Isolation and properties of a cellulosome-type multienzyme complex of the thermophilic Bacteroides sp. strain P-1

The extracellular form of cellulosome-type multienzyme complex of thermophilic Bacteroides sp. strain P-1 which was isolated from the anaerobic digester, is described. Multienzyme complex was isolated from the culture supernatant by an adsorption-desorption affinity chromatography on microcrystalline cellulose. The isolated multienzyme complex was found to form a complex that exhibited a high molecular weight (estimated at more than 1400 kDa) and was quite stable, requiring strong denaturing condition for dissociation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate resolved multienzyme complex into at least 12 subunits with the molecular weight range of 49 to 209 kDa, respectively. The isolated multienzyme complex showed cellulose-binding ability, cellulase and xylanase activities and effected the hydrolysis of crystalline cellulose and lignocellulosic materials in the form of corncob, corn hull, rice straw, and sugarcane bagasse.     

Purification and characterization of a multienzyme complex produced by Paenibacillus curdlanolyticus B-6

Paenibacillus curdlanolyticus B-6 showed effective degradation activities for xylan and cellulose and produced an extracellular multienzyme complex (approximately 1,450 kDa) containing several xylanases and cellulases. To characterize the multienzyme complex, we purified the complex from culture supernatants by four kind of chromatography. The purified multienzyme complex was composed of a 280-kDa protein with xylanase activity, a 260-kDa protein that was a truncated form on the C-terminal side of the 280-kDa protein, two xylanases of 40 and 48 kDa, and 60 and 65 kDa proteins having both xylanase and carboxymethyl cellulase activities. The 280-kDa protein resembled the scaffolding proteins of cellulosomes based on its migratory behavior in polyacrylamide gels and as a glycoprotein. Cloning of the 40-kDa major xylanase subunit named Xyn11A revealed that Xyn11A contained two functional domains which belonged to glycosyl hydrolase family-11 and to carbohydrate-binding module family-36, respectively, and a glycine- and asparagine-rich linker. However, an amino acid sequence similar to a dockerin domain, which is crucial to cellulosome assembly, was not found in Xyn11A. These results suggest that the multienzyme complex produced by P. curdlanolyticus B-6 should assemble by a mechanism distinct from the cohesin-dockerin interactions known in cellulosomes.     

Paenibacillus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzyme system that degrades insoluble polysaccharides

A facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6, isolated from an anaerobic digester produces an extracellular xylanolytic-cellulolytic enzyme system containing xylanase, beta-xylosidase, arabinofuranosidase, acetyl esterase, mannanase, carboxymethyl cellulase (CMCase), avicelase, cellobiohydrolase, beta-glucosidase, amylase, and chitinase when grown on xylan under aerobic conditions. During growth on xylan, the bacterial cells were found to adhere to xylan from the early exponential growth phase to the late stationary growth phase. Scanning electron microscopic analysis revealed the adhesion of cells to xylan. The crude enzyme preparation was found to be capable of binding to insoluble xylan and Avicel. The xylanolytic-cellulolytic enzyme system efficiently hydrolyzed insoluble xylan, Avicel, and corn hulls to soluble sugars that were exclusively xylose and glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a crude enzyme preparation exhibited at least 17 proteins, and zymograms revealed multiple xylanases and cellulases containing 12 xylanases and 9 CMCases. The cellulose-binding proteins, which are mainly in a multienzyme complex, were isolated from the crude enzyme preparation by affinity purification on cellulose. This showed nine proteins by SDS-PAGE and eight xylanases and six CMCases on zymograms. Sephacryl S-300 gel filtration showed that the cellulose-binding proteins consisted of two multienzyme complexes with molecular masses of 1,450 and 400 kDa. The results indicated that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes.     

Multicomplex cellulase-xylanase system of Clostridium papyrosolvens C7.

