Báo cáo Y học: Isolation, enzymatic properties, and mode of action of an exo-1,3-b-glucanase from Trichoderma viride doc

Isolation, enzymatic properties, and mode of action of an exo-1,3-b-glucanase from Trichoderma viride Anna A. Kulminskaya 1 , Karl K. Thomsen 2, *, Konstantin A. Shabalin 1 , Irina A. Sidorenko 1 , Elena V. Eneyskaya 1 , Andrew N. Savel’ev 3 and Kirill N. Neustroev 1 1 Petersburg Nuclear Physics Institute, Russian Academy of Science, Russia; 2 Carlsberg Laboratory, Department of Physiology, Copenhagen, Denmark; 3 St Petersburg Technical University, Biophysics Department, Russia An exo-1,3-b-glucanase has been isolated from cultural filtrate of Trichoderma viride AZ36. The N-terminal sequence of the purified enzyme (m ¼ 61 ^ 1kDa) showed no significant homology to other known glucanases. The 1,3-b-glucanase displayed high activity against laminarins, curdlan, and 1,3-b-oligoglucosides, but acted slowly on 1,3-1,4-b-oligoglucosides. No significant activity was detected against high molecular mass 1,3-1,4-b- glucans. The enzyme carried out hydrolysis with inversion of the anomeric configuration. Whereas only glucose was released from the nonreducing terminus during hydrolysis of 1,3-b-oligoglucosides, transient accumulation of gentiobiose was observed during hydrolysis of laminarins. The gentiobiose was subsequently degraded to glucose. The Michaelis constants K m and V max have been determined for the hydrolysis of 1,3-b-oligoglucosides with degrees of polymerization ranging from 2 to 6. Based on these data, binding affinities for subsites were calculated. Substrate binding site contained at least five binding sites for sugar residues. Keywords:exo-1;3-b-glucanase; Trichoderma viride; anomerity of hydrolysis. Enzymes hydrolyzing 1,3-b- D-glucans occur in a variety of organisms [1]. 1,3-b-Glucanases hydrolyze the O-glyco- sidic linkages of 1,3-b-linked glucans and are classified according to their mode of action. The exo-1,3-b-glucanases (EC 3.2.1.58) sequentially release glucose residues from the nonreducing terminus of a substrate while the endo-1,3-b- glucanases (EC 3.2.1.39) are capable of cleaving internal 1,3-b-linkages at random sites along the polysaccharide chain, releasing short oligosaccharides. 1,3-b-Glucanases have been isolated from bacteria [2], yeast and fungi [3–5], plants [6,7], and marine organisms [8,9]. It has been suggested that plant 1,3-b-glucanases may protect the germinating grain against pathogen attack [10]. Microbial 1,3-b-glucanases play an essential role in development and differentiation of saprophyte and mycoparasite cultures [11–13] while 1,3-b-glucanases from the filamentous fungi Coprinas seem to be involved in the process of stipe elongation [14]. In Saccharomyces cerevisiae the produc- tion of exo-1,3-b-glucanases is growth-associated and cell- cycle regulated, suggesting that their activities are required at specific stages during morphogenesis [15,16]. Most organisms synthesize multiple 1,3-b-glucanases rather than a single enzyme [17] and complete degradation of 1,3-b- glucans by fungi is often accomplished by synergistic action of endo- and exo-glucanases [18]. These enzymes have received attention in many fields of science and biotech- nology because many cultures of microorganisms widely used in industry produce 1,3-b-glucanases, which are essen- tial for cell-cycle functions [19,20] and due to their increasing importance in modification of b-glucans for pharmaceutical purposes [21,22]. Despite a number of reports describing exo-1,3-b-glucanases from different sources [19,20,23– 25], the subsite structure of the substrate binding site as well as the affinity and the number of subsites have not been analyzed for most of these enzymes. The present study describes the isolation and characterization of an exo-1,3-b- glucanase from the filamentous fungus Trichoderma viride AZ36. The subsite structure was evaluated by steady-state kinetics using 1,3-b-oligoglucosides with a different degree of polymerization. The mode of action and specificity as well as stereoselectivity of hydrolysis catalyzed by the exo- 1,3-b-glucanase were studied by NMR spectroscopy. MATERIALS AND METHODS Substrates Laminarin from Laminaria digitata, barley 1,3-1,4-b-glucan, lichenan from Cetraria islandica, gentiobiose, cellulose, Correspondence to K. N. Neustroev, Petersburg Nuclear Physics Institute, Gatchina, St Petersburg, 188350, Russia. Fax: 1 781271 32303, Tel.: 1 781271 32014, E-mail: neustk@omrb.pnpi.spb.ru Enzymes: 1,3-b-glucanase, 1,3-b- D-glucan glucanohydrolase, laminarinase (3.2.1.39); exo-b-1,3-glucanase, 1,3-b- D-glucan glucohydrolase (EC 3.2.1.58); a-glucosidase (EC 3.2.1.20); glucoamylase (EC 3.2.1.3); b- D-glucosidase, b-D-glucoside glucohydrolase (EC 3.2.1.21). Definition: G4G4G3G, b- D-Glcp-(1!4)-b-D-Glcp-(1 !4)-b- D-Glcp-(1 !3)-b-D-Glcp; G4G3G, b-D-Glcp-(1!4)-b-D-Glcp- (1!3)-b- D-Glcp; G3G3G3G3G, b-D-Glcp-(1 !3)-b-D-Glcp-(1!3)- b- D-Glcp-(1!3)-b-D-Glcp-b-D-Glcp-(1 !3)-b-D-Glcp; G3G3G3G, b- D-Glcp-(1!3)-b-D-Glcp-(1 !3)-b-D-Glcp-(1 !3)-b-D-Glcp; G3G3G, b- D-Glcp-(1!3)-b-D-Glcp-(1 !3)-b-D-Glcp; G3G, b- D-Glcp-(1!3)-b-D-Glcp; G6G, gentiobiose. *Present address: Fussingsvej 8, I, DK-8700 Horsens, Denmark. (Received 3 July 2001, revised 20 September 2001, accepted 27 September 2001) Abbreviations: DP, degree of polymerization; PHMB, p-hydroxymercuribenzoic acid sodium salt. Eur. J. Biochem. 