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Biochemical characterization and mechanism of action of a thermostable -glucosidase puried from Thermoascus aurantiacus
Neil J. PARRY*1, David E. BEEVER, Emyr OWEN, Isabel VANDENBERGHE, Jozef VAN BEEUMEN and Mahalingeshwara K. BHAT*2
*Food Materials Science Division, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, U.K., Department of Agriculture, The University of Reading, Earley Gate, P.O. Box 236, Reading RG6 6AT, U.K., and University of Ghent, Laboratory of Protein Biochemistry and Protein Engineering, K.L. Ledeganckstraat 35, B-9000, Ghent, Belgium
An extracellular -glucosidase from Thermoascus aurantiacus was puried to homogeneity by DEAE-Sepharose, Ultrogel AcA 44 and Mono-P column chromatography. The enzyme was a homotrimer, with a monomer molecular mass of 120 kDa ; only the trimer was optimally active at 80 mC and at pH 4.5. At 90 mC, the enzyme showed 70 % of its optimal activity. It was stable at pH 5.2 and at temperatures up to 70 mC for 48 h, but stability decreased above 70 mC and at pH values above and below 5.0. The enzyme hydrolysed aryl and alkyl --glucosides and cello-oligosaccharides, and was specic for substrates with a glycosidic linkage. The hydroxy groups at positions 2, 4 and 6 of a glucose residue at the non-reducing end of a disaccharide appeared to be essential for catalysis. The enzyme had the lowest Km towards p-nitrophenyl --glucoside (0.1137 mM) and the highest kcat towards cellobiose and ,-trehalose (17 052 minV"). It released one glucose unit at a time from the non-reducing end of cello-oligosaccharides, and the rate of hydrolysis decreased
with an increase in chain length. Glucose and --gluconolactone inhibited the -glucosidase competitively, with Ki values of 0.29 mM and 8.3 nM respectively, while methanol, ethanol and propan-2-ol activated the enzyme. The enzyme catalysed the synthesis of methyl, ethyl and propyl --glucosides in the presence of methanol, ethanol and propan-2-ol respectively with either glucose or cellobiose, although cellobiose was preferred. An acidic pH favoured hydrolysis and transglycosylation, but high concentrations of alcohols favoured the latter reaction. The stereochemistry of cellobiose hydrolysis revealed that glucosidase from T. aurantiacus is a retaining glycosidase, while N-terminal amino acid sequence alignment indicated that it is a member of glycoside hydrolase family 3.
Key words : mode of action, N-terminal sequencing, stereochemistry, substrate specicity, transglycosylation.
INTRODUCTION
Cellulose is the most abundant and renewable source of energy on Earth. Cellulose can be converted into soluble sugars by either acid or enzymic hydrolysis [1,2] ; the latter is preferred due to the high yields of desired products with fewer by-products, and in commercial situations it is considered to be economically favourable [2,3]. Micro-organisms capable of degrading crystalline cellulose extensively produce three main types of enzymes, namely endoglucanase (1,4---glucan glucanohydrolase ; EC 3.2.1.4), exoglucanase (1,4---glucan cellobiohydrolase ; EC 3.2.1.91) and -glucosidase (cellobiase or --glucoside glucohydrolase ; EC 3.2.1.21) [13]. Understanding the role and the mechanism of action of cellulase components during the solubilization of crystalline cellulose has been the subject of immense research for the past three decades [4,5]. Previous studies have demonstrated that endo- and exoglucanases act synergistically and promote the solubilization of crystalline cellulose into soluble sugars [4,5], while -glucosidase completes the hydrolysis by converting cellobiose and cellooligosaccharides into glucose [6]. -Glucosidase is also considered to be part of a cellulase system, since it stimulates the rate and extent of cellulose hydrolysis by relieving cellobiose-induced inhibition of endo- and exo-glucanases [7,8]. Furthermore, some
-glucosidases catalyse the synthesis of alkyl glycosides by transglycosylation and play an important role in improving the aroma of wines [9,10]. Thus a detailed study on the biochemical and catalytic properties, together with the mechanism of action, of -glucosidases is important in order to assess their full potential from both fundamental and applied standpoints. The thermophilic fungus Thermoascus aurantiacus produces relatively high levels of endoglucanase, exoglucanase and glucosidase when grown on a lignocellulosic carbon source such as corn cob [11]. Also, the cellulase components of this fungus are remarkably stable over a wide range of pH values and temperatures, and appear to be of tremendous commercial interest [12]. The present paper reports on the biochemical and catalytic properties of -glucosidase from T. aurantiacus, together with its transglycosylation activity, the stereochemistry of cellobiose hydrolysis and N-terminal amino acid sequencing.