The cellulase system of Clostridium papyrosolvens C7 was fractionated by means of ion-exchange chromatography into at least seven high-molecular-weight multiprotein complexes, each with different enzymatic and structural properties. The molecular weights of the complexes, as determined by gel filtration chromatography, ranged from 500,000 to 660,000, and the isoelectric points ranged from 4.40 to 4.85. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the complexes showed that each complex had a distinct polypeptide composition. Avicelase, carboxymethyl cellulase, and xylanase activity profiles differed from protein complex to protein complex. Three of the complexes hydrolyzed crystalline cellulose (Avicel). Activity zymograms of gels (following electrophoresis under mildly denaturing conditions) revealed different carboxymethyl cellulase-active proteins in all complexes but xylanase-active proteins in only two of the complexes. The xylanase specific activity of these two complexes was more than eightfold higher than that of the unfractionated cellulase preparation. A 125,000-M(r) glycoprotein with no apparent enzyme activity was the only polypeptide present in all seven complexes. Experiments involving recombination of samples eluted from the ion-exchange chromatography column indicated that synergistic interactions occurred in the hydrolysis of crystalline cellulose by the cellulase system. We propose that the C. papyrosolvens enzyme system responsible for the hydrolysis of crystalline cellulose and xylan is a multicomplex system comprising at least seven diverse protein complexes.     

Paenibacillus curdlanolyticus Strain B-6 Xylanolytic-Cellulolytic Enzyme System That Degrades Insoluble Polysaccharides

A facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6, isolated from an anaerobic digester produces an extracellular xylanolytic-cellulolytic enzyme system containing xylanase, β-xylosidase, arabinofuranosidase, acetyl esterase, mannanase, carboxymethyl cellulase (CMCase), avicelase, cellobiohydrolase, β-glucosidase, amylase, and chitinase when grown on xylan under aerobic conditions. During growth on xylan, the bacterial cells were found to adhere to xylan from the early exponential growth phase to the late stationary growth phase. Scanning electron microscopic analysis revealed the adhesion of cells to xylan. The crude enzyme preparation was found to be capable of binding to insoluble xylan and Avicel. The xylanolytic-cellulolytic enzyme system efficiently hydrolyzed insoluble xylan, Avicel, and corn hulls to soluble sugars that were exclusively xylose and glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a crude enzyme preparation exhibited at least 17 proteins, and zymograms revealed multiple xylanases and cellulases containing 12 xylanases and 9 CMCases. The cellulose-binding proteins, which are mainly in a multienzyme complex, were isolated from the crude enzyme preparation by affinity purification on cellulose. This showed nine proteins by SDS-PAGE and eight xylanases and six CMCases on zymograms. Sephacryl S-300 gel filtration showed that the cellulose-binding proteins consisted of two multienzyme complexes with molecular masses of 1,450 and 400 kDa. The results indicated that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes.     

An analysis of the extracellular xylanases and cellulases of Butyrivibrio fibrisolvens H17c

The extracellular xylanase and cellulase components of Butyrivibrio fibrisolvens H17c were investigated. Two major peaks of enzyme activity were eluted by hydroxylapatite chromatography and designated complex A (CA), having cellulase activity, and complex B (CB) having predominantly xylanase activity but with some activity on carboxymethyl cellulose (CMC). CB was further purified on a DE-52 column and subjected to gel filtration. The xylanase and CMCase activities eluted in a single peak with an apparent molecular mass greater than thyroglobulin (Mr 669,000). CMC xymograms of polyacrylamide gels electrophoresed under non-denaturing conditions indicated the presence of five bands with CMCase activity from CA and eight from CB. Xylan xymograms under the same conditions indicated the presence of four bands of activity in CB. Under mild denaturing conditions the xylanase activity in CB was found in 11 bands with molecular mass ranging from 45 to 180 and the CMCase activity in three bands with molecular mass ranging from 45 kDa to 60 kDa. This indicates that CB exists as a multi-subunit protein aggregate of xylanases, some of which also have cellulase activity.     

