Purification, Characterization, and Substrate Specificities of Multiple Xylanases from Streptomyces sp. Strain B-12-2

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1994, p Vol. 60, No /94/$ Copyright ) 1994, American Society for Microbiology Purification, Characterization, and Substrate Specificities of Multiple Xylanases from Streptomyces sp. Strain B-12-2 GRAZIANO ELEGIR,1 GEORGE SZAKACS,2 AND THOMAS W. JEFFRIES3* Institute for Microbial and Biochemical Technology, Forest Products Laboratory, Forest Service, U.S. Department of Agriculture, Madison, Wisconsin ; University of Technical Sciences, Budapest Institute of Agricultural Chemical Technology, Budapest, Hungary2; and Stazione Sperimentale per La Cellulosa, Carta e Fibre Tessili Vegetali ed Artificiali, Milan 20133, Italy' Received 22 February 1994/Accepted 9 May 1994 The endoxylanase complex from Streptomyces sp. strain B-12-2 was purified and characterized. The organism forms five distinct xylanases in the absence of significant cellulase activity when grown on oat spelt xylan. This is the largest number of endoxylanases yet reported for a streptomycete. On the basis of their physiochemical characteristics, they can be divided into two groups: the first group (xyl la and xyl lb) consists of low-molecular-mass (26.4 and 23.8 kda, respectively) neutral- to high-pi (6.5 and 8.3, respectively) endoxylanases. Group 1 endoxylanases are unable to hydrolyze aryl-o-d-cellobioside, have low levels of activity against xylotetraose (X4) and limited activity against xylopentaose, produce little or no xylose, and form products having a higher degree of polymerization with complex substrates. These enzymes apparently carry out transglycosylation. The second group (xyl 2, xyl 3, and xyl 4) consists of high-molecular-mass (36.2, 36.2, and 40.5 kda, respectively), low-pi (5.4, 5.0, and 4.8, respectively) xylanases. Group 2 endoxylanases are able to hydrolyze aryl-p-d-cellobioside, show higher levels of activity against X4, and hydrolyze xylopentaose completely with the formation of xylobiose and xylotriose plus limited amounts of X4 and xylose. The enzymes display intergroup synergism when acting on kraft pulp. Despite intragroup similarities, each enzyme exhibited a unique action pattern and physiochemical characteristic. xyl 2 was highly glycosylated, and xyl lb (but no other enzyme) was completely inhibited by p-hydroxymercuribenzoate. Xylan is the major component of the hemicellulose present in angiosperm cell walls (32). It is probably the second most abundant carbohydrate polymer of plants. Xylans are heterogeneous polysaccharides consisting of a backbone of 13-1,4- linked D-xylopyranosyl residues that often have O-acetyl, arabinosyl, and methylglucuronosyl substituents (35). They present a relatively complex substrate that varies greatly from plant to plant and from tissue to tissue. ao-arabinofuranosidases (EC ), acetylesterases (8) (EC ), cx-methylglucuronosidases (20, 26), and feruloyl esterases (15) act in concert to remove the side chains before xylanases reduce the backbone to xylooligosaccharides (17). Contemporary interest in xylan-degrading enzymes is due to new applications in the prebleaching of kraft pulps (34) and to their possible use in recovering fermentable sugars from hemicellulose (38). This attention has led to characterization of many new enzymes, some of which exhibit unusually high levels of thermal stability (16, 18, 28) or alkaline activity (2, 23). Most of those characterized are endoxylanases (EXs). EXs (1,4-13-D-xylan xylanohydrolase; EC ) attack the internal backbone of the polymer, producing xylooligosaccharides with different degrees of polymerization. 1-Xylosidases (EC ) remove successive D-xylose residues from the nonreducing termini (1, 25). Xylanases sometimes exhibit cooperativity in the hydrolysis of xylooligosaccharides and arabinosyl substituents (6, 37). Notwithstanding much study in recent years, it is not yet known why microorganisms exhibiting significant hemicellulase activity produce several EXs with different specificities (3, * Corresponding author. Mailing address: Forest Products Laboratory, One Gifford Pinchot Drive, Madison, WI Phone: (608) Fax: (608) Electronic mail address: , 37). The variety of these EXs could reflect the diverse structural features of hemicellulose, it could relate to the kinetics of hydrolysis, or it could reflect the various biophysical environments within