stocks stored at -80° C, were used to inoculate V-8 juice agar plates (Walton, 1987; Walton and Holden, 1988). Isolate SB111 was provided by S. P. Briggs, Pioneer Hi-Bred International, Inc., Johnston, Iowa. Isolate SB111 was used for all experiments unless otherwise noted. Isolate 1309 was supplied by K J. Leonard, North Carolina State University. Plates were grown at room temperature (21-23° C) under fluorescent lights and 0.5 cm2 squares were cut out and used to inoculate one-liter Erlenmeyer flasks containing 125 ml of liquid media. Liquid media was either modified Fries’ medium (Pringle and Braun, 1957) or mineral salts medium (Table 1) (Bateman et 31., 1973) with 1 or 2 gm/liter of sucrose (unless otherwise noted). 0.4% or 0.8% (w/v) oat-spelt xylan (Fluka Chemie AG, Switzerland) or 0.8% (w/v) washed corn cell walls was added to flasks individually. CI carbonum was grown for 7 or 9 days on an open laboratory bench (21-23° C). Washed corn cell walls were prepared by chopping whole mature corn plants into small pieces and from this a fine powder was prepared using a Waring blender with liquid nitrogen. 7 Table 1. Mineral salts medium. MgS 04 KC] (NH4)2SO4 KHZPO4 Sucrose trace element stock yeast extract Trace element stock (1000x) MI'ISO4'4H20 H3303 CUSO4 ZHSO4'7H20 F6504 m 0.18 gm 0.15 gm 1 gm 0.65 gm 1 or 2 gm (unless otherwise noted) 1ml 2gm 0.1 gm 0.1 gm 0.01 gm 0.01 gm 2.0 gm 8 This powdered corn plant preparation was suspended in 0.1 M KHZPO4, pH 7.0 buffer and stirred for one hour, strained through cheese cloth and then resuspended in chloroform, this was repeated twice with methanol after which the washed corn cell wall preparation was allowed to dry. Xylanase production versus time was determined by harvesting the culture filtrate of C. carbonum grown on modified Fries’ medium or mineral salts medium with 2 gm/liter of sucrose and 0.8% (w/v) xylan at varying times and assaying for xylanase activity. Inhibition of xylanase production by sucrose was tested by growing Cochliobolus carbonum for 7 days in modified Fries’ medium containing 0, 1, 2, 4, 8 or 12 gm/liter sucrose and 0.8% (w/v) xylan. Xylanase activities and the dry weights of the fungal mats were measured. I'll me sa S Viscometric assays for xylanase activity were done by measuring the decrease in relative viscosity of 1.5% (w/v) solution of oat-spelt xylan (Fluka) in 50 mM sodium acetate, pH 5.0, at 30° C. Reducing end groups were assayed using p-hydroxybenzoic acid hydrazide (H-9882, Sigma) (Lever, 1972). Aliquots of a 0.6% (w/v) solution of oat-spelt xylan (Fluka) were tested at different times to measure the free reducing ends liberated by xylanase activity in 50 mM 9 sodium acetate, pH 5.0. The p-hydroxybenzoic acid hydrazide working solution was prepared from a 5% (w/v) stock solution in 0.5 N HCl by diluting 1:4 in 0.5 N NaOH. 10 or 20 ul of xylan solution was added to 1.5 ml of the p-hydroxybenzoic acid hydrazide working solution and this was heated at 100°C for 10 min after which absorbance at 410 nm was measured. Xylose was used as a standard to quantify the amount of end reducing groups liberated in the digestions. B-xylosidase activity was determined by measuring the amount of p-nitrophenol released from the synthetic substrate p-nitrophenyl-B-D- xylopyranoside (N—2132, Sigma). The method used was adapted from Montreuil et a1., 1986. 0.1 ml of 10 mM p-nitrophenyl-B-D- xylopyranoside was added to 0.1 ml 200 mM sodium acetate, pH 5.0, and 0.2 m1 of each fraction of a cation exchange HPLC run of semipurified culture filtrate of C carbonum grown on corn cell walls. After incubating for 20 min, 0.6 ml of 1 M sodium carbonate was added and the absorbance at 400 nm was measured. B-glucosidase activity was assayed by the same method except p- nitrophenyl-B-D-glucopyranoside (N-7002, Sigma) was used as the substrate. 