Purification and Characterization of Aeromonas caviae ME-1 Xylanase V, Which Produces Exclusively Xylobiose from Xylan

A xylanase, which produces exclusively xylobiose from oat spelt and birch xylans, was isolated from the culture medium of Aeromonas caviae ME-1. The enzyme (xylanase V) was purified by ammonium sulfate fractionation, hydrophobic interaction, and ion-exchange and gel filtration chromatographies. The homogeneity of the final preparation was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel electrofocusing. The molecular mass and isoelectric point of the xylanase were 46 kDa and 5.4, respectively. Xylanase V had a maximum activity at a pH of 6.8 and at a temperature between 30 and 37 degrees C. It was relatively stable at a pH between 5.0 and 8.6 and a temperature between 25 and 37 degrees C. When soluble birch xylan was used as the substrate, the enzyme had a K(m) and V(max) of 2 mg/ml and 182 mumol of xylose equivalent liberated . min . mg of protein, respectively. By the action of xylanase V on xylans (from oat spelt and birch), only one product corresponding to xylobiose was observed by thin-layer chromatography. The xylanase V putative product was confirmed to be xylobiose by acid and enzymatic hydrolyses. The xylanase had neither beta-xylosidase, alpha-l-arabinofuranosidase, cellulase, nor beta-1,3-xylanase activities. Xylotriose was the shortest substrate which the enzyme could attack. These findings suggest that xylanase V is a novel enzyme that cleaves a xylobiose unit from one of the ends of xylans, probably by an exomechanism.     

Purification of xylanase from alkaliphilic Bacillus sp. K-8 by using corn husk column

The objective of this work was to apply low cost materials, agricultural residues, to the purification of xylanase. The results showed that crude extracellular, cellulase-free xylanase of an alkaliphilic Bacillus sp. strain K-8 could be purified in a single step by affinity adsorption–desorption on a corn husk column using a high flow rate, under the conditions 25 mM acetate buffer, pH 4.0, 4 °C, which prevented the hydrolysis of xylan by xylanase. After adsorption, the xylanase was eluted from the enzyme–corn husk complex with 500 mM Urea. The enzyme was purified 5.3-fold to homogeneity from culture supernatant. The molecular weight of the purified enzyme was 24 kDa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity and recovery yield after purification were 25.4 U/mg protein and 42.3%, respectively.     

Isolation and characterization of endocellulase-free multienzyme complex from newly isolated Thermoanaerobacterium thermosaccharolyticum strain NOI-1

An endocellulase-free multienzyme complex was produced by a thermophilic anaerobic bacterium, Thermoanaerobacterium thermosaccharolyticum strain NOI-1, when grown on xylan. The temperature and pH optima for growth were 60 degrees C and 6.0, respectively. The bacterial cells were found to adhere to insoluble xylan and Avicel. A scanning electron microscopy analysis showed the adhesion of xylan to the cells. An endocellulase-free multienzyme complex was isolated from the crude enzyme of strain NOI-1 by affinity purification on cellulose and Sephacryl S-300 gel filtration. The molecular mass of the multienzyme complex was estimated to be about 1,200 kDa. The multienzyme complex showed one protein on native PAGE, one xylanase on a native zymogram, 21 proteins on SDS-PAGE, and 5 xylanases on a SDS zymogram. The multienzyme complex consisted of xylanase, beta-xylosidase, alpha-L-arabinofuranosidase, beta-glucosidase, and cellobiohydrolase. The multienzyme complex was effective in hydrolyzing xylan and corn hulls. This is the first report of an endocellulase-free multienzyme complex produced by a thermophilic anaerobic bacterium, T. thermosaccharolyticum strain NOI-1.     

Purification and characterization of a cellulolytic multienzyme complex produced by Neocallimastix patriciarum J11

Understanding the roles of the components of the multienzyme complex of the anaerobial cellulase system, acting on complex substrates, is crucial to the development of efficient cellulase systems for industrial applications such as converting lignocellulose to sugars for bioethanol production. In this study, we purified the multienzyme complex of Neocallimastix patriciarum J11 from a broth through cellulose affinity purification. The multienzyme complex is composed of at least 12 comprised proteins, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Eight of these constituents have demonstrated β-glucanase activity on zymogram analysis. The multienzyme complex contained scaffoldings that respond to the gathering of the cellulolytic components. The levels and subunit ratio of the multienzyme complex from N. patriciarum J11 might have been affected by their utilized carbon sources, whereas the components of the complexes were consistent. The trypsin-digested peptides of six proteins were matched to the sequences of cellulases originating from rumen fungi, based on identification through liquid chromatography/mass spectrometry, revealing that at least three types of cellulase, including one endoglucanase and two exoglucanases, could be found in the multienzyme complex of N. patriciarum J11. The cellulolytic subunits could hydrolyze synergistically on both the internal bonds and the reducing and nonreducing ends of cellulose. Based on our research, our findings are the first to depict the composition of the multienzyme complex produced by N. patriciarum J11, and this complex is composed of scaffoldin and three types of cellulase.     