EXPERIMENTAL Materials
T. aurantiacus IMI 216529 was from the Centre for Agriculture and Biosciences International (CABI), Egham, Surrey, U.K. All chemicals and reagents were from either Sigma or BDH, while
Abbreviations used : pNP, p-nitrophenol or p-nitrophenyl ; pNPGlc, p-nitrophenyl -D-glucoside ; oNPGlc, o-nitrophenyl -D-glucoside ; pNPXyl, pnitrophenyl -D-xyloside ; oNPXyl, o-nitrophenyl -D-xyloside ; pNPGlc2, p-nitrophenyl -D-cellobioside ; pNPGlc3, p-nitrophenyl -D-cellotrioside ; pNPGlc4, p-nitrophenyl -D-cellotetraoside ; MeUmb, 4-methylumbelliferone or 4-methylumbelliferyl ; MeUmbGlc, 4-methylumbelliferyl -D-glucoside ; MeUmbGlc2, 4-methylumbelliferyl -D-cellobioside ; MeUmbMan, 4-methylumbelliferyl -D-mannoside ; MeUmbLac, 4-methylumbelliferyl -D-lactoside. 1 Present address : Unilever Research, Colworth Laboratory, Colworth House, Sharnbrook, Bedford MK44 1LQ, U.K. 2 To whom correspondence should be addressed (e-mail Mahalingeshwara.Bhat!bbsrc.ac.uk). # 2001 Biochemical Society
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N. J. Parry and others Biochemical characterization of -glucosidase Homogeneity and molecular mass
These were determined by SDS\PAGE [15] and gel ltration on Sephacryl 300. The time course of denaturation was studied by SDS\PAGE using 25 g of -glucosidase mixed with SDStreatment buer and boiled for 1, 2, 4, 6, 8 and 10 min. The possible quaternary structure was analysed under reduced and non-reduced conditions by incubating 25 g of -glucosidase at 100 mC for 0, 4 and 8 min. Activity staining was performed by incubating the SDS\polyacrylamide gel with 50 ml of 0.26 M 4-methylumbelliferyl --glucoside (MeUmbGlc) after washing the gel with 50 mM sodium acetate buer at pH 4.0. The -glucosidase activity was visualized under UV light.
electrophoresis solutions and chromatography media were from Anachem and Pharmacia respectively.
Stability
Stability was determined at pH 2.8, 4.0, 5.2 and 6.8 and at temperatures of 50, 60, 70 and 80 mC for 48 h. A 300 l portion of -glucosidase (2 g protein\ml) was incubated at dierent pH values, and a 15 l sample was withdrawn after 0.5, 1, 2, 4, 8, 18, 28 and 48 h and assayed for -glucosidase using pNPGlc.
Substrate specicity
This was determined using pNP glycosides, disaccharides, oligosaccharides and polysaccharides. Activity on pNP glycosides was determined by measuring pNP release, as described above. All compounds were used at 0.5, 1, 1.5 and 2 mM, at pH 4.5 and 60 mC, and were incubated with 20 ng of -glucosidase for 5 min. Hydrolysis of disaccharides and oligosaccharides was monitored by the release of glucose [16] using same reaction conditions as for pNP glycosides, except with a 2 ml reaction volume. -Glucosidase activity on polysaccharides was determined using 0.52.5 mg of substrate, 20 ng of enzyme and 50 mM sodium acetate buer, pH 4.5, in a 2 ml reaction volume at 60 mC for 5 min, and by measuring the reducing sugars by either the Somogyi [17] or the dinitrosalicylic acid [18] method.