10 Purification by Conventional Liquid Chromatography Culture filtrate was poured through cheesecloth and filtered through Whatman #1 filter paper by vacuum. The culture filtrate was then diluted two-fold with 25 mM sodium acetate buffer, pH 5.0, and loaded onto DEAE-cellulose (D—3764, Sigma) with a bed volume of 80 ml packed in two 60 ml-syringes in series. Following the DEAE- cellulose was a column of CM-cellulose (C-4146, Sigma), in series, with a bed volume of 60 m1. Both columns were washed with two bed volumes of 25 mM sodium acetate, pH 5.0. The CM-cellulose was eluted with a 250 ml gradient going from 25 mM sodium acetate, pH 5.0, to 25 mM sodium acetate plus 0.8 M KCl, pH 5.0. In some experiments the CM-cellulose was eluted stepwise with 25 mM sodium acetate plus 0.4 M KCl, pH 5.0. In earlier purifications the culture filtrate was not diluted and loaded directly. Instead, the culture filtrate was concentrated by rotary evaporation under reduced pressure at 37° C (Bfichi model 011) and then dialyzed (Spectrum Medical Industries, Inc., Spectrapor, 6,000 to 8,000 mw cutoff, 32 mm diameter) overnight in 25 mM sodium acetate before loading onto the DEAE and CM-cellulose columns. The eluate from the CM-cellulose column was dialyzed (same membrane as above) overnight against 12.5 mM sodium acetate, pH 5.0. In earlier purifications the CM-cellulose eluate was desalted by 11 membrane filtration (Amicon, 10,000 mw cutoff). f r'_ cat'o b 11: _ __’e fo a c icu'd Ch omato. .. _ g ’____C Further protein fractionation was done by HPLC on a Waters system with a model 680 automated gradient controller, two model 501 pumps and a model 440 UV detector (280 nm). Aqueous solvents were filtered through a 0.45 pm nitrocellulose membrane by vacuum and organic solvents were filtered through a 0.45 pm nylon membrane by vacuum. Ion exchange HPLC was done on a 200 x 4.6 mm polysulfoethyl aspartamide cation exchange column (The Nest Group Inc., Southboro, Massachusetts). Flow rate was 1 ml/min with a linear gradient from 0 to 100% B in 40 min. Buffer A was 25 mM sodium acetate, pH 5.0, and buffer B was 25 mM sodium acetate and 0.4 M KCl, pH 5.0. Fractions were collected at one min intervals. In some ion exchange runs 0.5 mM EDTA was added to buffers A and B. Also, phosphate buffer at pH 6.5 was used in one run. Buffer A was 12.5 mM KHZPO4, pH 6.5 and buffer B was 12.5 mM KHZPO4 and 0.4 M KCl, pH 6.5 the same gradient described above was used. Gel filtration was done on a 600 x 7.5 mm TSK-3000SW column with a 75 x 7.5 mm guard column (Beckman). The mobile phase was 100 mM KHZPO4, pH 6.5, and the flow rate was 0.5 mein. Molecular 12 weight standards to calibrate the gel filtration column were ovalbumin, mw 44,000 (Sigma A-7641), myoglobin, mw 17,000 (Sigma M-0630) and vitamin B-12, mw 1,350 (Sigma V-2876). Reverse phase HPLC was done on a 250 x 4.6 mm C-18 column (Beckman) and a 250 x 4.6 mm C-4 column (Vydac). A linear gradient from 0 to 100% B was run in 30 min at a flow rate of 1 ml/min. Solvent A was Milli-Q water plus 0.1% TFA and solvent B was acetonitrile plus 0.1% TFA or acetonitrile/isopropanol (1:1 v/v) plus 0.1% TFA. No xylanase was recovered from either column under either solvent condition. Hydrophobic interaction was on a 75 x 7.5 mm TSK-Phenyl-S-PW column (Bio-Rad). A 30 min linear gradient was run from 0 to 100% B. Solvent A was 100 mM KHZPO4 plus 1.5 M (NH4)2SO4, pH 6.5, and solvent B was Milli-Q water and the flow rate was 1 ml/min. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS- PAGE) SDS-PAGE was done using the method described by Hames and Rickwood (1981) with 12 or 15% acrylamide (Table 2). These gels were run on a Hoeffler Mini Protean 11 unit at 120 volts until the bromphenol blue tracking dye had just run off the bottom of the gel. Also, SDS- PAGE was done on a Pharmacia PhastSystem with 8-25% acrylamide Table 2. Recipe used for SDS-PAGE. 13 Running gel (10 ml) 12% 15% Acrylamide (30:0.8%) 4.0 5.0 ml 1.75 M Tris, pH 8.8 2.0 2.0 ml 10% SDS 0.1 0.1 ml Water 3.8 2.8 ml TEMED 10 10 pl 10% Ammonium persulfate 60 60 pl Stacking gel (6 ml) Acrylamide (30:0.8%) 1.0 ml 1.25 M Tris, pH 6.8 0.6 ml 10% SDS 60 pl Water 4.3 ml TEMED 5 pl 10% Ammonium persulfate 50 pl Acrylamide (30:0.8%) 30% acrylamide 0.8% Bis-acrylamide filtered through Whatman #1 filter paper 7 's H 8.8 1.75 M Tris base, titrated with HCl 1.25 M Tris. pI_-I 6.8 1.25 M Tris base, titrated with HCl M Tris base Glycine SDS Sample buffer (4x) (save at -20°C in 0.5 ml aliquots) 1.25 M Tris, pH 6.8 D'I'T SDS Sucrose Bromphenol blue Water per liter 3gm 14.4 gm 1gm 5 ml 0.15 gm 10 ml 20% 10 gm 1 mg to 25 ml 14 gradient gels by the method described in the Pharmacia PhastSystem manual. The size markers used were Bio-Rad low molecular weight SDS- PAGE standards. These were rabbit muscle phosphorylase