268, 6123–6131 (2001) q FEBS 2001 cellobiose, p-nitrophenyl cellotrioside, p-nitrophenyl b- D-glucopyranoside were from Sigma Chemical Co. (St Louis, MO, USA). Curdlan from Alcaligenes faecalis and pustulan from Umbilicaria popullosa were kindly donated by I. J.Goldstein (Michigan University, USA). Laminarin from Laminaria cichorioides was kindly donated by A. V. Kir’yanov (Institute of Organic Chemistry, Moscow, Russia). p-Hydroxymercuribenzoic acid sodium salt (PHMB) was from Merck (Germany). Mixed linkage oligosaccharides (for oligosaccharide definitions, see footnotes): G4G4G3G and G4G3G were produced by digestion of barley glucan with a 1,3-1,4-b- glucanase and purified [26]. The G3G3G3G3G3G, G3G3G3G3G, and G3G3G3G were prepared by formic acid hydrolysis of curdlan followed by purification of 1,3-b-oligoglucosides [26,27]. G3G and G3G3G were produced by digestion of laminarin with a commercially available laminarinase from Trichoderma sp. purchased from Sigma Chemical Co. Laminarin (80 mg) was dissolved in 2 mL of 20 m M sodium acetate buffer, pH 5.0, and digested using < 0.005 units of the enzyme per mg of laminarin. Incubation was at 37 8Cfor 60 min. The reaction mixture was fractionated on a Sephadex G-25 (Fine) column equilibrated in water, separating oligosaccharides of degree of polymerization (DP) 2–6 from the high molecular mass fraction. Following freeze drying the 1,3-b-oligoglucosides were fractionated on a TSK NH 2 -60 column (5 mm, 4.6 Â 250 mm) from Pharmacia Biotech (Uppsala, Sweden) in 80% acetonitrile in water (v/v). The purity of b-oligoglucosides was analyzed by TLC and 1 H and 13 C NMR spectroscopy as described below. Published values for 1 Hand 13 C chemical shifts of b-oligo- saccharides with different DP and linkage types [26,28–30] were used for structure determination of the obtained compounds. General methods SDS/PAGE was carried out according to Laemmli [31] and isoelectric focusing was on Servalyt PRECOATES plates 3–10 (Serva Electrophoresis GmbH, Heidelberg, Germany). Protein concentration was measured following the Lowry procedure using BSA as a standard [32]. 1 H-NMR spectra and 13 C-NMR spectra were recorded with an AMX-500 Bruker spectrometer. Prior to NMR analysis laminarin, buffer components, and the enzyme were freeze-dried twice from D 2 O. The measurements were made in 20 mM sodium phosphate buffer (pD 6.0) at room temperature and 50 m M 4,4-dimethyl-4-silapentane sodium sulfonate was used as an internal standard in 0.5 mL 99.8% D 2 O. The solvent reson- ance was presaturated for 0.5 s with a decoupler operating at 24 dB in the CW mode. Data were acquired after a 758 pulse into 16K points, with a spectral width of 10 kHz and 2053 scans, including first five dummy scans. The spectra were Lorentz-broadened by 1 Hz. The 4,4-dimethyl-4-silapen- tane sodium sulfonate signal was used for adjustment of phase and amplitude parameters in order to obtain correct differential spectra. Oligosaccharide substrates and products of enzymatic hydrolysis were analyzed by TLC on Kieselgel 60 plates from Merck (Darmstadt, Germany) with a mobile phase of ethyl acetate/acetic acid/water (2 : 1 : 1). Plates were developed at room temperature, air dried and sprayed with 5% H 2 SO 4 in 1-propanol followed by incubation at 120 8C for 8 min. N-Terminal amino-acid sequencing was conducted using the Edman degradation and phenylisothiocyanate amino-acid analysis. The Procise Protein Sequencing System (Applied Biosystems, Foster City, California 94404) was employed. Growth conditions The T. viride AZ36 from Petersburg Nuclear Physics Insti- tute strain collection was grown at 30 ^ 1 8C for 72 h in a 10-L fermentor with constant stirring. The growth medium contained (g per L) KH 2 PO 4 , 1; NaNO 3 , 1.5; (NH 4 ) 2 SO 4 , 1.5; MgSO 4 Â 7H 2 ), 0.5; wheat bran, 40. Purification of the exo-1,3-b-glucanase All steps were carried out at 4 8C. Mycelium was removed by centrifugation (3000 g, 30 min), and the supernatant was concentrated 20-fold by use of hollow fibers with a nominal molecular mass limit of 25 kDa (‘Kirishi’, Kirishi, Russia). During the process the buffer was changed to 20 m M Tris/HCl, pH 7.5 (buffer A). The crude 1,3-b-glucanase preparation was loaded on a DEAE-Sephadex column (50 Â 200 mm) equilibrated with buffer A and bound pro- tein was eluted with 1 M NaCl in the same buffer. Following concentration to 25 mL on an Amicon PM-30 membrane and dialysis against buffer A, the fraction was loaded onto a TSK column (21.5 Â 150 mm) equilibrated with buffer A. Elution was performed by using a linear 0–0.5 M NaCl gradient in buffer A. Fractions containing exo-1,3-b- glucanase activity were concentrated to 4 mL on an Amicon PM-30 membrane, dialyzed against 20 m M Tris/HCl, pH 7.2 (buffer B) and chromatographed on a Mono Q HR (5/5) (Pharmacia, Sweden). Bound protein was eluted by applying a linear 0–0.5 M NaCl gradient in buffer B. Finally, the exo-1,3-b-glucanase preparation was concen- trated to 3 mL using an Amicon PM-30 membrane, dialysed against 20 m M sodium acetate buffer, pH 5.0 (buffer C). Saturated (NH 4 ) 2 SO 4 in buffer C was added to a final concentration of 1.7 M before applying the enzyme prepar- ation onto a phenyl-Superose HR (5/5) column (Pharmacia, Sweden) equilibrated with 1.7 M (NH 4 ) 2 SO 4 in buffer C. Bound protein was eluted by using a linear gradient (0–100%) of 20 m M sodium acetate buffer, pH 5.0, and fractions with the exo-1,3-b-glucanase activity were dialyzed against buffer C, then against deionized water, and freeze dried. Enzyme assays Exo-1,3-b-glucanase activity was analyzed by measuring the amount of glucose released from laminarin. Standard assays (0.25 mL) were in 20 m M sodium acetate buffer, pH 4.5, and contained 0.5 mg of laminarin and crude or purified enzyme extract corresponding to at least 0.2 mg of pure exo-1,3-b-glucanase. Incubation was at 37 8C for 10–30 min and the reaction was stopped by boiling for 5 min. Glucose formation was measured by the glucose oxidase method [33]. Alternatively, enzyme activity was evaluated by measuring the formation of reducing sugars according to the Somogyi-Nelson method [34]. One unit of the enzyme activity produced 1 mmol of glucose : min 21 at 6124 A. A. Kulminskaya et al. (Eur. J. Biochem. 