Assay of -glucosidase
A microtitre plate method was developed using p-nitrophenyl -glucoside (pNPGlc). A 25 l portion of culture ltrate or 20 ng (25 l) of pure enzyme was mixed with 25 l of 200 mM sodium acetate buer (pH 5.0) and 50 l of distilled water, and preincubated at 50 mC for 5 min. The reaction was initiated by adding 25 l of 10 mM pNPGlc and continued for 15 min at 50 mC, before being terminated by the addition of 100 l of glycine buer, pH 10.8. The colour developed was read at 405 nm, and translated to mol of p-nitrophenol (pNP) using a standard graph prepared under the same conditions. The unit of -glucosidase activity is expressed as the amount of enzyme required to release 1 mol of pNP per min under the above assay conditions.
Kinetics
Km, kcat and the Km\kcat ratio for the hydrolysis of pNPGlc, o-nitrophenyl --glucoside (oNPGlc), gentiobiose, sophorose, cellobiose, ,-trehalose and n-octyl --glucopyranoside by -glucosidase were determined using 04 mM substrate and by analysing the data by reciprocal plots using an Enztter program.
Determination of protein
Protein was determined using bicinchoninic acid [14]. Absorbance at 280 nm was used to monitor the protein content in the column fractions.
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Eects of alcohols
The eects of methanol, ethanol and propan-2-ol (0100 %, v\v) on the hydrolysis of pNPGlc were studied using the pNPGlc assay, with 20 ng of -glucosidase.
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products were determined by one-dimensional "H and "$C NMR spectroscopy. The reaction products were also analysed by HPLC. For this purpose, a 100 l sample was diluted 2-fold with acetonitrile, and 10 l of this was injected on to an NH -Spherisorb column # (15 cmi0.4 cm), and eluted using acetonitrile\water (8 : 2, v\v) V at a ow rate of 250 l:min ". The products were identied using standard and methyl glucosides.
Mode of action
This was studied using unsubstituted and pNP-substituted cellooligosaccharides. A 300 l reaction mixture containing 100 g of substrate, 20 ng of -glucosidase, 150 l of 20 mM sodium acetate buer (pH 5.0) and distilled water was incubated at 50 mC for 2 h. Samples of 25 l were withdrawn at dierent time intervals, mixed with 25 l of acetonitrile and analysed on an NH # Spherisorb column connected to a Gynkotech HPLC system using acetonitrile\water (7 : 3, v\v) as a mobile phase. Initial hydrolytic rates for pNP cello-oligosaccharides were determined relative to the release of glucose.
Transglycosylation activity
This was monitored using either glucose or cellobiose as a donor and methanol as an acceptor. A 300 l reaction mixture containing 100 ng of -glucosidase, 50 l of glucose or cellobiose (2 mg:mlV"), 25 or 50 % (v\v) methanol and distilled water was incubated at 50 mC. Samples of 25 l were withdrawn at dierent time intervals (0.2524 h) and mixed with 25 l of acetonitrile, and 20 l of this mixture was analysed on an NH -Spherisorb # column connected to a Gynkotech HPLC system, using acetonitrile\water (7 : 3, v\v) as the mobile phase. The data were analysed using a Gynkosoft program. The eect of pH (3.5, 5.0 and 6.5) on the synthesis of alkyl glycosides was studied using 40 and 80 % (v\v) methanol, ethanol or propan-2-ol. A 1 ml reaction mixture containing 100 ng of glucosidase, 1 mg of cellobiose, 0.25 ml of 100 mM buer of the required pH value, the required amount of alcohol and distilled water was incubated at 50 mC for 10 h. Samples of 25 l were taken at dierent time intervals and analysed by HPLC as described above.
