Enzymatic Properties and Intracellular Localization of the Novel Trichoderma reesei β-Glucosidase BGLII (Cel1A)

This paper describes the characterization of an intracellular β-glucosidase enzyme BGLII (Cel1a) and its gene (bgl2) from the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). The expression pattern of bgl2 is similar to that of other cellulase genes known from this fungus, and the gene would appear to be under the control of carbon catabolite repression mediated by the cre1 gene. The BGLII protein was produced in Escherichia coli, and its enzymatic properties were analyzed. It was shown to be a specific β-glucosidase, having no β-galactosidase side activity. It hydrolyzed both cellotriose and cellotetraose. BGLII exhibited transglycosylation activity, producing mainly cellotriose from cellobiose and sophorose and cellobiose from glucose. Antibodies raised against BGLII showed the presence of the enzyme in T. reesei cell lysates but not in the culture supernatant. Activity measurements and Western blot analysis of T. reesei strains expressing bgl2 from a constitutive promoter further confirmed the intracellular localization of this β-glucosidase.     

Molecular cloning and expression of the novel fungal beta-glucosidase genes from Humicola grisea and Trichoderma reesei

A novel fungal beta-glucosidase gene (bgl4) and its homologue (bgl2) were cloned from the cellulolytic fungi Humicola grisea and Trichoderma reesei, respectively. The deduced amino acid sequences of H. grisea BGL4 and T. reesei BGL2 comprise 476 and 466 amino acids, respectively, and share 73.1% identity. These beta-glucosidases show significant homology to plant beta-glucosidases belonging to the beta-glucosidase A (BGA) family. Both genes were expressed in Aspergillus oryzae, and the recombinant beta-glucosidases were purified. Recombinant H. grisea BGL4 is a thermostable enzyme compared with recombinant T. reesei BGL2. In addition to beta-glucosidase activity, recombinant H. grisea BGL4 showed a significant level of beta-galactosidase activity, while recombinant T. reesei BGL2 showed weak beta-galactosidase activity. Cellulose saccharification by Trichoderma cellulases was improved by the addition of recombinant H. grisea BGL4.     

The impact of a single-nucleotide mutation of <Emphasis Type="Italic">bgl2</Emphasis> on cellulase induction in a <Emphasis Type="Italic">Trichoderma reesei</Emphasis> mutant

Background

The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.

Results

To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.

Conclusion

We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

     

Expression in Trichoderma reesei and characterisation of a thermostable family 3 beta-glucosidase from the moderately thermophilic fungus Talaromyces emersonii

The gene encoding a thermostable beta-glucosidase (cel3a) was isolated from the thermophilic fungus Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus Trichoderma reesei. The cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59 kDa. Tal. emersonii beta-glucosidase falls into glycosyl hydrolase family 3, showing approximately 56 and 67% identity with Cel3b (GenBank ) from T. reesei, and a beta-glucosidase from Aspergillus Niger (GenBank ), respectively. The heterologously expressed enzyme, Cel3a, was a dimer equal to 130 kDa subunits with 17 potential N-glycosylation sites and a previously unreported beta-glucosidase activity produced extracellularly by Tal. emersonii. Cel3a was thermostable with an optimum temperature of 71.5 degrees C and half life of 62 min at 65 degrees C and was a specific beta-glucosidase with no beta-galactosidase side activity. Cel3a had a high specific activity against p-nitrophenyl-beta-D-glucopyranoside (Vmax, 512 IU/mg) and was competitively inhibited by glucose (k(i), 0.254 mM). Cel3a was also active against natural cellooligosacharides with glucose being the product of hydrolysis. It displayed transferase activity producing mainly cellobiose from glucose and cellotetrose from cellobiose.     