which the organisms grow. Earlier studies have shown that some EXs produce mainly xylose and xylobiose (X2), while others produce mainly oligosaccharides with higher degrees of polymerization (DPs) (12, 24). These groupings may or may not coincide with other classifications based on nucleotide sequences (11) or molecular weights and pl values (37). Because of the difficulties in purifying multiple xylanases and determining their action patterns, there have been relatively few characterizations of all of the EXs produced by any one organism (5, 12, 30, 33). Recent advances in protein purification and product characterization techniques have facilitated this task. In this paper we report the purification and characterization of five EXs from a highly xylanolytic organism, Streptomyces sp. strain B-12-2, and their action patterns. The enzymes can be divided into two broad groups on the basis of their molecular weights, isoelectric points, and substrate specificities; however, their action patterns-considered in total-appear to be distinct from one another. MATERIALS AND METHODS Organism and culture conditions. Streptomyces sp. strain B-12-2 was isolated from soil (Vesima, Italy) by incubation at 45 C. It was cultivated in the medium of Morosoli et al. (22) at 45 C in Erlenmeyer flasks (50 or 250 ml) with 1% oat spelt or birch xylan as the sole carbon source. For enzyme production, a spore suspension prepared from a 1-week-old plate culture was primed by growth in Trypticase soy broth (Difco) at 45 C with shaking at 250 rpm for 24 h. A portion (5% [vol/vol]) of this culture was used to inoculate 500 ml of xylanase produc-

2 2610 ELEGIR ET AL. tion medium in a 2-liter Erlenmeyer flask. Xylanase production medium employs defined mineral salts with oat spelt xylan as the sole carbon source. Medium components were autoclaved separately from the xylan (13, 31). Maximum xylanase activity was generally detected after 48 h. Medium optimization. Several carbon sources were tested at 1% (wt/vol) each by using Morosoli's basal medium (22). The medium was inoculated with a spore suspension in sterile water (2 x 107 spores per ml), and xylanase activity was determined after 2 and 3 days. Shake flask fermentations were carried out at 45 C and 300 rpm. Four replicate flasks were used for each medium. Five inexpensive organic nitrogen sources (defatted soybean meal, rapeseed meal, sunflower meal, yellow pea meal, and corn steep liquor with 50% dry matter) were also tested at 0.3% (wt/vol) to replace yeast extract and Bacto Proteose Peptone in xylanase production medium. Experiments were performed as described for carbon sources. Substrates. Acetylglucuronoxylan was prepared from birchwood holocellulose by dimethyl sulfoxide extraction (14). Arabinoxylan from oat spelt, birch xylan, carboxymethyl cellulose, Remazol brilliant blue-xylan, p-nitrophenol (PNP)-,B-Dxyloside, and PNP-,B-D-cellobioside were obtained from Sigma. Xylooligosaccharides (X2 to xylopentaose [X5]) were from Megazyme (North Rocks, Australia). Unbleached kraft pulp from southern red oak was kindly supplied by Consolidated Paper Inc. (Wisconsin Rapids, Wis.). Enzymatic assays. Xylanase activity was routinely determined by measuring the release of reducing sugars from either 1% (wt/vol) alkali-soluble or water-soluble oat spelt xylan by the Somogyi modification of the Nelson method (29) as previously described (13). Crude or purified enzyme preparations were diluted appropriately to obtain maximal activity consistent with a linear response. For comparative purposes, xylanase activities against 1% (wt/vol) water-soluble and waterinsoluble xylan from either oat spelts or birch were measured. The dinitrosalicylic acid method of Miller (21) was used to assay xylanase activity during initial screening. Carboxymethyl cellulase activity was assayed by replacing 1% xylan with 1% low-viscosity carboxymethyl cellulose.,-xylosidase was assayed as described by Bachmann and McCarthy (1) by using 5 mm p-nitrophenyl-3-d-xylopyranoside in 50 mm potassium phosphate buffer at ph 7.0. The values of the Michaelis constant (Kin) and the maximum velocity (Vmax) were determined from a Lineweaver-Burk plot of assayed activities over a range of substrate concentrations. Suitably diluted xylanases were incubated with alkali-soluble birchwood or oat spelt xylan at concentrations ranging from 0.5 to 10 mg/ml under the assay conditions described above. Xylanase