Figure 1
(a) Denaturation of -glucosidase at 100 mC as a function of time ; (b) mobility of glucosidase, denatured under reduced and non-reduced conditions for dierent time periods ; (c) activity staining of -glucosidase, denatured under reduced and non-reduced conditions for dierent time periods. Standard protein markers in the order of increasing molecular mass are : carbonic anhydrase (31.0 kDa), lactate dehydrogenase (36.5 kDa), glutamate dehydrogenase (55.4 kDa), BSA (66.3 kDa), phosphorylase b (97.4 kDa), -galactosidase (116.3 kDa) and myosin (200 kDa). Arrows in (a) indicate (from bottom to top) the monomer, dimer and trimer of -glucosidase, and that in (c) indicates -glucosidase activity staining. # 2001 Biochemical Society
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Activity was assayed using 04 mM substrate, at pH 4.5 and 60 mC, with 20 ng of enzyme. The release of pNP, o-nitrophenol or glucose was measured as described in the Experimental section. The kinetic constants were determined by analysing the data by reciprocal plots using an Enztter program. All values are the averages of two separate experiments done in duplicate. Substrate pNPGlc oNPGlc n-Octyl -D-glucoside ,-Trehalose Cellobiose Gentiobiose Sophorose Km (mM) 0.1137 0.2500 0.2718 0.4340 0.6370 0.2095 1.3390 kcat (minV1) 14 532 14 112 14 088 17 052 17 052 8444 8184 10V3ikcat/Km (mMV1:minV1) 127.81 56.45 51.83 39.29 26.77 41.01 6.11
Figure 2
Activity was determined using pNPGlc, at pH values between 2.8 and 6.8 and at temperatures between 40 and 90 mC, as described in Experimental section. The activity at dierent temperatures is plotted as a function of pH. All values are the averages of two separate experiments done in duplicate.
was achieved by chromatofocusing on a Mono-P column in the pH region from 3.4 to 5.5. A total of 1 mg of -glucosidase was obtained with a specic activity of 190i10$ mol:minV":mg of proteinV", when measured at pH 4.5 and 60 mC.
Stability
-Glucosidase was stable up to 70 mC at pH 5.2. At 80 mC and pH 5.2, the enzyme lost 90 % of its original activity in 30 min. At all other pH and temperatures tested the enzyme was less stable, except at 50 mC and pH 6.8, under which conditions it was stable for up to 48 h.
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Activity on polysaccharides
The -glucosidase hydrolysed CM-cellulose and laminarin, but the specic activity was considerably lower than that with disaccharides. The enzyme showed the highest activity on
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Figure 3
For details, see the Experimental section. Panels (A), (B), (C) and (D) show the products released from cellobiose, cellotriose, cellotetraose and cellopentaose respectively, as a function of time. The solid and broken lines in (A) correspond to reaction products released in the presence of distilled water and in sodium acetate buer (pH 5.0) respectively. Each point is the mean of two separate experiments done in duplicate. Symbols : , glucose ; #, cellobiose ; $, cellotriose ; =, cellotetraose ; 4, cellopentaose.
at 20 % (v\v) ; at a concentration of 30 % (v\v), methanol and ethanol activated the enzyme by 30 % and 40 % respectively. The enzyme was more active in methanol than in ethanol and propan2-ol at concentrations higher than 60 %. Propan-2-ol at between 90 and 100 % (v\v) resulted in the greatest inhibition (50 %) of this enzyme.
Mode of action
This was studied using unsubstituted and pNP-substituted cellooligosaccharides.
Effects of alcohols
Methanol, ethanol and propan-2-ol activated the -glucosidase at concentrations below 50 % (v\v), and were inhibitory at higher concentrations. Propan-2-ol activated the enzyme 2-fold
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Figure 4
For details, see the Experimental section. Panels (A), (B), (C) and (D) show the products released from pNPGlc, pNPGlc2, pNPGlc3 and pNPGlc4 respectively as a function of time. Each point is the mean of two separate experiments done in duplicate. Symbols : , glucose ; , pNPGlc ; 5, pNPGlc2, 4, pNPGlc3 ; W, pNPGlc4.
level of glucose increased. Thus the -glucosidase appeared to hydrolyse cello-oligosaccharides in a stepwise manner, releasing one glucose unit at a time.
pNP cello-oligosaccharides from the non-reducing end, by releasing one glucose unit at a time. The rate of hydrolysis of pNP cello-oligosaccharides by glucosidase was determined by measuring the glucose released as a function of time. The enzyme released 1.7, 1.4, 0.2 and 0.1 g of glucose per h from pNPGlc, pNPGlc , pNPGlc and # $ pNPGlc respectively. Thus the rate of hydrolysis of pNP cello% oligosaccharides decreased markedly as the chain length increased.