Lactic fermentation of cellobiose by a yeast strain displaying β-glucosidase on the cell surface

The Aspergillus aculeatus beta-glucosidase 1 (bgl1) gene was expressed in a lactic-acid-producing Saccharomyces cerevisiae strain to enable lactic fermentation with cellobiose. The recombinant beta-glucosidase enzyme was expressed on the yeast cell surface by fusing the mature protein to the C-terminal half region of the alpha-agglutinin. The beta-glucosidase expression plasmids were integrated into the genome. Three strong promoters of S. cerevisiae, the TDH3, PGK1, and PDC1 promoters, were used for beta-glucosidase expression. The specific beta-glucosidase activity varied with the promoter used and the copy number of the bgl1 gene. The highest activity was obtained with strain PB2 that possessed two copies of the bgl1 gene driven by the PDC1 promoter. PB2 could grow on cellobiose and glucose minimal medium at the same rate. Fermentation experiments were conducted in non-selective-rich media containing 95 g l(-1) cellobiose or 100 g l(-1) glucose as a carbon source under microaerobic conditions. The maximum rate of L-lactate production by PB2 on cellobiose (2.8 g l(-1) h(-1)) was similar to that on glucose (3.0 g l(-1) h(-1)). This indicates that efficient fermentation of cellobiose to L-lactate can be accomplished using a yeast strain expressing beta-glucosidase from a mitotically stable genomic integration plasmid.     

Purification and partial characterization of a cellodextrin glucohydrolase (beta-glucosidase) from Trichoderma reesei strain QM 9414

A beta-glucosidase (E.C. 3.2.1.21) was isolated from the culture filtrate of fungus Trichoderma reesei QM 9414 grown in continuous culture with biomass retention. The crude extracellular enzyme preparation was fractionated by a three-step purification procedure [chromatography on Fractogel HW-55 (S) and Bio-Gel A 0.5 plus final preparative isoelectric focusing] to yield three beta-glucosidases with isoelectric points at pH 8.4, 8.0, and 7.4. Only one enzyme (pi 8.4) met the stringent criterion of being homogeneous according to titration curve analysis. This enzyme was then characterized not to be a glycoprotein, although the native protein contained 35% carbohydrate (as glucose). It was found to have an apparent molar mass of 7 x 10(4) g/mol (SDS-PAGE), exhibited its optimum activity towards cellobiose at pH 4.5 and 70 degrees C (30 min test), and lost less than 3% activity at 50 degrees C over a period of 7 h. The K(M) values towards cellobiose and p-nitrophenyl-beta-D-glucopyranoside were determined to be 0.5mM and 0.3mM, respectively. The enzyme hydrolyzed cellodextrins (cellotriose to cellooctaose) by sequentially splitting off glucose units from the nonreducing end of the oligomers. The extent of the observed transfer reactions varied with the initial substrate concentration. No enzyme activity towards microcrystalline cellulose or carboxymethylcellulose could be detected. The classification of the enzyme as beta-glucosidase or exo-beta-1,4-glucan glucohydrolase is discussed with respect to the exhibited hydrolytic activities.     

Construction of the industrial ethanol-producing strain of Saccharomyces cerevisiae able to ferment cellobiose and melibiose

The gene mel1, encoding alpha-galactosidase in Schizosaccharomyces pombe, and the gene bgl2, encoding and beta-glucosidase in Trichoderma reesei, were isolated and co-expressed in the industrial ethanol-producing strain of Saccharomyces cerevisiae. The resulting strains were able to grow on cellobiose and melibiose through simultaneous production of sufficient extracellular alpha-galactosidase and beta-glucosidase activity. Under aerobic conditions, the growth rate of the recombinant strain GC 1 co-expressing 2 genes could achieve 0.29 OD600 h(-1) and a biomass yield up to 7.8 g l(-1) dry cell weight on medium containing 10.0 g l(-1) cellobiose and 10.0 g l(-1) melibiose as sole carbohydrate source. Meanwhile, the new strain of S. cerevisiae CG 1 demonstrated the ability to directly produce ethanol from microcrystalline cellulose during simultaneous saccharification and fermentation process. Approximately 36.5 g l(-1) ethanol was produced from 100 g of cellulose supplied with 5 g l(-1) melibose within 60 h. The yield (g of ethanol produced/g of carbohydrate consumed) was 0.44 g/g, which corresponds to 88.0% of the theoretical yield.     

Peptide fragmentation suitable for solid-phase microsequencing. Use of N-bromosuccinimide and BNPS-skatole (3-bromo-3-methyl-2-[(2-nitrophenyl)thio]-3H-indole).