purification. The purification was performed according to a strategy similar to that of Grabski et al. (12). Xylanases from Streptomyces sp. strain B-12-2 were purified from crude extracellular broth by concentration, clarification, anion-exchange chromatography, and hydrophobic interaction chromatography. Chromatography was performed with a Pharmacia fast protein liquid chromatography system. Cells were harvested by centrifugation (6,000 x g, 30 min), and the supernatant solution was concentrated 10-fold by using a 10,000-molecular-weight cutoff membrane (Minitan; Millipore). The retentate ('100 ml) was treated with 2 to 3% (vol/vol) bioprocessing acid-1000 (Rohm and Haas Co.) to precipitate pigments and other contaminants (12). BPA-1000 is a cross-linked polymer that has strong basic quaternary ammonium functional groups on its surface. The clear supernatant solution was diafiltered with 10 mm bis-tris buffer (ph 6.5) in a stirred ultrafiltration cell (Amicon Division, Grace & Co., Danvers, Mass.) equipped with a YM-3 Amicon disc APPL. ENVIRON. MICROBIOL. membrane (3,000-molecular-weight cutoff). The retentate was centrifuged (10,000 x g, 10 min). The supernatant (50 to 60 mg of protein, -1,200 IU of xylanase) was applied to a Mono Q HR 10/10 (Pharmacia) column. Proteins were separated by using a discontinuous gradient of buffer A (10 mm bis-tris, ph 6.5) and buffer A plus 1.0 M NaCl (buffer B). The flow rate was 4 ml/min, and 4-ml fractions were collected. Elution was monitored at 280 nm by using a UV detector. Active fractions were pooled, concentrated, and diafiltered into 50 mm potassium phosphate (ph 7.0). Ammonium sulfate was added to a final concentration of 1.25 M. Samples were microcentrifuged at 10,000 x g for 5 min. The supernatant solution (20 mg of protein per load) was applied (0.5 ml/min) to a Phenyl Superose (Pharmacia) column and eluted with a continuous gradient of buffer C (50 mm potassium phosphate [ph 7.0], 1.25 M ammonium sulfate) and buffer D (50 mm potassium phosphate, ph 7.0). Elution was monitored at 280 nm, and 1-ml fractions were collected. Fractions were pooled, concentrated, and diafiltered into 50 mm potassium phosphate (ph 7.0) by using a Centricon-3 microcentrifuge diafiltration unit (Amicon). Purity of the samples was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) and isoelectric focusing. Electrophoretic analysis. SDS-PAGE was performed on a Phast system (Pharmacia, Piscataway, N.J.) by using 10 to 15% gradient polyacrylamide gels. A low-molecular-weight standard mixture (Bio-Rad) containing six proteins in the 14,400- to 97,400-molecular-weight range was used in order to determine the apparent molecular weights (Mrs) of the samples. Protein bands were stained with Coomassie brilliant blue G-250. Isoelectric focusing was performed on a Bromma 2117 Multiphor horizontal slab-gel system (Pharmacia LKB, Piscataway, N.J.) by using Servalyt Precotes (ph 3 to 10; Serva, Heidelberg, Germany), as recommended by the supplier. Protein bands were revealed by staining with Serva Blue W. Zymogram analysis of EX activity in isoelectric focusing gels was done according to a modification of the method of Biely et al. (7) by using Remazol brilliant blue-xylan (Sigma). Phosphate buffer (0.1 M, ph 7.0) was used instead of acetate, and the incubation period for overlaid gels was 15 min at 60 C. Effect of ph and temperature on xylanase activity. Xylanase activity was measured at phs from 4 to 10 under standard assay conditions with oat spelt xylan as the substrate. Buffers used were 100 mm sodium acetate (ph 4 to 5.5), 100 mm potassium phosphate (ph 6 to 8), and 100 mm glycine-naoh (ph 9 to 10). Enzyme activities were also assayed at temperatures from 30 to 70 C at ph 7.0. Effect of the temperature on xylanase stabilities. Each purified enzyme (0.5 IU) was incubated at 60 C in 100 mm potassium phosphate buffer at ph 7.0 in the absence of substrate. Aliquots were removed at different times between 0 and 3 h and immediately cooled on ice. Residual activity was assayed under standard conditions. Hydrolysis studies. The extent of hydrolysis of different xylans was evaluated by measuring the release of reducing sugars from 0.25% substrate solutions in 50 mm phosphate buffer (ph 7.0) at 40 C in the presence of 0.1 IU of purified enzyme in a final volume of 1.0 ml. Time course experiments were terminated when an increase in reducing sugars was no longer detected. Hydrolyses of aryl-o-d-xyloside (PNP-r3-Dxyloside) and aryl-p-d-cellobioside (PNP-1-D-cellobioside) were performed at 50 C by using 0.1 IU of purified enzyme and 5 mm substrate in a final volume of 1.0 ml of the abovedescribed buffer. The release of PNP groups was determined spectrophotometrically at 400 nm. Synergism among purified xylanases was determined by using alkali-soluble oat spelt