268) q FEBS 2001 pH 4.5, 37 8C, with laminarin as substrate. b-Glucosidase activity was measured using p-nitrophenyl b- D-glucopyr- anoside as substrate according to [35]. The influence of pH and temperature on activity of the enzyme was studied by the glucose oxidase method. The effect of pH on activity was measured at 37 8C for 10 min in the range pH 3–9 in 100 m M sodium phosphate/citrate buffers. Reaction mixture: 400 mL of buffer and 10 mLof enzyme (0.01 U) in 2 m M sodium acetate buffer, pH 4.5, was mixed with 50 mL of laminarin (20 mg : mL 21 in water) or with 20 mL of G3G3G3G (40 m M in water). The influence of pH on stability of the enzyme was studied by incubating samples of the enzyme at 20 8C for 16 h in 100 m M sodium phosphate/citrate buffers ranging from pH 3 to pH 9, followed by measuring the residual activity of the enzyme using standard conditions. The effect of temperature on activity was measured at pH 4.5 in 100 m M sodium citrate buffer over a temperature range of 25–80 8C. The reaction mixture (0.4 mL) con- taining 1 mg of laminarin was preincubated at various temperatures in the range mentioned above. Then 50 mL (0.005 U) of enzyme solution in the same buffer was added and incubated for 10 min. The reaction was stopped by addition of 1 mL of copper reagent (Somogyi reagent) and boiling for 5 min. Liberated glucose was measured by the Somogyi-Nelson method [34]. To study stability in relation to temperature, the purified exo-1,3-b-glucanase was incubated at various temperatures for 10 min, in 100 m M sodium citrate buffer, pH 4.5. After cooling, the residual activity was determined by the glucose-oxidase method as described above. Effect of metal ions on activity was determined in 50 m M sodium acetate buffer, pH 4.5, at 37 8C after 15 min of incubation with different ions. Kinetic parameters TheMichaelis–MentenconstantsK m and V max were deter- mined from the Lineweaver–Burk representation of data obtained by measuring the initial rate of substrate hydrolysis. The purified exo-1,3-b-glucanase (0.01–1 U) was incubated with different substrates in 0.5 mL of 20 m M sodium acetate buffer, pH 4.5. After the reaction was stopped by boiling, liberated glucose was measured as described above. Concentrations of laminarins from 1 to 0.05 mg : mL 21 , curdlan concentrations from 15 to 1 mg : mL 21 ,and b-oligoglucoside concentrations from 12 to 0.1 m M were used. Enzyme specificity The specificity of the exo-1,3-b-glucanase was studied by analyzing the products of hydrolysis of different substrates. Laminarins with different degrees of ramification as well as curdlan, pustulan, and b-oligoglucosides differing in DP and linkage type were used. Hydrolysis was stopped after appropriate time intervals by boiling for 5 min. The resulting products were analysed qualitatively by TLC and quantitatively by HPLC using a TSK-NH 2 -60 column (5 mm, 4.6 Â 250 mm) from Pharmacia Biotech (Uppsala, Sweden) in 80% acetonitrile in water (v/v) with refracto- metric detection. Glucose and 1,3-b-oligoglucosides of DP 2–6 were used as standards. To investigate activity of the exo-1,3-b-glucanase towards different soluble and insoluble b-glucans, substrates at a concentration of 5 mg : mL 21 were incubated with 2–3 U of enzyme at 37 8C for various time intervals. For studies of the mode of action in the hydroysis of laminarins, intermediate products were fractionated by HPLC on a Dextro-Pak cartrige column (8 Â 10 mm) WATO85650 from Millipore-Waters (Bedford, MA, USA) using isocratic elution with water. The purified products of hydrolysis were analyzed by 1 H and 13 C NMR spectroscopy as described previously [30]. Determination of the stereochemical course of hydrolysis The enzyme was dissolved in 20 m M sodium phosphate buffer, pH 6.0, made up in D 2 O. The reaction mixture of 0.6 mL contained 20 mg : mL 21 laminarin in sodium phosphate buffer. All NMR measurements were performed at ambient temperature (<17 8C). After accumulation of the initial spectrum, 80 U of the exo-1,3-b-glucanase was added and the reaction mixture was brought to 37 8C within 5 min. The stereochemical course of hydrolysis was monitored by collecting spectra at 6, 20, and 40 min after addition of the enzyme. Direct 1 H NMR analysis of the exo-1,3-b-glucanase action on 1,3-b-oligoglucosides was carried out at a substrate concentration of <10 m M in 0.6 mL of the same buffer. RESULTS AND DISCUSSION Enzyme purification The T. viride AZ36 used in this study is a producer of mainly exo-1,3-b-glucanase. An exo-1,3-b-glucanase was purified 25-fold from concentrated culture supernatants of T. viride in four chromatographic steps. About 40% of the initial activity was recovered and the purified enzyme Table 1. Purification of the exo-1,3-b-glucanase. Purification step Volume (mL) Total protein (mg) Total activity (U) Specific activity (U : mg 21 ) Purification (fold) Yield (%) Cultural liquid 1000 250 630 2.5 1 100 Ultrafiltration 50 125 570 4.6 1.8 90 DEAE-Sephadex chromatography 50 70 440 6.3 2.5 70 DEAE-5PW chromatography 25 15 315 21 8.4 50 Mono Q chromatography 4 6 283 47 18.8 45 Phenyl Superose chromatography 3 4 252 63 25.2 40 q FEBS 2001 Exo-1,3-b-glucanase from T. viride (Eur. J. Biochem. 