A new intracellular beta-glucosidase was isolated from Trichoderma reesei. It was sequentially purified by (NH4)2SO4 precipitation and chromatography and rechromatography on Sephadex G-150. The enzyme has a mol.wt. of 98 000, optimal activity at pH 6.5, pI 4.4 and Km values of 6.7 mM and 3.3 mM for sophorose and cellobiose respectively. Possible functions of the enzyme may be regulation of cellulase induction and/or to serve as a proenzyme.     

Characterization of a cellodextrin glucohydrolase with soluble oligomeric substrates: experimental results and modeling of concentration-time-course data

A previously isolated cellodextrin glucohydrolase (beta-glucosidase) from Trichoderma reesei QM 9414 is characterized using beta-1,4-glucose oligomers with defined degrees of polymerization as soluble substrates. The enzyme splits off glucose units from the nonreducing chain ends of cellooligomers. Besides this hydrolytic activity there is also evidence for transfer activity depending on the concentration and degree of polymerization of substrates. Concentration-time-course data have been gathered for the degradation of cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose covering a wide range of enzyme and substrate concentrations. A Michaelis-Menten type kinetic model has been developed, which is able to satisfactorily describe the complex system of parallel and series reactions during the conversion of oligomers to glucose. The only kind of inhibition considered is competitive inhibition by the final product glucose. The model takes into account the formation of multiple enzyme-substrate complexes and is limited to those conditions, in which no transglucosylation products are observed. Cellodextrins with higher degrees of polymerization are found to be better substrates for this enzyme than is the dimer cellobiose.     

Kinetic properties and contribution to cellulose saccharification of a cloned Pseudomonas beta-glucosidase

The plasmid pND71, which encodes beta-glucosidase (cellobiase) activity, cloned from the cellulolytic Pseudomonad, PS2-2, was mobilized by conjugation into 10 Pseudomonas strains. The highest specific activity was produced by 17498 (pND71) and the properties of the enzyme produced from this transconjugant were studied. The enzyme was shown to be cell associated, to have a temperature optimum of 37 degrees C, a pH optimum of 7.0 and Km values of 1.33 and 2.94 mM for pNPG and cellobiose respectively. It was competitively inhibited by glucose, with a Ki of 30 mM. Evidence was obtained which suggested that the enzyme was produced constitutively and that synthesis was not repressed by glucose. When culture preparations were used in combination with Trichoderma reesei QM9414 and C30 enzyme preparations to saccharify cellulose, 17498 (pND71) was more effective than preparations of PS2-2 in acting synergistically with T. reesei to solubilize more carbohydrate and produce more glucose.     

Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata.

Candida peltata (NRRL Y-6888) produced beta-glucosidase when grown in liquid culture on various substrates (glucose, xylose, L-arabinose, cellobiose, sucrose, and maltose). An extracellular beta-glucosidase was purified 1,800-fold to homogeneity from the culture supernatant of the yeast grown on glucose by salting out with ammonium sulfate, ion-exchange chromatography with DEAE Bio-Gel A agarose, Bio-Gel A-0.5m gel filtration, and cellobiose-Sepharose affinity chromatography. The enzyme was a monomeric protein with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. It was optimally active at pH 5.0 and 50 degrees C and had a specific activity of 108 mumol.min-1.mg of protein-1 against p-nitrophenyl-beta-D-glucoside (pNP beta G). The purified beta-glucosidase readily hydrolyzed pNP beta G, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, with Km values of 2.3, 66, 39, 35, 21, and 18 mM, respectively. The enzyme was highly tolerant to glucose inhibition, with a Ki of 1.4 M (252 mg/ml). Substrate inhibition was not observed with 40 mM pNP beta G or 15% cellobiose. The enzyme did not require divalent cations for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol at an optimal concentration (0.75%, vol/vol) stimulated the initial enzyme activity by only 11%. Cellobiose (10%, wt/vol) was almost completely hydrolyzed to glucose by the purified beta-glucosidase (1.5 U/ml) in both the absence and presence of glucose (6%). Glucose production was enhanced by 8.3% when microcrystalline cellulose (2%, wt/vol) was treated for 24 h with a commercial cellulase preparation (cellulase, 5 U/ml; beta-glucosidase, 0.45 U/ml) that was supplemented with purified beta-glucosidase (0.4 U/ml).     