3 VOL. 60, I XYLANASES FROM STREPTOMYCES SP. STRAIN B TABLE 1. Physiochemical properties of EXs from Streptomyces sp. strain B-12-2 pl H Residual Temperature Residual Enzyme Mra optimum activity at optimum activity at otmm ph 9 (%) OC) 70-C(% Group 1 xyl 1a xyl1 b Group 2 xyl xyl xyl a Determined by SDS-PAGE. FIG. 1. SDS-PAGE analysis of purified EXs. Lanes 4 and 8, low-molecular-mass standards (Bio-Rad; in kda); lane 1, crude supernatant; lane 2, xyl la; lane 3, xyl lb; lane 5, xyl 2; lane 6, xyl 3; lane 7, xyl 4. xylan (1% [wt/vol]) or washed unbleached kraft pulp from southern red oak (Quercus falcata) (10% [wt/vol]). In the case of oat spelt xylan, hydrolyses were carried out for 24 h at 40 C; in the case of pulp, hydrolyses were carried out for 3 h at 60 C. Turbidity-clearing assays were performed at 50 C by using 0.5 IU of each enzyme preparation (as described by Somogyi [29]) plus 2 mg of water-insoluble xylan in a final volume of 1.0 ml of 50 mm phosphate buffer, ph 7.0. Turbidities (620 nm) were read directly for up to 4 h. Action pattern studies. The hydrolysis of xylans was performed as previously reported (24) by using soluble and insoluble xylan from oat spelt and birchwood. Hydrolysis products of xylooligosaccharides were determined by incubating 0.05 IU of purified EXs with 1 mg of X2, xylotriose (X3), xylotetraose (X4), or X5 in 1.0 ml of 50 mm phosphate buffer (ph 7.0) overnight at 50 C. Enzymes were inactivated by boiling for 10 min. Hydrolysates were analyzed by highperformance liquid chromatography with a CARBOPAC PAl column equipped with a pulsed amperometric detector (Dionex). The standard mixture contained 20,uM (each) xylooligosaccharide (X2 to X5), xylose, and L-arabinose. Other analyses. Protein concentration was measured by the method of Lowry et al. (19) with bovine serum albumin as a standard. Proteins were precipitated with trichloroacetic acid to eliminate interfering substances (4). Glycoproteins were detected on nitrocellulose blots with a glycan detection kit which is based on an enzyme immunoassay (Boehringer Mannheim Corporation, Indianapolis, Ind.). In this method the hydroxyl groups of the sugar moieties are oxidized to aldehyde and linked to digoxigenin. Subsequently the digoxigenin-labeled glycoproteins are detected by using a digoxigenin-specific antibody conjugated to alkaline phosphatase. Total carbohydrates were determined by the phenol-h2so4 method described by Dubois et al. (10). RESULTS Induction and optimization of xylanase production. Various carbon sources induced Streptomyces strain B-12-2 to produce extracellular EX. The average titer, 74 IU/ml (as assayed by the dinitrosalicylic acid method) or 20 IU/ml (as assayed by the Somogyi modification of the Nelson method), was obtained after 48 h of cultivation on 1% oat spelt xylan. Since oat spelt xylan is expensive, various crude xylan-rich substrates were evaluated for enzyme production. Several lignocellulosic materials induced xylanase to various extents. Among these, ground, sieved cornstalks and wheat straw (<200,um) were the best substrates. Xylanase activity could not be induced by cellulosic substrates (Avicel PH 102 and Solka Floc), glucose, or xylose. However, a small amount of xylanase activity was detected when Solka Floc SW 40 was used. This activity was probably due to contamination of the cellulose with xylan (27). Defatted rapeseed meal was a good replacement for both yeast extract and Bacto Peptone. When 2% ground and sieved cornstalks was used in combination with defatted rapeseed meal, xylanase productivity was comparable to that obtained with pure xylan. The level of cellulase activity in the crude preparation was very low (0.08 IU/ml) even when raw lignocellulosic materials were used as a carbon source. Purification. Xylanases were purified from supernatant solutions of cultures grown in oat spelt xylan. Activity staining after isoelectric focusing was performed by using Remazol brilliant blue-xylan. This analysis showed the presence of both acidic and basic xylanases in the crude broths. No