Inexpensive, rapid procedure for bulk purification of cellulase-free beta-1,4-D-xylanase of high specific activity

A process has been developed for the bulk purification of cellulase-free beta-1,4-D-xylanase from the fungus Trichoderma harzianum E58. The process involved the primary step of ultrafiltering the culture filtrate via a 10,000-molecular-weight cut-off membrane to separate the cellulase (retentate) and xylanase (permeate) fractions. The cellulase component was concentrated by 40- to 60-fold, resulting in an enzyme complex that could effectively hydrolyze high concentrations of cellulose and xylan to glucose and xylose. The xylanase was concentrated and solvent exchanged by adsorption to a cationic exchanger, SP-ZetaPrep 250, followed by elution with a pH change in the buffer to give a purified and concentrated xylanase complex dissolved in a low-salt buffer. The resultant xylanase system was pure by the criteria of sodium dodecyl sulfate polyacrylamide electrophoresis, had a very high specific activity of 2400 IU/mg protein, was virtually free of filter paper activity, and had a ratio of contaminating filter paper activity of 2 x 10(-6) (0.009% endoglucanase activity). Approximately 3.3 g protein, which contained in excess of 7 x 10(6) IU xylanase activity, was obtained from 17 L original culture filtrate. The process scheme was designed to facilitate scale-up to an industrial level of production.     

Production, Purification, and Characterization of beta-(1-4)-Endoxylanase of Streptomyces roseiscleroticus

Twelve species of Streptomyces that formerly belonged to the genus Chainia were screened for the production of xylanase and cellulase. One species, Streptomyces roseiscleroticus (Chainia rosea) NRRL B-11019, produced up to 16.2 IU of xylanase per ml in 48 h. A xylanase from S. roseiscleroticus was purified and characterized. The enzyme was a debranching beta-(1-4)-endoxylanase showing high activity on xylan but essentially no activity against acid-swollen (Walseth) cellulose. It had a very low apparent molecular weight of 5,500 by native gel filtration, but its denatured molecular weight was 22,600 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had an isoelectric point of 9.5. The pH and temperature optima for hydrolysis of arabinoxylan were 6.5 to 7.0 and 60 degrees C, respectively, and more than 75% of the optimum enzyme activity was retained at pH 8.0. The xylanase had a K(m) of 7.9 mg/ml and an apparent V(max) of 305 mumol . min . mg of protein. The hydrolysis rate was linear for xylan concentrations of less than 4 mg/ml, but significant inhibition was observed at xylan concentrations of more than 10 mg/ml. The predominant products of arabinoxylan hydrolysis included arabinose, xylobiose, and xylotriose.     

Production, characterization, and partial amino acid sequence of xylanase A from Schizophyllum commune.

Xylanase A, one of several extracellular xylanases produced by Schizophyllum commune strain Delmar when grown in submerged culture with spruce sawdust as carbon source, was purified 43-fold in 25% yield with respect to total xylanase activity. Although some polysaccharide was strongly bound to the purified enzyme, the complex could be dissociated by sodium dodecyl sulfate and appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the protein, calculated from the electrophoretic mobility, was 33,000. The molecular activity of the purified xylanase A, determined with soluble larch xylan as substrate, was 1.4 X 10(5) min-1, with xylobiose and xylose as the major products. The enzyme had a pH optimum of 5.0 and a temperature optimum of 55 degrees C in 10-min assays. The acid hydrolysate of xylanase A was rich in aspartic acid and aromatic amino acids. The sequence of 27 residues at the amino terminus showed no homology with known sequences of other proteins.     

Production, characterization, and partial amino acid sequence of xylanase A from Schizophyllum commune.