268) 6125 had a specific activity of 63 U : mg 21 (Table 1). In SDS/ PAGE analysis the protein appeared as a single polypeptide with an apparent molecular mass of 61 ^ 1 kDa (Fig. 1). Small amounts of endo-1,3-b-glucanase were separated from the main fraction during the first steps of purification. During the purification process, the exo-1,3-b-glucanase and b-glucosidase activities were efficiently separated, and the purified exo-1,3-b-glucanase preparation possessed less than 0.1% b-glucosidase activity. Analytical gel filtration on a Superose 12 column demonstrated that the exo-1,3-b-glucanase had an apparent molecular mass of 60 ^ 5 kDa suggesting that the enzyme was a monomer (data not shown). Multiple forms of Trichoderma harzianum exo-1,3-b- glucanases with a wide range of molecular masses up to 35 kDa have been reported [13]. The multiplicity of secreted forms of the Trichoderma enzymes might be caused by postsecretory proteolysis by acid proteases also secreted by the fungus [36,37]. The strain T. viride AZ36 employed in the present study has a lower level of secreted proteases than the other Trichoderma species (data not shown), which is likely to be one of the reasons that a homogenous exo-1,3-b-glucanase preparation could be obtained. General properties The purified exo-1,3-b-glucanase displayed optimal activity in hydrolysis of laminarin from L. digitata and 1,3-b- oligoglucosides at pH 4.5. The optimal temperature was 55 8C. Activity decreased rapidly at temperatures above 60 8C. At room temperature the enzyme was stable for 24 h in the pH range 3.5 to 7.5. The isolelectric point was 4.2 ^ 0.05 and the N-terminal amino-acid sequence was: AVDDFAPNTKQTPIALNNVLL. There are a number of reports providing amino-acid sequence data for fungal exo- 1,3-b-glucanases such as Cochliobolus carbonum [38], yeast C. albicans [39], Saccharomyces cerevisiae [40], Yarrowia lipolitica [41] but none of these display significant homology in their N-terminal amino-acid sequence to the exo-1,3-b-glucanase described in here. Whereas some divalent metal ions as well as EDTA had a slight inhibitory effect on the exo-1,3-b-glucanase, MnSO 4 and MnCl 2 increased the activity towards laminarin (Table 2). Similar dependence of the enzymic activity on metal ions was reported for endo-1,6-a-mannanase from a soil bacterium [42]. Based on the observation that neither Hg 21 nor PHMB caused any inhibition of enzyme activity, it is assumed that no essential SH-groups are involved as functional groups. Most exo-1,3-b-glucanases of fungal origin typically display optimal enzymatic activity in the pH range between 4 and 6 [19,23,43]. Similarly the enzyme from T. viride AZ36 displayed optimal activity at pH 4.5. The stability in relation to pH and temperature was also similar to that of other fungal exo- and endo-glucanases [11], and like most exo-1,3-b-glucanases that have been studied, the enzyme described here was not inhibited by heavy metal ions and PHMB. The mode of action of the exo-1,3-b-glucanase was analyzed employing 1,3-b-oligoglucosides with DP 3–6 and curdlan from A. faecalis [26] as substrates. Using TLC and HPLC analyses it was demonstrated that glucose was the only product formed during the enzyme-catalysed reaction. 1 H NMR techniques was also used to study the mode of action of purified exo-1,3-b-glucanase on curdlan and reduced G3G3G3G3G. A signal at d ¼ 5.248 p.p.m. corresponding to reducing ends [26] did not increase during hydrolysis. This observation unequivocally demonstrates the absence of endo-type hydrolysis. Consequently, this glucanase should be classified as an exo-glucohydrolase (Fig. 2). The enzyme displayed only very low activity against 1,3-1,4-b-glucans, lichenan, and barley glucan, and Fig. 1. SDS/PAGE of the exo-1,3-b-glucanase from T. viride. Lane 1, purified exo-1,3-b-glucanase from T. viride;lane2,protein standards – phosphorylase a (98 kDa), BSA (67 kDa), ovalbumin (45 kDa), a-chymotrypsin (25 kDa). Table 2. Effect of metal ions, PHMB, and EDTA on the exo-1,3-b- glucanase activity towards laminarin. Effector (1 m M) Relative activity (%) None 100 Ca 21 97 Mn 21 136 Mg 21 89 Zn 21 83 Hg 21 86 PHMB 90 EDTA 81 6126 A. A. Kulminskaya et al. (Eur. J. Biochem. 268) q FEBS 2001 no activity was detected against 1,4-b-glucan, 1,4-b-oligo- glucosides, pustullan (1,6-b-glucan), cellulose, and p-nitro- phenyl b-glucoside. Whereas the K m values for mixed linked 1,3-1,4-b-oligoglucosides and the corresponding 1,3-b-oligoglucosides were similar (Tables 3 and 4), the V max value for hydrolysis of G4G3G was <260-fold lower than for G3G3G. For the corresponding tetraoses the difference was < 4000-fold. The mode of action of the T. viride exo-1,3-b-glucanase in hydrolysis of 1,3-b-oligoglucosides and curdlan is typical of fungal exo-1,3-b-glucanases [17,19,23]. The enzyme also displayed some activity towards 1,3-1,4-b-oligoglucosides. In contrast to barley exo-1,3-b-glucanase [44] and exo- 1,3-b-glucanase from C. albicans [45], the enzyme was inactive against cellooligosaccharides and p-nitrophenyl b-glucopyranoside. Subsite stucture of the active center For determination of the active center subsite structure the kinetic parameters K m and V max of the hydrolytic reaction were measured using 1,3-b-oligoglucosides of DP 2 –6 as substrates. The initial rate of hydrolysis was measured as a function of substrate concentration. For all 1,3-b-oligo- glucosides the reaction follows Michaelis–Menten kinetics. Values for K m , V max and V max /K m for different substrates shown in Table 4 indicate that oligoglucosides with lower DP have lower V max . Because this enzyme exclusively displays exo-activity in the hydrolysis of 1,3-b-oligogluco- sides, the subsite theory of Hiromi [46,47] was employed for construction of a subsite map for the estimation of affinities and number of subsites in the active center of the enzyme. According to this theory the kinetic parameters can be expressed in a unified way in terms of subsite affinities A i of m subsites and the