significant differences in enzyme activity profiles were found when cultures grown with cornstalks as the substrate were used (data not shown). The supernatant solution of oat spelt xylan cultures exhibited a dark coloration which could be eliminated with BPA Following anion-exchange chromatography (Mono Q), xylanase activity was detected primarily in the unbound fraction and in the fractions eluting between 120 and 200 mm (i.e., xylanase 2 [xyl 2], xyl 3, and xyl 4). Fractions showing EX activity were further purified by hydrophobic interaction chromatography. The unbound fraction was resolved into two distinct xylanase peaks (xyl la and xyl lb). Following purification by hydrophobic interaction chromatography, the proteins were found to be more than 95% homogeneous by SDS-PAGE (Fig. 1). Total recovery levels were remarkably good. Approximately 55% of the original activity could be accounted for by summing the activities in fractions following Mono Q separation, and 45% was present following FIG. 2. Glycosylation analysis by enzyme immunoassay (glycan detection kit; Boehringer) of purified EXs. Lane 1, xyl la; lane 2, xyl lb; lane 3, xyl 2; lane 4, xyl 3; lane 5, xyl 4; lane 6, transferrin (positive control); lane 7, creatinase (negative control).

4 2612 ELEGIR ET AL. APPL. ENVIRON. MICROBIOL. TABLE 2. Kinetic properties of EXs from Streptomyces sp. strain B-12-2 Oat spelt xylan Birchwood xylan Enzyme Sp act (nmol Km Vmax Kcata Sp act (nmol K Vmax Kcata min-' mg-1) (mg/ml) (U mg-1) (s-') min-' mg-') (U mg-,) (U mg-) (s') xyl la xyl lb xyl xyl xyl a Molar turnover number. phenyl superose separation. xyl 3 accounted for approximately half of the total recovered activity. Characterization of the multiple endoxylanases. Table 1 summarizes the physiochemical characteristics of five purified isoenzymes. Xylanases la and lb (group 1) were low-molecular-weight enzymes with neutral and basic pis, respectively. Xylanases 2, 3, and 4 (group 2) had higher molecular weights and acidic pls. These latter enzymes, although they exhibited similar temperature and ph optima, retained greater activity at higher temperatures and more activity at alkaline ph than did xyl la and xyl lb (Table 1). xyl 2, xyl 3, and xyl 4 retained about 40% of their original activity after 3 h at 60 C (ph 7.0), while xyl la and xyl lb were almost completely inactivated after 1 h of incubation. The group 2 enzymes were glycosylated to varying degrees. xyl 2 is apparently strongly glycosylated, while xyl 3 and xyl 4 are only weakly glycosylated (Fig. 2). Group 1 enzymes are not glycosylated. Substrate affinities. The kinetic parameters were determined by using both arabinoxylan (oat spelt) and a lessersubstituted glucuronoxylan (birchwood xylan). All isoenzymes were more active on birch xylan (Table 2). We observed substrate inhibition at higher xylan concentrations (>2.5 mg/ ml)-especially with the acidic EXs-when we plotted the data by the Lineweaver-Burk method. We obtained the kinetic parameters by extrapolating from the linear region of the data set, and we did not include the rate data obtained at higher substrate concentrations. Effectors. We investigated the effects of metal ions (1 mm) and other agents on the activities of purified EXs (Table 3). The isoenzymes were not significantly influenced by Ca2" or Mg2+ but were totally inactivated by Hg2' and N-bromosuccinamide. Other compounds such as EDTA, SDS, and compounds of Cu2+ and Fe2+ showed only partial inhibition depending on the enzymes. p-hydroxymercuribenzoate affected only xyl lb, which was completely inactivated. The TABLE 3. Reagent Effects of various reagents on the activity of purified EXs Relative activity (%) of enzyme: xyl la xyl lb xyl 2 xyl 3 xyl 4 CaC MgCl CuC FeSO HgCl EDTA SDS p-hydroxymercuribenzoate Phenylmethylsulfonyl fluoride N-Bromosuccinamide different levels of inhibition by HgCl and p-hydroxymercuribenzoate suggest the presence of a relatively hydrophobic pocket at the catalytic site of xyl lb. Phenylmethylsulfonyl fluoride, a classical inhibitor of serine proteases, did not influence the activity of any enzyme, indicating that serine residues are not involved in the active site. Total inactivation due to Hg2" and N-bromosuccinamide has already been reported for xylanases of different origins (3, 9, 23) and, as suggested by Deshpande et al. (9), could be due to the presence of a tryptophan residue which may be conserved among all