Transglycosylation activity
This was demonstrated using either glucose or cellobiose as a donor and methanol as an acceptor. With 25 % (v\v) methanol, low levels of glucose coincided with an increase in methyl -glucoside formation over the 24 h incubation period (Figure 5A). With 50 % (v\v) methanol, the level of glucose uctuated, and only small amounts of methyl --glucoside were formed during the 24 h reaction period (Figure 5B). When cellobiose was used with 25 % (v\v) methanol, there was a rapid decrease in the cellobiose concentration, with a steady increase in the concentrations of methyl --glucoside and glucose in equimolar amounts (Figure 5C). With 50 % (v\v) methanol, as the cellobiose concentration decreased, an increase in the concentrations of both methyl --glucoside and glucose
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Figure 5
For details, see the Experimental section. Panels (A) and (B) show transglycosylation activity with glucose at 25 % and 50 % (v/v) methanol respectively. Panels (C) and (D) show transglycosylation activity with cellobiose at 25 % and 50 % (v/v) methanol respectively. Each point is the mean of two separate experiments done in duplicate. Symbols : , glucose ; $, cellobiose ; #, methyl -D-glucoside.
was noted, with the concentration of methyl --glucoside being higher than that of glucose (Figure 5D).
Effect of pH on the transglycosylation activity of -glucosidase with methanol, ethanol and propan-2-ol Methanol
The eect of pH (3.5, 5.0 and 6.5) on transglycosylation activity was studied using cellobiose with 40 and 80 % (v\v) methanol ; these concentrations activated and inhibited the hydrolytic activity of -glucosidase respectively. With 40 % (v\v) methanol, a rapid increase in the production of methyl --glucoside was noted at all pH values. The highest amount of methyl -glucoside (1200 g\ml) was produced at pH 5.0, and the lowest amount (200 g\ml) at pH 6.5. The amount of methyl -glucoside formed in the presence of 80 % (v\v) methanol was low
compared with that formed with 40 % (v\v) methanol at all pH values. The amounts of glucose released in the presence of 40 and 80 % (v\v) methanol were highest at pH 3.5 and 5.0, and lowest at pH 6.5. At pH 3.5 and 5.0, other transglycosylated products with retention times higher than that of glucose and lower than that of cellobiose were also formed. At pH 6.5, no such products were found with either 40 or 80 % (v\v) methanol. Nevertheless, 40 % (v\v) methanol and pH 5.0 were found to be ideal for the production of high levels of methyl --glucoside from cellobiose.
Ethanol
The eects of 30 % and 80 % (v\v) ethanol on the production of ethyl --glucoside were studied at pH 3.5, 5.0 and 6.5 using
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Figure 6
Time-course 1H NMR spectra of cellobiose hydrolysis by -glucosidase from T. aurantiacus in the presence of methanol
For details, see the Experimental section. T is reaction time (h). Notations : 2-Glc2, proton signal at the C-1 position of the terminal glucose residue of cellobiose in an -conformation (chemical shift 5.3 p.p.m.) ; 21-Glc2, proton signals at the C-1 positions of both glucose residues of cellobiose in the -conformation (chemical shifts of 4.62 and 4.5 p.p.m. respectively) ; 1-MeDGlc, proton signal at the C-1 position of methyl -D-glucoside in the -conformation (chemical shift 4.35 p.p.m.).
cellobiose as a donor. These two concentrations of ethanol respectively activated and inhibited -glucosidase activity. With 30 % (v\v) ethanol, the enzyme produced the highest amount of ethyl --glucoside (250 g\ml) at pH 5.0 ; at pH 3.5 and 6.5, the amounts formed were 125 and 200 g\ml respectively. The amount of glucose produced from cellobiose with 30 % (v\v) ethanol at all pH values was at least four times the amount of ethyl --glucoside formed. The -glucosidase produced the highest amount of ethyl --glucoside (400 g\ml) with 80 % (v\v) ethanol, at pH 3.5 ; however, at pH 5.0 ethyl --glucoside production was decreased to 50 g\ml, while at pH 6.5 the enzyme was totally inactive. In addition, the amounts of glucose produced at pH 3.5 and 5.0 with 80 % (v\v) ethanol were comparable with the amount of ethyl --glucoside formed. Thus the low concentrations of ethanol favoured the hydrolysis
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of cellobiose, while high concentrations promoted the transglycosylation reaction. Also, 80 % (v\v) ethanol and pH 3.5 were found to be ideal conditions for the production of high levels of ethyl --glucoside from cellobiose. The formation of other transglycosylated products was observed only at pH 3.5 and 5.0, at both ethanol concentrations.