intrinsic rate constant K int for glucosyl bond cleavage [46]. As shown in [47], the subsite affinities for sites A 225 of the exo-1,3-b-glucanase according to Davies et al. numbering system for exo-glycanases [48]can be calculated from the Eqn (1): lnðV max /K m Þ n 11 2 lnðV max /K m Þ n ¼ 2ðA n 11 /RTÞð1Þ where K m and V max are the kinetic parameters of the exo-1,3- b-glucanase-catalyzed hydrolysis of 1,3-b-oligoglucosides of DP 3–6. Assuming that there is only one nonproductive complex A 21 can be obtained from the Hiromi dependence of 1/V max on exp[–A n11 /(RT )] [47]. The value for A 1 can be estimated from the Eqn (2) based on preliminary calculated affinities A 2 –A 5 : ðK m Þn ¼ 55 exp ÿ X nÿ1 i¼ÿ1 A i RT 1 exp ÿ X n i¼1 A i RT 1 … ! ð2Þ The calculated affinities A 21 and A 1 –A 5 are represented in Fig. 3A. The diagram shows that negative values for binding energy were at subsites 21to1 4, so that the exo-1,3-b- glucanase from T. viride strain AZ336 possesses at least five binding subsites of monomeric units at its binding site. A Fig. 2. HPLC analysis of the hydrolytic products resulting from the exo-1,3-b-glucanase action on curdlan. Separation was performed on a Lichrosorb NH 2 -60 column in 80% acetonitrile in water. Elution rate was 1 mL : min 21 : 1, glucose. Inset: elution profile for a Lichrosorb NH 2 -60 column separation of a mixture of 1,3-b-oligoglucosides: 1, glucose; 2, laminariobiose; 3, laminariotriose; 4, laminariotetraose; 5, laminariopentaose. Table 3. Relative rate of hydrolysis of b -glucans, and oligosaccharides by the exo-1,3-b-glucanase. Substrate K m (mM) 10 3 : V max (mmol : min 21 : mg 21 ) Laminarin from L. digitata 0.12 mg : mL 21 17 Laminarin from Laminaria cichorioides 0.14 mg : mL 21 38 Curdlan 12 mg : mL 21 8.3 G4G3G 6.06 0.021 G4G4G3G 0.9 0.0034 G6G 2.5 0.185 q FEBS 2001 Exo-1,3-b-glucanase from T. viride (Eur. J. Biochem. 268) 6127 schematic model of the enzyme active centre is represented in Fig. 3B. The Hiromi subsite theory [46,47] is based on two cardinal assumptions: firstly, each subsite has its own proper affinity for a glucose residue of the oligoglucoside substrate and there is no interaction between subsites; secondly, the subsite affinities are additive. The important thing is regularity of a value for the intrinsic rate constant K int . Accordingly, the substrate binding affinity becomes the sum of the affinities for glucose residues of the substrate accessible for binding; the distinction of V max (k cat ) values is conditioned by different quota of productive complexes for various substrates. Employing this model kinetic analysis of the hydrolysis of oligosaccharides with different DP is an effective tool for characterization of subsite structure of the active center of exo-glucanases and glucosidases. The Hiromi subsite theory has initially been applied for glycoamylase [47]. Further it was employed in the determination of binding affinities for various exo-glycosidases: a-glucosidase [49], b-glucosidases [50,51] and glucoamylases [47,52]. The results obtained for a-glucosidase [49] as well as for b-glucosidases from Aspergillus niger [50] and Candida wickerhamii [51] demonstrated that for these enzymes the active center affinity for substrates decreases as the DP of the substrate increases: consequently, A 21 to A 3 have posi- tive values. A b-glucosidase from germinated barley has been reported to have negative affinity at site 12 and positive at the rest [53]. This is in contrast to the results obtained for the exo-1,3-b-glucanase described here where the substrate affinity increases with increasing DP of the substrate. Thus, for this enzyme the affinities, A 21 to A 4 , have negative values (Fig. 3A). Negative values for A i of enzymes where V max increases with increasing DP of oligosaccharide substrates is typical for glucoamylases from several sources [47,52] and has also been described for an exo-1,3-b-glucanase from C. albicans [45]. The latter enzyme has similar structure of subsites at its active center where A 21 has a negative value for affinity energy in contrast to values for all other sites that are positive. Stereochemical course of action 1 H NMR provides a direct method for determining the stereochemical course of hydrolysis catalyzed by glyca- nases. Chemical shifts and coupling constants of the anomeric protons in a- and b-glycosides as well as in the product hemiacetals are distinct and readily observed. When sufficient exo-1,3-b-glucanase was used to complete hydrolysis within 5 min the initially formed anomer accu- mulated in sufficient amounts to permit detection before mutarotation had occured to a significant extent. NMR spectra reflecting the time course of laminarin hydrolysis catalyzed by exo-1,3-b-glucanase from T. viride AZ36 are presented in Fig. 4A. Spectrum 1 shows the anomeric proton region of laminarin in buffer. The large resonance peak at d 4.7 corresponds to the H 2 O protons. Spectra 2 and 3 were recorded at different time intervals after addition of the Fig. 3. Subsite structure structure of the exo-1,3-b-glucanase (A) and schematic model of the exo-1,3-b-glucanase-binding site (B). (A) The subsite affinities A i are represented by the histogram. (B) Schematic model of the exo-1,3-b-glucanase-binding site: X, nonreducing terminus glucosyl residue; W, glucosyl residues; veritcal arrow, catalytic amino acids. Table 4. Kinetic parameters of the hydrolysis of laminarioligosaccarides effected by the exo-1,3-b-glucanase. Substrate (G n ) K m (mM) 10 3 : V max (mmol : min 21 : mg 21 ) 10 3 : (V max /K m ) (min 21 : mg 21 ) ln (V max /K m ) G3G 16.5 0.36 0.02 210.73 G3G3G 5.0 5.5 1.1 26.80 G3G3G3G 1.9 14 7.4 24.91 G3G3G3G3G 2.0 16 8.0 24.83 G3G3G3G3G3G 2.1 18 8.6 24.85 6128 A. A. Kulminskaya et al. (Eur. J. Biochem. 