xylanases. Substrate specificity. None of the purified EXs could release reducing sugars from carboxymethyl cellulose, filter paper, avicel, or galactomannan (locust bean gum). Basic (group 1) EXs did not hydrolyze PNP-xyloside or PNP-cellobioside. In contrast, all the acidic (group 2) EXs were able to catalyze the hydrolysis of PNP-cellobioside as was already reported for xylanase A from Streptomyces lividans (5), but none of the xylanases from Streptomyces sp. strain B-12-2 could hydrolyze PNP-xyloside. Extent of hydrolysis. Acidic EXs (xyl 2, xyl 3, and xyl 4) achieved a higher degree of hydrolysis than basic EXs when acting on acetylglucuronoxylan, oat spelt arabinoxylan, or birch glucuronoxylan (Table 4). Among the acidic EXs no significant differences were found in extents of hydrolysis. In contrast, basic EXs showed different capacities to hydrolyze acetylxylan. In fact, xyl la released almost as much sugar from this substrate as the acidic EXs did. Synergism. The cooperative activity among the different EXs was investigated by using both oat spelt xylan and a more complex substrate (unbleached red oak pulp). When substratelimiting conditions were used (36), no synergism was detected among EXs in the degradation of the oat spelt arabinoxylan. Sugar release was additive and did not exceed the amount observed with group 2 (xyl 2, xyl 3, and xyl 4) enzymes. In contrast, experiments carried out with unbleached red oak pulp showed that the isoenzymes have different capacities to release sugars from this substrate (Table 5). The level of total sugars released from pulp by a combination of xyl la or xyl lb and xyl 3 increased 15 to 25% (as measured by the phenol- H2SO4 method) over that obtained with either enzyme individually, demonstrating that synergism between these two TABLE 4. Substrate Extent of hydrolysis of various xylans % of substrate hydrolyzed by enzyme: xyl la xyl lb xyl 2 xyl 3 xyl 4 Acetylglucuronoxylan Oat spelt Birchwood

5 VOL. 60, 1994 XYLANASES FROM STREPTOMYCES SP. STRAIN B TABLE 5. Sugar release from hardwood pulp by purified EXs from Streptomyces sp. strain B-12-2 Amt of reducing Amt of total Enzyme added sugars released sugars released Ratio (mg/g of oven- (mg/g of ovendried pulp) dried pulp) xyl laa xyl lb' xyl 3' xyl la + xyl lbb xyl la + xyl 3b xyl lb + xyl 3b a A 5-IU portion of purified enzyme was added per g of oven-dried pulp. b A 2.5-IU portion of enzyme was added per g of oven-dried pulp. C a- 'a..2 o xi a X4 B 5 El DP >5 groups of enzymes exists. The ratio of total sugars to reducing sugars revealed that group 1 (xyl la and xyl lb) tends to produce larger xylooligosaccharides. When a mixture of xyl la and xyl 3 or xyl lb and xyl 3 was used, the ratio of total sugars to reducing sugars decreased to the value obtained with only xyl 3, even though the level of total sugars released was greater. This fact suggests that xylooligosaccharides produced by xyl la and xyl lb can be further degraded by xyl 3 (Table 5). Solubilization. Group 1 enzymes cleared the turbidity of a suspension of insoluble oat spelt xylan approximately twice as rapidly as group 2 enzymes did (data not shown). Action patterns. We characterized the action patterns of each of the purified enzymes by using water-soluble and water-insoluble oat spelt arabinoxylan and birch xylan (Fig. 3 and 4). The experimental conditions used were selected to minimize the extent of hydrolysis and thus to enable the identification of any intermediates. Although none of the enzymes were able to release arabinose, remarkable differences were noted when different substrates were used. The hydrolysis of both soluble and insoluble oat spelt arabinoxylan resulted in larger amounts of xylooligosaccharides with DPs of >5, and X3 was the main product regardless of the enzyme used. X2 was the major product following hydrolysis of soluble birchwood xylan for all enzymes except xyl la. xyl la produced a greater amount of xylooligosaccharides with DPs of >5 than the other xylanases on soluble oat Substrate/Enzyme FIG. 4. Hydrolysis of insoluble birch (IB) xylan and insoluble oat (10) arabinoxylan by the five purified EXs (xyl la, xyl lb, xyl 2, xyl 3, and xyl 4). The relative quantities of oligosaccharide products are shown as the moles percent of the total soluble sugars recovered from each analysis. arabinoxylan or insoluble birch xylan. In contrast, xyl lb and xyl 4 tend to accumulate small amounts of these products when