Propan-2-ol
The eects of 20 and 80 % (v\v) propan-2-ol on propyl -glucoside production were studied at pH 3.5, 5.0 and 6.5 with cellobiose as the donor. At 20 % (v\v) propan-2-ol, the amount of propyl --glucoside formed was less than 40 g\ml at all three pH values, and the enzyme hydrolysed cellobiose rapidly irrespective of pH. With 80 % (v\v) propan-2-ol, the enzyme
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the C-1 position of glucose residues of cellobiose were observed. After 12 h, the depletion of the conguration of cellobiose, together with a rapid decrease in the -glucosidic bond signal, were observed. Also, a new signal corresponding to methyl -glucoside was detected. As the reaction progressed, the signal of the -glycosidic bond ( ) of the cellobiose decreased, while # " that of the -cellobiose ( ) did not change. In addition, the # analysis of samples by "$C NMR at times 0 h and 36 h demonstrated the production of methyl --glucoside only.
HPLC analysis
HPLC analysis of the and forms of methyl -glucoside showed only a partial separation with a double peak when the two forms were present in the reaction mixture. However, HPLC analysis of reaction samples taken at various time intervals contained a single peak, corresponding to methyl --glucoside. In addition, the levels of methyl --glucoside and glucose increased simultaneously as the level of cellobiose decreased (Figure 7). Figure 7 Quantitative HPLC analysis of cellobiose hydrolysis by glucosidase from T. aurantiacus in the presence of methanol as a function of time
For details, see the Experimental section. Each value is the mean of two separate experiments done in duplicate. Black, grey and white bars correspond to methyl -D-glucoside, glucose and cellobiose respectively.
N-terminal sequencing
The N-terminal sequence of 20 amino acid residues of T. aurantiacus -glucosidase showed a high degree of identity with the N-terminal sequences of ve other -glucosidases belonging to family 3 of glycoside hydrolases (Figure 8).
synthesized higher amounts of propyl --glucoside at pH 3.5 (250 g\ml) and pH 5.0 (150 g\ml). The amount of glucose released from cellobiose was only marginally higher than the amount of propyl --glucoside formed at both pH 3.5 and pH 5.0. The formation of other transglycosylated products was noted at pH 3.5 and pH 5.0. At pH 6.5 and in the presence of 80 % (v\v) propan-2-ol, the enzyme was inactive. Thus conditions of pH 3.5 and 80 % (v\v) propan-2-ol were found to be ideal for the synthesis of relatively high levels of propyl --glucoside from cellobiose.