268) q FEBS 2001 enzyme. The resonance at d 5.23 (d, J ¼ 3.6 Hz) comes from the equatorial anomeric proton of a- D-glucose. In the latter spectra (Figs 4A, 2 and 3), a resonance peak appears at d 4.65 (d, J ¼ 7.9 Hz) from the axial anomeric proton of b- D-glucose which is the product of mutarotation of the initially formed a-glucose. Mutarotation was considered complete when the anomeric ratio (< 42% a, < 58% b) had been established (about 40 min). Similar results were obtained by 1 H NMR analysis of the hydrolysis of reduced laminariotetraose (Fig. 4B). The data unequivocally demon- strate that enzymatic hydrolysis proceeds with inversion of the anomeric configuration, presumably as a result of a single displacement reaction [54]. Moreover, as seen from Fig. 4B, the enzyme released only glucose from 1,3-b- oligoglucosides attacking the substrates in an exo-pattern action. The anomer specificity of hydrolysis has been thoroughly investigated for products of exo-1,3-b-glucanases from Basidiomycete aphyllophorales [19] and barley malt 1,3- and 1,3-,1,4-b-glucanases [20]. The first enzyme showed inversion of the anomer configuration in contrast to the barley enzymes. Transglycosylating activity has been demonstrated for 1,3-b-glucanases performing hydrolysis with retention of anomeric configuration [20,26,45], but as expected no such activity was observed for the inverting exo-1,3-b-glucanase from T. viride AZ36. Mode of action in the hydrolysis of laminarins The mode of action of the exo-1,3-b-glucanase was studied using laminarin from L. digitata with a ratio of 1,3-b-and 1,6-b-linkages of 1 : 7 [55] and laminarin from L. cichor- ioides, which has fewer side 1,6-b-Glc residues (<1 : 17) [56]. The exo-1,3-b-glucanase exhibited a higher rate of hydrolysis of the glucan with the lower degree of ramification (Table 3). Kinetic parameters of the exo-1,3-b-glucanase in hydrolysis of curdlan from A. faecalis, an essentially unbranched 1,3-b-glucan, are presented in Table 3. For this substrate, K m is 10-fold higher and the rate of hydrolysis is < twofold lower than for laminarin from L. digitata. The mode of action of the exo-1,3-b-glucanase on laminarin from L. digitata was studied qualitatively by TLC and quantitatively by HPLC. HPLC analysis of the reaction mixture revealed that glucose and gentiobiose (6-O-b- D-glucopyranosyl-b-D-glucose) were liberated during hydrolysis (Fig. 5). The latter was isolated from the reaction mixture and characterized by 1 H and 13 C NMR spectroscopy [30]. Thus, the enzyme, although typically exo- in its mode of attack, can initiate endo-type cleavage of 1,3-b- bonds adjacent to 1,6-b-linkages. At subsequent stages of hydrolysis, gentiobiose was degraded to glucose (Fig. 5). Gentiobiose appeared to be a real substrate for the exo- 1,3-b-glucanase. The kinetic parameters are presented in Table 3. The values for K m and V max for hydrolysis of G6G are similar to those of G3G (Tables 3 and 4). Therefore, it may be assumed that the enzyme has a mixed mode of action towards laminarins and that it is capable of degrading such branched b-glucans completely to glucose without syner- gistic action with a b-glucosidase. Intermediate accumu- lation of gentiobiose was also observed during hydrolysis of scleroglucan by the exo-1,3-b-glucanase from Basidio- mycetes aphyllophorales [19]. Similar action on laminarin from Eisenia bicyclis was reported for the exo-1,3-b- glucanase from Basidiomycetes sp. QM 806 which released glucose, gentiobiose and gentiotriose [30], and the intra- cellular b-1,3-glucan hydrolase from Euglena gracilis [57]. In conclusion, it should be noted that Trichoderma species secrete a complex mixture of carbohydrases that are able Fig. 5. Kinetics of product formation during hydrolysis of laminarin from L. digitata. X, glucose; B, gentiobiose. Laminarin from L. digitata (4 mg) was incubated with exo-1,3-b-glucanase (5 U) at 37 8Cin20m M sodium acetate buffer, pH 4.5. After 25 min (left) and after 10 h (right) of the incubation the reaction was stopped by boiling. The products were separated on a TSK-NH 2 -60 column (5 mm, 4.6 Â 250 mm) with elution rate 0.8 mL : min 21 . Fig. 4. Time-course of a- and b-glucose formation. (A) 1 H NMR spectra of anomeric proton region of glucose showing the stereochemical course of the hydrolysis of laminarin from L. digitata by the exo-1,3-b- glucanase. 1, 1 H NMR spectrum of laminarin; 2, the spectrum of the reaction mixture at an intermediate stage of hydrolysis (2 min); 3, after 40 min when mutarotation was close to completion. (B) Kinetics of a/b-glucose formation during the hydrolysis of reduced laminariote- traose. X, a-glucose; B, b-glucose; O, sum of a- and b-glucose. q FEBS 2001 Exo-1,3-b-glucanase from T. viride (Eur. J. Biochem. 268) 6129 to affect components of the fungal cell wall such as chitin and 1,3-b-glucan [58,59]. Therefore, lytic enzymes including endo- and exo-1,3-b-glucanases, exo-b-N acetyl- glucosaminidases and chitinases may be important in protecting plants against pathogenes. Future detailed investigation of different enzyme systems, exo-1,3-b- glucanases, in particular, is essential for evaluating the effectiveness of such multienzyme complex. ACKNOWLEGMENTS We thank Professor Irwin J. Goldstein, Michigan University, for his helpful assistance in sequencing the protein. The research was made possible by a grant 00-04-48878 from the Russian Foundation for Basic Research. REFERENCES 1. Stone, B.A. & Clarke, A.E. (1992) Chemistry and Biology of (1 !3)- b -Glucans, La Trobe University Press, La Trobe, Melbourne, Australia. 