Xylanase A, one of several extracellular xylanases produced by Schizophyllum commune strain Delmar when grown in submerged culture with spruce sawdust as carbon source, was purified 43-fold in 25% yield with respect to total xylanase activity. Although some polysaccharide was strongly bound to the purified enzyme, the complex could be dissociated by sodium dodecyl sulfate and appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the protein, calculated from the electrophoretic mobility, was 33,000. The molecular activity of the purified xylanase A, determined with soluble larch xylan as substrate, was 1.4 X 10(5) min-1, with xylobiose and xylose as the major products. The enzyme had a pH optimum of 5.0 and a temperature optimum of 55 degrees C in 10-min assays. The acid hydrolysate of xylanase A was rich in aspartic acid and aromatic amino acids. The sequence of 27 residues at the amino terminus showed no homology with known sequences of other proteins.     

Ethylene biosynthesis-inducing protein from cellulysin is an endoxylanase

The proteinaceous ethylene biosynthesis-inducing factor (EIF) that was purified from Cellulysin was also shown to contain a xylanase activity. In all nondenaturing protein separation methods employed (Sephacryl S-200 chromatography, and preparative isoelectric focusing and agarose electrophoresis), xylanase activity copurified with the ethylene biosynthesis-inducing activity. Treatment with heat (60°C) or proteases in 8 molar urea inhibited both ethylene-inducing and xylanase activities. Antibodies raised against purified EIF, which contains three polypeptides of 18, 14, and 10 kilodaltons, immunoprecipitated both ethylene biosynthesis-inducing and xylanase activities. The purified EIF contained no detectable cellulase, polygalacturonase, or protease activity. Other hydrolytic activities as estimated by using p-nitrophenyl derivatives of several sugars as substrates also were not detected. Different commercially available hydrolytic enzyme preparations were tested for both ethylene biosynthesis-inducing and xylanase activities. All enzymes tested contained xylanase activity, but only a few induced ethylene biosynthesis. Western blots of proteins separated by SDS-PAGE, using antibodies prepared against the non-denatured purified EIF, revealed two major bands of about 18 and 14 kilodaltons in EIF. These antibodies seem to be specific for these proteins from Trichoderma viride, because there was little cross-reactivity with the other proteins in Cellulysin and other commercial enzyme preparations. Based on these data, we suggest that EIF contains a specific xylanase activity which is involved in inducing ethylene biosynthesis.     

A High-Molecular-Weight, Cell-Associated Xylanase Isolated from Exponentially Growing Thermoanaerobacterium sp. Strain JW/SL-YS485

An unusual cell-associated (beta)-1,4-xylanase was purified to gel electrophoretic homogeneity from a cell extract of the bacterium Thermoanaerobacterium sp. strain JW/SL-YS485 harvested at the late exponential growth phase. The molecular mass of the xylanase was 350 kDa as determined by gel filtration and 234 kDa as determined by native gradient gel electrophoresis. The enzyme contained 6% carbohydrates. Heterosubunits of 180 and 24 kDa were observed for the xylanase on sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis gels. The xylanase had a pI of 4.37 and a half-life of 1 h at 70(deg)C. Using a 5-min assay, we observed the highest level of activity at pH 6.2 and 80(deg)C. The K(infm) and k(infcat) values when oat spelt xylan was used were 3 mg/ml and 26,680 U/(mu)mol, respectively. The Arrhenius energy was 41.8 kJ/mol. The purified enzyme differed in size, subunit structure, and location from other xylanases that have been described. The cell-associated enzyme activity appeared in the S-layer fraction.     

Seven mammalian aminoacyl-tRNA synthetases co-purified as high molecular weight entities are associated within the same complex.

Seven aminoacyl-tRNA synthetases from sheep liver were co-purified as high mol. wt. entities to constant specific activities. The purified multienzyme preparation displayed an apparent mol. wt. of approximately 10(6) and was composed of 11 distinct polypeptides, as revealed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). To test the assumption that all of these components were physically associated within the same complex, the purified preparation was subjected to immunoprecipitation by antibodies raised against its lysyl- or methionyl-tRNA synthetase component. Depending on the limiting concentrations of the specific antibodies used, from 5 to 40% of the input protein was recovered in the immunoprecipitate. Its polypeptide composition, as revealed by SDS-PAGE, was indistinguishable from that of the original material. The immunoprecipitation reaction was highly specific, as attested by the observation that IgG from nonimmunized rabbit failed to precipitate any of the 11 polypeptides, even when used in 30-fold molar excess over input protein. We conclude that co-precipitation of all of these polypeptides by antibodies directed against a single component of the purified preparation is a consequence of their physical association within the same multienzyme complex.     