DISCUSSION
An extracellular -glucosidase puried from T. aurantiacus was a homotrimer, with a monomeric molecular mass of 120 kDa. The high molecular mass of this -glucosidase is in agreement with those of many extracellular -glucosidases characterized from other fungal sources [8,2022]. Under non-reduced conditions, the enzyme retained the high-molecular-mass conformation even after boiling, but the protein was no longer active. Only the native protein was fully active in its non-reduced form. This suggested that the enzyme was structurally conferred to some degree by cysteine residues, and thus exhibited a low molecular mass under reduced conditions. Nevertheless, under such conditions and without boiling, the enzyme was present as a high-molecular-mass protein, but showed low activity. Hence both boiling and the presence of a reducing agent were essential to denature and separate the monomers of -glucosidase, which implied the presence of heat-labile cysteines. The temperature optimum (80 mC) of this -glucosidase was higher than that reported for low-molecular-mass -glucosidases
The time course of the hydrolysis of cellobiose by -glucosidase in the presence of methanol over 36 h was analysed by "H NMR ; the data are presented in Figure 6. Assignment of the proton signals to specic hydroxy groups and the glucosidic bond was performed according to the standard analysis, with cross-referencing to published Aldrich standards. At 0 h, the proton signals corresponding to and conformations at # # "
Figure 8
N-terminal sequence alignment of T. aurantiacus -glucosidase with -glucosidases of glycoside hydrolase family 3
Amino acid residues common to all -glucosidases are highlighted in bold. Database numbers and microbial sources of -glucosidases are given on the right and left sides respectively. # 2001 Biochemical Society
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from T. aurantiacus [23,24], as well as for other fungal cellulases [25,26]. Also, this -glucosidase showed greater thermostability than other -glucosidases from mesophilic and thermophilic fungi that have been studied [8,11,27,28]. Based on substrate specicity, -glucosidases can be classied as aryl --glucosidases (which hydrolyse exclusively aryl --glucosides), cellobiases (which hydrolyse only cellooligosaccharides) or broad-specicity -glucosidases (which hydrolyse both aryl --glucosides and cello-oligosaccharides). The -glucosidase from T. aurantiacus was active on aryl --glucosides as well as on cello-oligosaccharides, and was found to have a broad substrate specicity. Nevertheless, this enzyme exhibited highest activity towards pNPGlc. Plant et al. [29] suggested that the preference of -glucosidases for aryl glycosides is due to the high electrophilicity of the aglycone moiety, which enhances the stability of the ortho or para nitrophenoxide anion generated during the rst step of catalysis. A low activity obtained with the nitro group in the ortho position suggests steric hindrance with a hydroxy group at C-6. The importance of this hydroxy group was demonstrated by the very low activity of -glucosidase towards pNPXyl. Furthermore, the enzyme was more active on oNPXyl than on pNPXyl, indicating that the nitro group of oNPXyl was interacting favourably at the active site, since the steric hindrance with a hydroxy group at C6 was then no longer a problem. The importance for catalysis of hydroxy groups at the C-2 and C-4 positions of glucose, as well as the specicity of this -glucosidase for a 1-4-glucosidic bond, was evident from its negligible activity on pNP --galactoside, pNPmannose, pNPgalactosamine, pNPgentiobiose, pNP --lactoside, pNP 1-thio---glucoside and pNP 1-thio--galactoside. The specicity of T. aurantiacus -glucosidase towards linked diglycosides was, however, broad. The enzyme hydrolysed disaccharides with 1-1, 1-6 and 1-2 glucosidic bonds. The glucose derivatives arbutin, esculin and salicin were hydrolysed to a limited extent, indicating the occurrence of steric hindrance with these compounds, as observed by Fadda et al. [30]. A high activity on n-octyl --glucoside is probably due to favourable interactions between the alkyl group and a hydrophobic residue in the active site of the enzyme. The kinetics of the hydrolysis of preferred substrates showed that the enzyme was more specic for pNP glycosides than for normal disaccharides such as cellobiose and gentiobiose. Although the studies on mode of action suggested that this -glucosidase attacks the terminal glucosidic bond of cellooligosaccharides, it was not possible to demonstrate the precise mode of action using these substrates. However, using pNP cellooligosaccharides, it was demonstrated that this enzyme cleaved one glucose unit at a time from the non-reducing end. In contrast, the -glucosidase from Fusarium oxysporum hydrolysed MeUmb cello-oligosaccharides from both ends [28]. The rate of hydrolysis of cello-oligosaccharide by -glucosidase from T. aurantiacus decreased with increasing chain length, indicating its -glucosidase character. Also, a large decrease in hydrolysis rates from pNPGlc and pNPGlc to pNPGlc and pNPGlc demon# $ % strates the specicity of this enzyme for short-chain cellooligosaccharides. Determination of the transglycosylation activity of glucosidase in the presence of alcohols and with cellobiose as a donor provided some interesting results. With 25 % (v\v) methanol, the yields of glucose and methyl --glucoside were equimolar, indicating simultaneous hydrolysis of the glucosidic bond and transfer of glucose to the alkyl group. In the presence of 50 % (v\v) methanol, the partitioning of products occurred, with more methyl --glycoside formed than glucose. Methanol,
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