2. Krah, M., Misselwitz, R., Politz, O., Thomsen, K.K., Welfle, H. & Borriss, R. (1998) The laminarinase from thermophilic eubac- terium Rhodothermus marinus – conformation, stability, and iden- tification of active site carboxylic residues by site-directed mutagenesis. Eur. J. Biochem. 257, 101–111. 3. Hien, N.H. & Fleet, G.H. (1983) Separation and characterization of six (1–3)-b-glucanases from Saccharomyces cerevisiae. J. Bacteriol. 156, 1204–1213. 4. Reichelt, B.Y. & Fleet, G.H. (1981) Isolation, properties, function and regulation of endo-(1–3)-b-glucanases in Schizosaccharo- myces pombe. J. Bacteriol. 147, 1085–1094. 5. Jirku, V., Kraxnerova, B. & Krumphanzl, V. (1980) The extra- cellular system of b-1,3-glucanases of Altermaria tenuissima and Aspergillus versicolor. Folia Microbiol. 25, 24–31. 6. Hrmova, M. & Fincher, G.B. (1993) Purification and properties of three (1–3)-b- D-glucanase isoenzymes from young leaves of barley (Hordeum vulgare ). Biochem. J. 289, 453–461. 7. Høj, P.B., Hartman, D.J., Morrice, N.A., Doan, D.N.P. & Fincher, G.B. (1989) Purification of (1–3)-beta-glucan endohydrolase isoenzyme II from germinated barley and determination of its primary structure from a cDNA clone. Plant Mol. Biol. 13, 31–42. 8. Lepagnol-Descamps, V., Richard, C., Lahaye, M., Potin, P., Yvin, J C. & Kloareg, B. (1998) Purification and determination of the action pattern of Haliotis tuberculata laminarinase. Carbohydr. Res. 310, 283–289. 9. Seki, N., Muta, T., Oda, T., Iwaki, D., Kuma, K., Miyata, T. & Iwanaga, S. (1994) Horseshoe crab (1,3)-beta- D-glucan-sensitive coagulation factor G. A serine protease zymogen heterodimer with similarities to beta-glucan-binding proteins. J. Biol. Chem. 269, 1370–1374. 10. Skriver, K., Olsen, F.L., Rogers, J.C. & Mundy, J. (1991) cis-acting DNA elements responsive to gibberellin and its antagonist abscisic acid. Proc. Natl Acad. Sci. USA 88, 7266–7270. 11. Fontane, T., Hartland, R.P., Beauvais, A., Diaquin, M. & Latge, J P. (1997) Purification and characterization of an endo-1,3-b- glucanase from Aspergillus fumigatus. J. Biochem. 243, 315–321. 12. Cabib, E., Roberts, R. & Bowers, B. (1982) Synthesis of the yeast cell wall and its regulation. Annu. Rev. Biochem. 51, 763–793. 13. Vazquez-Garciduenas, S., Leal-Morales, C.A. & Herrera-Estrella, A. (1998) Analysis of the b-1,3-glucanolytic system of the biocontrol agent Trichoderma harzianum. Appl. Environ. Micro- biol. 64, 1442–1446. 14. Kamada, T., Fujii, T., Nakagawa, T. & Takenaru, T. (1985) Changes in (1–3)-b-glucanase activities during stipe elongation in Coprinus cinereus. Curr. Microbiol. 12, 257–260. 15. Jiang, B., Ram, A.F.R., Sheraton, J., Klis, F.M. & Bussey, H. (1995) Regulation of the cell wall b-glucan assembly: PTC1 negatively affects PBS2. Action in a pathway that includes modulation of EXG1 transcription. Mol. Gen. Genet. 248, 260–269. 16. del Rey, F.J., Garcia Acha, I. & Nombela, C. (1979) The regulation of b-glucanase synthesis in fungi and yeast. J. Gen. Microbiol. 110, 83–89. 17. Jones, D., Gordon, A.H. & Bacon, J.S.D. (1973) Co-operative action by endo-exo-b-1,3-glucanases from parasitic fungi in the degradation of cell wall glucans of Sclerotina Sclerotiorum. Biochem. J. 140, 47–55. 18. Larriba, G., Andaluz, E., Cueva, R. & Basco, R.D. (1995) Molecular biology of yeast exoglucanases. FEMS Microbiol. Lett. 125, 121–126. 19. Tsujisaka, Y., Hamada, N. & Kobayashi, R. (1981) Purification and some properties of an exo-1,3-b-glucanase from Basidiomycete species. Agric. Biol. Chem. 45, 1201–1208. 20. Chen, L., Sadek, M., Stone, B.A., Brownlee, R.T.C., Fincher, G.B. & Høj, P.B. (1995) Stereochemical course of glucan hydrolysis by barley (1–3)- and (1–3,1–4)-b-glucanases. Biochim. Biophys. Acta 1253, 112–116. 21. Wasser, S.P. & Weis, A.L. (1999) Therapeutic effects of substances occuring in higher Basidiomycetes mushrooms: a modern perspective. Crit. Rev. Immunol. 19, 65–96. 22. Ooi, V.E. & Liu, F. (2000) Immunomodulation and anti-cancer activity of polysaccharide-protein complexes. Curr. Med. Chem. 7, 715–729. 23. Bamforth, C.W. (1980) The adaptability, purification and properties of exo-b1,3-glucanase from the fungus Trichoderma reesei. Biochem. J. 191, 863–866. 24. del Rey, F., Villa, T.G., Santos, T., Garcia-Acha, I. & Nombela, C. (1982) Purification and partial characterization of a new, sporu- lation specific, exo-b-glucanase from Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 105, 1347–1353. 25. Pitson, S.M., Seviour, R.J., McDougall, B.M., Woodward, J.R. & Stone, B.A. (1995) Purification and characterization of three extracellular (1!3)-b- D-glucan glucohydrolases from the filamen- tous fungus Acremonium persicinum. Biochem. J. 308, 733–741. 26. Petersen, B.O., Krah, M., Duus, J.O. & Thomsen, K.K. (2000) A transglycosylating 1,3(4)-beta-glucanase from Rhodothermus marinus NMR analysis of enzyme reactions. Eur. J. Biochem. 267, 361–369. 27. Broberg, A., Thomsen, K.K. & Duus, J.Ø. (2000) Application of nano-probe NMR for structure determination of low nanomole amounts of arabinoxylan oligosaccharides fractionated by analyti- cal HPAEC-PAD. Carbohydr. Res. 328, 375–382. 28. Bock, K., Pedersen, C. & Pedersen, H. (1984) Carbon-13 nuclear magentic resonance data for oligosaccharides. Adv. Carbohydr. Chem. Biochem. 42, 193–225. 29. Bock, K., Duus, J.Ø., Norman, B. & Pedersen, S. (1991) Assign- ment of structures to oligosaccharides produced by enzymic degradation of a b- D-glucan from barley by 1 H- and 13 C-NMR spectroscopy. Carbohydr. Res. 211, 219–233. 30. Nanjo, F., Usui, T. & Suzuki, T. (1984) Mode of action of an exo-b- 1,3-D- glucanase on the laminarin from Eisenia bicyclis. Agric. Biol. Chem. 48, 1523–1532. 31. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. 32. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. & Randall, R.J. (1951) Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. 