Induction of Cellulase by Gentiobiose and Its Sulfur-Containing Analog in Penicillium purpurogenum

Cellulase induction by beta-glucodisaccharides was investigated by using non-cellulase-induced mycelia of Penicillium purpurogenum P-26, a highly-cellulase-producing fungus. Gentiobiose induced significant amounts of cellulase compared with cellobiose when nojirimycin was added to the induction medium to inhibit extracellular beta-glucosidase activity. Thiogentiobiose (6-S-beta-d-glucopyranosyl-6-thio-d-glucose), a sulfur-containing analog of gentiobiose, was more effective for cellulase induction than gentiobiose even in the absence of nojirimycin. Thiogentiobiose appeared to be a gratuitous inducer since it was not metabolized during cellulase induction. Gentiobiose was formed from cellobiose by the intracellular beta-glucosidase of P. purpurogenum. These findings indicate that gentiobiose is an active inducer of cellulase for this fungus and may possibly be formed by intracellular beta-glucosidase from cellobiose.     

Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular beta-glucosidases as expressed in Saccharomyces cerevisiae

We isolated two genes for extracellular beta-glucosidase, BGL1 and BGL2, from the genomic library of the yeast Saccharomycopsis fibuligera. Gene products (BGLI and BGLII) were purified from the culture fluids of Saccharomyces cerevisiae transformed with BGL1 and BGL2, respectively. Molecular weights of BGLI and BGLII were estimated to be 220,000 and 200,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two beta-glucosidases showed the same enzymatic characteristics, such as thermo-denaturation kinetics and dependencies on pH and temperature, but quite different substrate specificities: BGLI hydrolyzed cellobiose efficiently, but BGLII did not. This result is consistent with the observation that the S. cerevisiae transformant carrying BGL1 fermented cellobiose to ethanol but the transformant carrying BGL2 did not. Southern blot analysis revealed that the two beta-glucosidase genes were derived from Saccharomycopsis fibuligera and that the nucleotide sequences of the two genes are closely related. The complete nucleotide sequences of the two genes were determined. BGL1 and BGL2 encode 876- and 880-amino-acid proteins which were shown to be highly similar to each other. The putative precursors begin with hydrophobic segments that presumably act as signal sequences for secretion. Amino acid analysis of the purified proteins confirmed that BGL1 and BGL2 encode BGLI and BGLII, respectively.     

Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular beta-glucosidases as expressed in Saccharomyces cerevisiae.

We isolated two genes for extracellular beta-glucosidase, BGL1 and BGL2, from the genomic library of the yeast Saccharomycopsis fibuligera. Gene products (BGLI and BGLII) were purified from the culture fluids of Saccharomyces cerevisiae transformed with BGL1 and BGL2, respectively. Molecular weights of BGLI and BGLII were estimated to be 220,000 and 200,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two beta-glucosidases showed the same enzymatic characteristics, such as thermo-denaturation kinetics and dependencies on pH and temperature, but quite different substrate specificities: BGLI hydrolyzed cellobiose efficiently, but BGLII did not. This result is consistent with the observation that the S. cerevisiae transformant carrying BGL1 fermented cellobiose to ethanol but the transformant carrying BGL2 did not. Southern blot analysis revealed that the two beta-glucosidase genes were derived from Saccharomycopsis fibuligera and that the nucleotide sequences of the two genes are closely related. The complete nucleotide sequences of the two genes were determined. BGL1 and BGL2 encode 876- and 880-amino-acid proteins which were shown to be highly similar to each other. The putative precursors begin with hydrophobic segments that presumably act as signal sequences for secretion. Amino acid analysis of the purified proteins confirmed that BGL1 and BGL2 encode BGLI and BGLII, respectively.     