Purification and properties of an acetylxylan esterase from Thermobifida fusca

An acetylxylan esterase from Thermobifida fusca NTU22 was purified 51-fold as measured by specific activity from crude culture filtrate by ultrafiltration concentration, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. The overall yield of the purified enzyme was 14.4%. The purified enzyme gave an apparent single protein band on an SDS-PAGE. The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sepharose CL-6B was found to be 30 and 28kDa, respectively, indicating that the acetylxylan esterase from T. fusca NTU22 is a monomer. The pI value of the purified enzyme was estimated to be 6.55 by isoelectric focusing gel electrophoresis. The N-terminal amino acid sequence of the purified esterase was ANPYERGP. The optimum pH and temperature for the purified enzyme were 8.0 and 80°C, respectively. The Zn(2+), Hg(2+), PMSF and DIPF inhibited the enzyme activity. The K(m) value for p-nitrophenyl acetate and acetylxylan were 1.86μM and 0.15%, respectively. Co-operative enzymatic degradation of oat-spelt xylan by purified acetylxylan esterase and xylanase significantly increased the acetic acid liberation compared to the acetylxylan esterase action alone.     

Production, purification, and characterization of xylanase from a hyperxylanolytic mutant of Aspergillus ochraceus

Enhancement of the productivity of xylanase and beta-xy-losidase of Aspergillus ochraceus was investigated by multistep mutagenesis. The spores of the wild strain were subjected to UV and N-methyl-N-nitro-N-nitro-soguanidine (NTG). The hyperxylanolytic mutant (NG-13), which showed good clearing on the surface of the xylan-agar plate, secretes xylanase and beta-xylosidase at high levels during growth on commercial xylan and on agricultural wastes. Both liquid and solid state cultures were employed in the study for enzyme production. The xylanase from NG-13 was purified to homogeneity by ammonium sulfate precipitation and gel filtration. This purified enzyme showed a pH optimum of 6.0 and was stable in the range of pH 5 to 10. Prolonged stability of the enzyme was observed at 45 degrees C though its activity was maximal at 50 degrees C. The molecular weight of the enzyme was estimated to be 4.3 x 10(4) by SDS-polyacrylamide gel electrophoresis and 5 x 10(4) by gel filtration on Sephadex G-75. The kinetic data showed that the K(m) and V(max) values for xylan were 1 x 10(-3)M and 19.6 mumol/ min/mg protein, respectively. The enzyme was both more active and thermostable in the presence of K(+)and was inactivated by thiol reagents such as Hg(2+), p-hydroxymercuribenzoate (PHMB), 3', 5'-dithiobis (2'-nitrobenzoic acid) (DTNB), and N-ethylmaleimide (NEM).     

Purification and characterization of a cellulase from the ruminal fungus Orpinomyces joyonii cloned in Escherichia coli

A cellulase from the ruminal fungus Orpinomyces joyonii cloned in Escherichia coli was purified 88-fold by chromatography on High Q and hydroxyapatite. N-terminal amino acid sequence analyses confirmed that the cellulase represented the product of the cellulase gene Cel B2. The purified enzyme possessed high activity toward barley beta-glucan, lichenan, carboxymethyl cellulose (CMC), xylan, but not toward laminarin and pachyman. In addition, the cloned enzyme was able to hydrolyze p-nitrophenyl (PNP)-cellobioside, PNP-cellotrioside, PNP-cellotetraoside, PNP-cellopentaoside, but not PNP-glucopyranoside. The specific activity of the cloned enzyme on barley beta-glucan was 297 units/mg protein. The purified enzyme appeared as a single band in SDS-polyacrylamide gel electrophoresis and the molecular mass of this enzyme (58000) was consistent with the value (56463) calculated from the DNA sequence. The optimal pH of the enzyme was 5.5, and the enzyme was stable between pH 5.0 and pH 7.5. The enzyme had a temperature optimum at 40 degrees C. The K(m) values estimated for barley beta-glucan and CMC were 0.32 and 0.50 mg/ml, respectively.