33. Bruss, M.L. & Black, A.L. (1978) Enzymatic microdetermination of glycogen. Anal. Biochem. 84, 309–312. 34. Somogyi, M. (1952) Notes on sugar determination. J. Biol. Chem. 195, 19–23. 35. He, S. & Withers, S.G. (1997) Assignment of sweet almond 6130 A. A. Kulminskaya et al. (Eur. J. Biochem. 268) q FEBS 2001 b-glucosidase as a family 1 glycosidase and identification of its active site nucleophile. J. Biol. Chem. 272, 24864–24867. 36. Eneyskaya, E.V., Kulminskaya, A.A., Savel’ev, A.N., Savel’eva, N.V., Shabalin, K.A. & Neustroev, K.N. (1999) Acid protease from Trichoderma reesei: limited proteolysis of fungal carbohydrases. Appl. Microbiol. Biotechnol. 52, 226–231. 37. Mischak, H., Hofer, F., Messner, R., Weissinger, E., Hayn, M., Tomme, P., Esterbauer, H., Ku ¨ chler, E., Claeyssens, M. & Kubicek, C.P. (1989) Monoclonal antibodies against different domains of cellobiohydrolase I and II from Trichoderma reesei. Biochim. Biophys. Acta 990, 1–7. 38. Schaeffer, H.J., Leykam, J. & Walton, J.D. (1994) Cloning and targeted gene disruption of EXG1, encoding exo-beta 1,3-gluca- nase, in the phytopathogenic fungus Cochliobolus Carbonum. Appl. Environ. Microbiol. 60, 594–598. 39. Chambers, R.S., Broughton, M.J., Cannon, R.D., Carne, A., Emerson, G.W. & Sullivan, P.A. (1993) An exo-beta-(1,3)- glucanase of Candida albicans: purification of the enzyme and molecular cloning of the gene. J. Gen. Microbiol. 139, 325–334. 40. Klebl, F. & Tanner, W. (1989) Molecular cloning of a cell wall exo- beta-1,3-glucanase from Saccharomyces cerevisiae. J. Bacteriol. 171, 6259–6264. 41. Esteban, P.F., Casare ´ gola, S., Vazquez de Alana, C.R. & Del Rey, F. (1999) Cloning and characterization of the EXG1 gene from the yeast Yarrowia lipolitica. Yeast. 15, 1631–1644. 42. Nakajima, T., Maitra, S.K. & Ballou, C.E. (1976) An endo- a1!6- D-mannanase from a soil bacterium. Purification, proper- ties, and mode of action. J. Biol. Chem. 251, 174–181. 43. Mackenzie, L.F., Brooke, G.S., Cutfield, J.F., Sullivan, P.A. & Withers, S.G. (1997) Identification of Glu-330 as the catalytic nucleophile of Candida albicans exo-b-(1,3)-glucanase. J. Biol. Chem. 272, 3161–3167. 44. Hrmova, M., Harvey, A.J., Wang, J., Shirley, N.J., Jones, G.P., Stone, B.A., Hoj, P.B. & Fincher, G.B. (1996) Barley b- D-glucan exohydrolases with b- D-glucosidase activity. Purification, charac- terization and determination of primary structure from a cDNA clone. J. Biol. Chem. 271, 5277–5286. 45. Subbs, H.J., Brasch, D.J., Emers on, G.W. & Sullivan, P.A. (1999) Hydrolase and transferase activities of the b-1,3-exoglucanase of Candida albicans. Eur. J. Biochem. 263, 889 –895. 46. Hiromi, K. (1970) Interpretation of dependency of rate parameters on the degree of polymerization of substrate in enzyme-catalyzed reactions. Evaluation of subsite affinities of exo-enzyme. Biochem. Biophys. Res. Commun. 40, 1–6. 47. Hiromi, K., Nitta, Y., Numata, C. & Ono, S. (1973) Subsite affinities of glucoamylase: examination of the validity of the subsite theory. Biochim. Biophys. Acta 302, 362–375. 48. Davies, G.J., Wilson, K.S. & Henrissat, B. (1997) Nomenclature for sugar-binding subsites in glycosyl hydrolases. Biochem. J. 321, 557–559. 49. Kimura, A., Takewaki, S., Matsui, H., Kubota, M. & Chiba, S. (1990) Allosteric properties, substrate specificity, and subsite affinities of honeybee a-glucosidase I. J. Biochem. 107, 762–768. 50. Yazaki, T., Ohnishi, M., Rokushika, S. & Okada, G. (1997) Subsite structure of the b-glucosidase from Aspergillus niger, evaluated by steady-state kinetics with cello-oligosaccharides as substrates. Carbohydr. Res. 298, 51–57. 51. Freer, S.N. (1993) Kinetic characterization of a beta-glucosidase from a yeast, Candida wickerhamii. J. Biol. Chem. 268, 9337–9342. 52. Savelyev, A.N., Sergeev, V.R. & Firsov, L.M. (1982) Study of the active center of glucoamylase from Aspergillus awamori. Biochemstry (Moscow) 47, 390–397. 53. Hrmova, M., McGregor, E.A., Biely, P., Stewart, R.J. & Fincher, G.B. (1998) Substrate binding and catalytic mechanism of a barley b- D-glucosidase/(1,4)-b-D-glucan exohydrolase. J. Biol. Chem. 273, 11134–11143. 54. Withers, S.G. & Aebersold, R. (1995) Approaches to labeling and identification of active site residues in glycosidases. Protein Sci. 4, 361–372. 55. Chizhov, A.O., Dell, A., Morris, H.R., Reason, A.J., Haslam, S.M., McDowell, R.A., Chizhov, O.S. & Usov, A.I. (1998) Structural analysis of laminarans by MALDI and FAB mass spectrometry. Carbohydr. Res. 310, 203–210. 56. Elyakova, L.A. & Zvyagintseva, T.N. (1974) A study of the laminarins of some Far-Eastern, brown seaweeds. Carbohydr Res. 34, 241–248. 57. Barras, D.R. & Stone, B.A. (1969) Beta-1,3-glucan hydrolases from Euglena gracilis. I. The nature of the hydrolases. Biochim. Biophys. Acta. 191, 329 –341. 58. de la Cruz, J., Rey, M., Lora, J.M., Hidalgo-Gallero, A., Dominguez, F., Pintor-Toro, J.A., Llobell, A. & Benitez, T. (1993) Carbon source control on b-glucanases, chitobiase and chitinase from Trichoderma harzianum. Arch. Microbiol. 159, 1–7. 59. Lorito, M., Hayes, C.K., Di Pietro, A., Woo, S.L. & Harman, G.E. (1994) Purification, characterization, and synergetic activity of a glucan 1,3-b-glucosidase and an N-acetyl-b-glucosaminidase from Trichoderma harzianum. Phytopathology 84, 398–405. q FEBS 2001 Exo-1,3-b-glucanase from T. viride (Eur. J. Biochem. 268) 6131 . Isolation, enzymatic properties, and mode of action of an exo-1,3-b-glucanase from Trichoderma viride Anna A. Kulminskaya 1 , Karl K. Thomsen 2, *,. 4000-fold. The mode of action of the T. viride exo-1,3-b-glucanase in hydrolysis of 1,3-b-oligoglucosides and curdlan is typical of fungal exo-1,3-b-glucanases