b, 97,400 mw; bovine serum albumin, 66,200 mw; hen egg white ovalbumin, 42,699 mw; bovine carbonic anhydrase, 31,000 mw; soybean trypsin inhibitor, 21,500 mw; and hen egg white lysozyme, 14,400 mw. Standards were loaded at concentrations of 0.5 or 1.0 pg of each protein per lane in the Hoeffler mini-gel unit and 10 ng of each protein per lane for the Pharmacia PhastSystem. Hoffler mini-gels were stained with coomassie blue R-250 (Eastman Kodak Co.) 0.1% in methanolzwaterzacetic acid (5:5:2), and destained by several washes in 7% acetic acid heated to 90° C. Pharmacia PhastSystem gels were silver stained according to the manual except the concentration of formaldehyde used in the development step was decreased by one half. soelectric Focusin IEF Isoelectric focusing was done on an LKB Ampholine PAGplate, pH 3.5 to 9.5. The gel was run for 2 hours at 4°C at a current of 10 mA. The gel was fixed in 10% (w/v) trichloroacetic acid plus 3% (w/v) sulfosalicylic acid for 1 hour and stained with Coomassie blue R-250 15 (Eastman Kodak Co.) in methanol, water and acetic acid (5:5:2) and destained in the same solvent minus the dye. Isoelectric focusing was also done on a Pharmacia PhastSystem using a pH 3 to 9 IEF gel and silver stained according to the Pharmacia PhastSystem manual. Pharmacia isoelectric focusing markers were used to calibrate the resulting pH profile. Mesurement of Xylanase Activity versus pH angl Temperature Xylanase I, II and III activity was measured in 30 min assays using 50 mM KHZPO4 adjusted to pH 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0. Activity versus temperature of xylanase I was measured in a 30 min reaction at 30, 37, 45, 54 and 65° C. in C romato ra h o X lanase Reactio ucts Thin layer chromatography (TLC) was done with ethanol soluble (67% v/v) products of an overnight digestion of oat-spelt xylan (Fluka). The solvent was butanol, water and acetic acid (4:1:5) and the plates used were cellulose on polyester (T-6890, Sigma). Free xylose was added to some samples prior to ethanol precipitation to determine whether components of the reaction mixture interfered with the migration of xylose. 16 Plates were developed by the method of Trevelyan et 31., 1950, which is specific for end reducing groups. 0.1 ml of saturated AgNO3 was added to 20 m1 of acetone and water was added until the AgNO3 had dissolved. The AgNO3 in acetone was sprayed on the plates and allowed to dry. 0.5 N NaOH in ethanol was prepared by adding 20 M NaOH to 95% ethanol and water was added until the NaOH dissolved. The plates were sprayed with this and then submerged in 6 N NH4OH for less than 1 min and washed in water for 1 hour. Standards used were xylose (X-1500, Sigma), arabinose (Uninted States Biochemical), glucuronic acid (ICN Biochemicals), glucose (Mallinckrodt) and xylobiose (X-1501, Sigma). n atic Di estion of X lanase I The xylanase sample was desalted by Centricon membrane filtration (3,000 mw cutoff) (Amicon) and then dried by vacuum centrifugation (Savant). The trypsin digestion followed the procedure by Stone et a]. (1989). 50 pl 8 M urea, 0.4 M NH4HCO3 was added to the dried protein. The pH was checked by spotting 1 pl on pH paper and was determined to be between 7.5 and 8.5. 5 pl were removed for amino acid analysis. 5 pl of 45 mM dithriothreitol (DTT) were added and the sample was incubated at 50°C for 15 min to denature it. After cooling to room temperature 5 pl of 100 mM iodoacetamide was added 17 to alkylate the cysteine residues so the xylanase became permanently denatured. The sample was incubated at room temperature for 15 min to allow the iodoacetamide to react with the cysteine residues. 140 pl of water was added and trypsin was added equal to 1/25th the amount of xylanase as determined by a coomassie blue R-250 (Eastman Kodak Co.) stained SDS-polyacrylamide gel. Trypsin (TPCK-treated, Worthington Enzymes) was prepared as a 0.2 mg/ml solution in 0.001 N HCl and stored at 20° C in 0.5 ml aliquots. The reaction was incubated at 37°C for 4 hours and then frozen at -80°C until the peptides were separated by reverse phase HPLC. Other attempts were made with digestions 24 hours in length but these resulted in very short peptides. Additionally, alkylation of cysteines by 4-vinylpryidine was tried unsuccessfully. Enzymatic digestion by endoproteinase lys-C (Boehringer Mannheim Biochemica) was attempted by the method of Stone et a1. (1989) but the details were not sufficiently worked out to get the enzyme to cleave the substrate. ar tio ofT t'c e tides eve se a e HP C The first attempt at fractionation of peptides from a 24 hour trypsin digestion by reverse phase HPLC was done on a Beckman system with a model 421 controller, two model 114 pumps and a model 18 163 UV detector (215 nm). The column used was a 250 x 4.6 mm Ultrasphere C-18 (Beckman). A linear gradient was run from 0 to 100% B in 60 min. Solvent A was water (Burdick and Jackson) and solvent B was acetonitrile (Burdick and Jackson), both solvents contained 0.1% trifloroacetic acid (TFA) (Pierce Chemical Co.). Peaks were collected manually and were either sequenced or repurified by microbore HPLC. Microbore HPLC was used to further resolve peptides purified on the Beckman system and to fractionate tryptic peptides generated from 4 hour digestions. Microbore HPLC was performed on an Applied Biosystems model 130 microbore HPLC system with a Spectroflow 783 detector (Kratos Analytical Instruments) set at 214 nm using a 200 nl flow cell. The column used was a 250 x 1 mm C-8 (Applied Biosystems). Peaks were collected manually. Data collection was by Beckman System Gold computer software. Gradients were linear going from water to 90% acetonitrile in 60 min. Water contained 0.1% TFA and the 90% acetonitrile slightly less TFA so the absorbance at 214 nm was identical for both solvents. Peaks that were not homogenous as determined by the computer software were run again using 0.1% hexaflorobutaric acid (HFBA) instead of TFA in the solvents. Peptides were dried by vacuum centrifugation (Savant) and stored at -80°C until they could be sequenced. 19 at' lef - 'aSucin Four separate attempts to sequence the N—terminus of xylanase I and one attempt to sequence the N-terminus of B-xylosidase were made. Two of the attempts made with xylanase I used cation exchange and gel filtration purified samples. A third xylanase I sample was purified by cation exchange HPLC, run on a preparatory gel (SDS-PAGE) and then transferred to immobilon membrane (Millipore) using a semi-dry electrophoresis transfer unit (LKB-Produkter AB, Sweeden). After transfer the immobilon membrane was stained with 0.2% (w/v) ponceau- S (P-3504, Sigma) in 1% (v/v) acetic acid for 1 min and destained by washing in water for several min. The xylanase band was cut out and direct N-terminal sequencing of the xylanase bound to the membrane was attempted. The fourth attempt to sequence the N-terminus of xylanase I was made using a preparation that was purified by cation exchange HPLC and hydrophobic interaction HPLC. B-xylosidase was purified by cation exchange HPLC and gel filtration HPLC before N- terminal sequencing was attempted. Salts were removed from all samples, except the immobilon membrane sample, by centricon membrane filtration (3,000 mw cutoff) (Amicon). Samples were dried by vacuum centrifugation (Savant) and stored at -80°C until they could be sequenced. 20 Amine Aeigl Seqpeneing Amino acid sequencing was performed by Joseph Leykam, Macro- Molecular Facility, Michigan State University, using Edman degradation on an Applied Biosystems model 477A protein sequencer using an Applied Biosystems model 120A analyzer. W A sample of xylanase was hydrolyzed with acid and the free amino acids were derivatized with phenylthiohydantoin (PTH) and separated by HPLC. HPLC was on a Waters system with a model 680 automated gradient controller, two model 510 pumps and a model 440 UV detector (254 nm). The column used was a Waters 150 x 3.9 mm C-18 and data collection and analysis was by Beckman System Gold computer software. The system was calibrated using PTH derivatized amino acid standards. Qligepueleetide Synthesis Two peptides, 13 and 22, were chosen to make degenerate oligonucleotides, 1 and 2, respectively (Table 3). Both oligonucleotides were 17 bases long. Oligonucleotide 1 was 32-fold degenerate and in the complementary orientation and Oligonucleotide 2 was 128-fold 21 38.5: bfiaoEEQEOO ZOEMMHQ wmfi 3 Nm 5 >U>>>m> ohoiiaioioioozéooo H om=o <305 Eooxo 823mg m 8.58 mm EoEtomxo 086m 05 Bob 0.53 325% m 8.50m 0.33 893 86>»: wHO an Bursa cosmomfi 59596 on Bob 8m BEER“ < 8.50m mzoaomaazotohnzgmimz 65% O S >zzoomoam>m>mooAm§moo