they act on soluble or insoluble birch xylan. In the case of insoluble birchwood xylan, most enzymes formed X3 as the major hydrolysis product along with X2, X4, and a minor amount of X.. However, xyl lb produced primarily X2 and no X5 from the soluble and insoluble birch substrates. Xylose represented only a small percentage of the total but was present as a product of all enzymes when they were acting on polymeric substrates. The action patterns of the EXs were further investigated by using purified xylooligosaccharides as substrates. None of the EXs showed activity on X2 or X3. They were able to cleave X4 and X5 but to different extents. Figure 5 shows the molar percentages of the products obtained from the hydrolyses of these latter substrates. Acidic (group 2) EXs completely hydrolyzed X5s producing mostly X2 and X3 but also X4 and small 11 0 U) 2U 0 a. O x1 a x GI DP:,5 CO)CO Co U)V CO cn c CJ o c co co co Substrate/Enzyme FIG. 3. Hydrolysis of soluble birch (SB) xylan and soluble oat (SO) arabinoxylan by the five purified EXs (xyl la, xyl lb, xyl 2, xyl 3, and xyl 4). The relative quantities of oligosaccharide products are shown as the moles percent of the total soluble sugars recovered from each analysis. C0 U 0 >x> S >btaeezm Substrate/Enzyme ElxG 0 X4 Ea x FIG. 5. Hydrolysis of xylooligosaccharides (xylotetraose, X4; xylopentaose, X5) by five purified EXs (xyl la, xyl lb, xyl 2, xyl 3, and xyl 4). The concentrations of the individual products (Xl, X29 X3, X4, and X5) are given as the moles percent of the total soluble sugars recovered from each analysis.

6 2614 ELEGIR ET AL. amounts of xylose. The hydrolysis of X4 and X5 by basic (group 1) EXs was much slower, and insignificant amounts of xylose were formed under the conditions used. The production of X3 without the formation of xylose from X4 suggested that the basic (group 1) enzymes, xyl la and xyl lb, carry out transglycosylation. DISCUSSION Analysis of the purified proteins by SDS-PAGE and isoelectric focusing indicated that the EXs produced by Streptomyces strain B-12-2 may be classified as low-mr, basic (group 1) and high-mr, acidic (group 2) xylanases (Table 1) as suggested by Wong et al. (37). None of the purified isoenzymes was able to release arabinose from oat spelt xylan. This is consistent with a higher affinity (lower Kin) for less-branched substrate (birchwood xylan) than for the highly substituted oat spelt arabinoxylan (Table 2). Nevertheless, in the case of xyl 2, specific activity and V..ax were higher when oat spelt xylan was used as the substrate. This suggests that a substituted substrate may be required for maximal activity of xyl 2. It is noteworthy that arabinoxylans (oat spelt and cornstalk) were the best inducers for overall xylanase activity. In general, group 2 enzymes showed higher affinities for their substrates than did group 1 enzymes. The xylanases isolated from Streptomyces strain B-12-2 are all endo-acting enzymes as demonstrated by their hydrolysis products (Fig. 3 through 5). However, the type of substrate used clearly influences their action patterns and the ratios of the main products. The greater amount of higher-dp xylooligosaccharides obtained from oat spelt xylan hydrolysis compared with that obtained from birchwood hydrolysis is probably due to the higher degree of branching of the former substrate. The results obtained from the hydrolysis of the X4 and X5 substrates allowed a clear classification of the isoenzymes based on their action patterns. We discerned differences in the ratios among the products with polymeric substrates, but quantitative analysis was possible only up to a DP of 5. The group 1 EXs (xyl la and xyl lb) appear to have a "more endo" action pattern in that they did not degrade X4 to a significant extent and did not fully hydrolyze X5. The group 2 EXs (xyl 2, xyl 3, and xyl 4) degraded X4 to various extents and completely hydrolyzed X5, indicating that they are able to act on lower-dp substrates. Moreover, the mode of action of the group 2 EXs seemed to be different in that they formed small amounts of xylose. This is probably due to a lower specificity in the bond-cleavage frequency of the acidic proteins of group 2 (33). The acidic group 2 EXs in the present study produced higher degrees of hydrolysis with all tested xylans (Table 4). The group 1 enzymes could be clearly distinguished from those of group 2 by their different action patterns on X4 and X5. In