Cloning and characterization of the glucooligosaccharide catabolic pathway beta-glucan glucohydrolase and cellobiose phosphorylase in the marine hyperthermophile Thermotoga neapolitana

Characterization in Thermotoga neapolitana of a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-beta-D-glucan glucohydrolase (GghA) and cellobiose phosphorylase (CbpA), and the apparent absence of a cellobiohydrolase (Cbh) suggest a nonconventional pathway for glucan utilization in Thermotogales. GghA purified from T. neapolitana is a 52.5-kDa family 1 glycosyl hydrolase with optimal activity at pH 6.5 and 95 degrees C. GghA releases glucose from soluble glucooligomers, with a preference for longer oligomers: k(cat)/K(m) values are 155.2, 76.0, and 9.9 mM(-1) s(-1) for cellotetraose, cellotriose, and cellobiose, respectively. GghA has broad substrate specificity, with specific activities of 236 U/mg towards cellobiose and 251 U/mg towards lactose. With p-nitrophenyl-beta-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrate- and end product-directed activation. Its capacity for transglycosylation is a factor in this activation. Cloning of gghA revealed a contiguous upstream gene (cbpA) encoding a 93.5-kDa cellobiose phosphorylase. Recombinant CbpA has optimal activity at pH 5.0 and 85 degrees C. It has specific activity of 11.8 U/mg and a K(m) of 1.42 mM for cellobiose, but shows no activity towards other disaccharides or cellotriose. With its single substrate specificity and low K(m) for cellobiose (compared to GghA's K(m) of 28.6 mM), CbpA may be the primary enzyme for attacking cellobiose in Thermotoga spp. By phosphorolysis of cellobiose, CbpA releases one activated glucosyl molecule while conserving one ATP molecule per disaccharide. CbpA is the first hyperthermophilic cellobiose phosphorylase to be characterized.     

Purification and characterization of a Bacillus polymyxa beta-glucosidase expressed in Escherichia coli.

The beta-glucosidase encoded by the bglA gene from Bacillus polymyxa was overproduced in Escherichia coli by using a plasmid in which bglA is under control of the lacI promoter. Induction with isopropyl-beta-D-thiogalactopyranoside allowed an increase in the specific activity of the enzyme of about 100 times the basal level of gene expression. The enzyme was purified by a two-step procedure involving salting out with ammonium sulfate and ion-exchange chromatography with DEAE-cellulose. Fractions of beta-glucosidase activity recovered by this procedure, after electrophoresis in an acrylamide gel and staining with silver nitrate, yielded a single band of protein. This band was shown by a zymogram to correspond to beta-glucosidase activity. The purified protein showed an apparent molecular mass of 50 kDa and an isoelectric point of 4.6, values in agreement with those expected from the nucleotide sequence of the gene. Km values of the enzyme, with either cellobiose or p-nitrophenyl-beta-D-glucoside as the substrate, were determined. It was shown that the enzyme is competitively inhibited by glucose. The effects of different metallic ions and other agents were studied. Hg2+ was strongly inhibitory, while none of the other cations tested had any significant effect. Ethanol did not show the stimulating effect observed with other beta-glucosidases. The mechanism of enzyme action was investigated. High-pressure liquid chromatography analysis with cellobiose as the substrate confirmed previous data revealing the formation of two products, glucose and another, unidentified, compound. Results presented here indicate that this compound is cellotriose formed by transglycosylation.     

Purification and Properties of beta-Glucosidase from Aspergillus terreus

A beta-glucosidase (EC 3.2.1.21) from the fungus Aspergillus terreus was purified to homogeneity as indicated by disc acrylamide gel electrophoresis. Optimal activity was observed at pH 4.8 and 50 degrees C. The beta-glucosidase had K(m) values of 0.78 and 0.40 mM for p-nitrophenyl-beta-d-glucopyranoside and cellobiose, respectively. Glucose was a competitive inhibitor, with a K(i) of 3.5 mM when p-nitrophenyl-beta-d-glucopyranoside was used as the substrate. The specific activity of the enzyme was found to be 210 IU and 215 U per mg of protein on p-nitrophenyl-beta-d-glucopyranoside and cellobiose substrates, respectively. Cations, proteases, and enzyme inhibitors had little or no effect on the enzyme activity. The beta-glucosidase was found to be a glycoprotein containing 65% carbohydrate by weight. It had a Stokes radius of 5.9 nm and an approximate molecular weight of 275,000. The affinity and specific activity that the isolated beta-glucosidase exhibited for cellobiose compared favorably with the values obtained for beta-glucosidases from other organisms being studied for use in industrial cellulose saccharification.