general, group 2 enzymes-having low pis, high Mrs, and lower Kms4formed lower-dp products from X4 and X5 (Fig. 5). In addition, basic (group 1) EXs clarified a suspension of insoluble xylan much more rapidly and to a greater extent than did acidic (group 2) EXs (data not shown). When group 2 enzymes hydrolyzed birchwood glucuronoxylan, their action patterns were not clearly distinguishable from those of group 1 enzymes. xyl lb showed the highest specific activity (Vmax) against both birch and oat spelt substrates, but xyl 3 showed the highest turnover number. When activities were normalized on the basis of production of reducing groups as determined by the Somogyi modification of the Nelson method, xyl lb was found to produce the most X2 from birch xylan; xyl la generally APPL. ENVIRON. MICROBIOL. exhibited the most extreme endo action pattern by producing oligosaccharides with higher DPs-especially on insoluble substrates. It had the lowest Vmax and the highest Km. Within group 1, xyl la and xyl lb were clearly different. xyl lb was inactivated byp-hydromercuribenzoate, and its catalytic turnover rate was approximately two to three times higher than that of xyl la. Moreover, xyl lb degraded X4 to a greater extent and produced equal amounts of X2 and X3. In contrast, xyl la produced mostly X3. No xylose was detected among the products. This suggests to us that a transglycosylation reaction occurred. In fact, the production of X3 from X4 is not possible without the formation of xylose. However, when we tested xyl la and xyl lb on X2 and X3, transglycosylation was not detected. Further research using end-labeled substrates would be necessary to resolve the question, but transglycosylation has been reported previously for EXs (33). The acidic xylanases (xyl 2, xyl 3, and xyl 4) were remarkably similar in most physiochemical and kinetic characteristics. xyl 2 could be distinguished from xyl 3 and xyl 4 by its high degree of glycosylation (Fig. 2), its lower Vmax value on oat spelt or birch xylan (Table 2), and its greater ability to degrade X4 (Fig. 5). xyl 4 was much less active at ph 9 and less thermostable. These three enzymes showed similar product profiles when used with polymeric xylan substrates, but xyl 4 exhibited a much lower capability to degrade X4. In previous work we classified the xylanase isoenzymes from Streptomyces roseiscleroticus as endo-1 and endo-2 depending on their hydrolysis products (24). Endo-1 xylanases tended to hydrolyze oat spelt xylan more completely, while endo-2 xylanases produced greater amounts of larger oligosaccharides. Our current results seem to partially confirm the previous data. However, with the enzyme complex from Streptomyces strain B-12-2, using linear birch xylan in place of branched oat spelt arabinoxylan leads to a very different pattern with respect to unhydrolyzed substrate. The endo-i action pattern could be attributed to a greater ability to bind shorter oligosaccharides and an ability to hydrolyze them (5). The different affinities in binding could be especially important in the degradation of complex substrates. Indeed, with increased substrate complexity, the accessibility of some moieties could be limited to xylanases having a smaller catalytic active site. In this respect we found a synergistic action between acidic EXs and basic EXs only when a complex substrate (unbleached red oak pulp) was used. There are very few reports dealing with the complete characterization of xylanase isoenzymes produced by streptomycetes. S. lividans is probably the best-characterized streptomycete in this respect (5). The results reported herein confirm the greater catalytic versatility of the high-molecular-mass EXs compared with the low-molecular-mass EXs when they react with the substrates tested here. The action patterns and the catalytic properties are similar among group 2 enzymes. This suggests that their multiplicity may be due to other properties. Only one acidic (group 2) enzyme (xyl A) is produced by S. lividans, and it appears to have properties somewhat different from those of the acidic xylanases reported here. In contrast to that of S. lividans, none of the Streptomyces strain B-12-2 acidic EXs is able to cleave X3 or PNP-xyloside. The shortest xylooligosaccharides hydrolyzed by both low-molecular-mass basic EXs and high-molecular-mass acidic EXs from B-12-2 are X4 and X5, respectively. 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