Characterization of endo-1,4-beta-D-glucanases purified from Trichoderma viride

Four electrophoretically distinct endo-1,4-beta-D-glucanases (EC 3.2.1.4) from Trichoderma viride have been identified and named as isozymes, Endoglucanases I, II, III and IV, according to their electrophoretic mobilities on polyacrylamide gels. Endoglucanases II, III and IV, the homogeneity of each of which was established by discontinuous gel electrophoresis and ultracentrifugation, had specific activities on CM-cellulose of 1010, 60 and 250 specific fluidity units/mg protein, respectively. These enzymes have similar pH optima (pH 4.0-4.5) and are labile at pH values greater than 8.0. The endoglucanases are high in acidic and hydroxylated amino acids and glycine, but low in basic amino acids. Values of 12.0, 10.3 and 13.1 have been determined for the epsilon 1%280 of purified Endoglucanases II, III and IV, respectively. Sedimentation equilibrium analysis has established the molecular weights of Endoglucanases II, III and IV to be 37 200, 52 000 and 49 500, respectively. The three endoglucanases contain mannose, galactose, glucose and glucosamine. Mannose is the principal neutral sugar in each enzyme. Endoglucanase II is distinguished by its low carbohydrate content, 4.5% (w/w), compared to Endoglucanases III and IV which contain 15.0% and 15.2% carbohydrate, respectively.     

Biochemical characterization and mode of action of a thermostable endoglucanase purified from Thermoascus aurantiacus

A major extracellular endoglucanase purified to homogeneity from Thermoascus aurantiacus had a M(r) of 34 kDa and a pI of 3.7 and was optimally active at 70-80 degrees C and pH 4.0-4.4. It was stable at pH 2.8-6.8 at 50 degrees C for 48 h and maintained its secondary structure and folded conformation up to 70 degrees C at pH 5.0 and 2.8, respectively. A 33-amino acid sequence at the N terminus showed considerable homology with 14 microbial endoglucanases having highly conserved 8 amino acids (positions 10-17) and Gly, Pro, Gly, and Pro at positions 8, 22, 23, and 32, respectively. The enzyme is rich in Asp (15%) and Glu (10%) with a carbohydrate content of 2.7%. Polyclonal antibodies of endoglucanase cross-reacted with their own antigen and with other purified cellulases from T. aurantiacus. The endoglucanase was specific for polymeric substrates with highest activity toward carboxymethyl cellulose followed by barley beta-glucan and lichenan. It preferentially cleaved the internal glycosidic bonds of Glc(n) and MeUmbGlc(n) and possessed an extended substrate-binding site with five subsites. The data indicate that the endoglucanase from T. aurantiacus is a member of glycoside hydrolase family 5.     

Purification of two immunologically distinct endoglucanases without affinity for microcrystalline cellulose from Cellulomonas sp. ATCC 21399

Two endoglucanases (endoglucanase B and endoglucanase C) without affinity for cellulose were purified from the culture broth of Cellulomonas sp. ATCC 21399 using gelfiltration and ion exchange chromatography. Fused rocket immunoelectrophoresis was used to select the fractions with the highest content of endoglucanase and lowest content of contaminating proteins. The endoglucanases were purified to immunological homogeneity. In addition both endoglucanases were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular weights of endoglucanase B and endoglucanase C were 67000 and 25000, respectively). Endoglucanase B was homogeneous when studied by isoelectric focusing showing one protein band at pl 4.3. Both endoglucanases lacked activity against microcrystalline cellulose (Avicel) and showed similar endo action on carboxymethylcellulose (CMC). Endoglucanase B had a high specific activity against CMC, H(3)PO(4)-swollen Avicel and xylan, but showed no activity against galactomannan. In contrast, endoglucanase C showed activity against both CMC, xylan, and galactomannan all being polysaccharide substrates linked with beta-1-4-D-glucoside bonds. The specific activity of endoglucanase C against H(3)PO(4)-swollen Avicel was low.     

Purification and characterization of an endoglucanase from Streptomyces lividans 66 and DNA sequence of the gene.

The endoglucanase isolated from culture filtrates of Streptomyces lividans IAF74 was shown to have an Mr of 46,000 and a pI of 3.3. The specific enzyme activity of 539 IU/mg, determined by the reducing assay method on carboxymethyl cellulose, is among the highest reported in the literature. The cellulase showed typical endo-type activity when reacting on oligocellodextrins. Optimal enzyme activity was obtained at 50 degrees C and pH 5.5. The kinetic constants for this endoglucanase, determined with carboxymethyl cellulose as the substrate, were a Vmax of 24.9 IU/mg of enzyme and a Km of 4.2 mg/ml. Activity was found against neither methylumbelliferyl- nor p-nitrophenyl-cellobiopyranoside nor with xylan. The DNA sequence contains one possible reading frame validated by the N terminus of the mature purified protein. However, neither ATG nor GTG starting codons were identified near the ribosome-binding site. A putative TTG codon was found as a good candidate for the start codon. Comparison of the primary amino acid sequence of the endoglucanase of S. lividans revealed that the N terminus contains a bacterial cellulose-binding domain. The catalytic domain at the C terminus showed similarity to endoglucanases from a Bacillus sp. Thus, the endoglucanase CelA belongs to family A of cellulases as described before (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991.     

Purification and characterization of an endoglucanase from Streptomyces lividans 66 and DNA sequence of the gene.

The endoglucanase isolated from culture filtrates of Streptomyces lividans IAF74 was shown to have an Mr of 46,000 and a pI of 3.3. The specific enzyme activity of 539 IU/mg, determined by the reducing assay method on carboxymethyl cellulose, is among the highest reported in the literature. The cellulase showed typical endo-type activity when reacting on oligocellodextrins. Optimal enzyme activity was obtained at 50 degrees C and pH 5.5. The kinetic constants for this endoglucanase, determined with carboxymethyl cellulose as the substrate, were a Vmax of 24.9 IU/mg of enzyme and a Km of 4.2 mg/ml. Activity was found against neither methylumbelliferyl- nor p-nitrophenyl-cellobiopyranoside nor with xylan. The DNA sequence contains one possible reading frame validated by the N terminus of the mature purified protein. However, neither ATG nor GTG starting codons were identified near the ribosome-binding site. A putative TTG codon was found as a good candidate for the start codon. Comparison of the primary amino acid sequence of the endoglucanase of S. lividans revealed that the N terminus contains a bacterial cellulose-binding domain. The catalytic domain at the C terminus showed similarity to endoglucanases from a Bacillus sp. Thus, the endoglucanase CelA belongs to family A of cellulases as described before (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991.     

endAFS, a novel family E endoglucanase gene from Fibrobacter succinogenes AR1.

The complete nucleotide sequence of endAFS, an endoglucanase gene isolated from the ruminal anaerobe Fibrobacter succinogenes AR1, was determined. endAFS encodes two overlapping open reading frames (ORF1 and ORF2), and it was proposed that a -1 ribosomal frameshift was required to allow contiguous synthesis of a 453-amino-acid endoglucanase. A proline- and threonine-rich region at the C terminus of ORF1 and rare codons for arginine and threonine were coincident with the proposed frameshift site. ENDAFS is proposed to be a member of subgroup 1 of family E endoglucanases, of which endoglucanases from Thermomonospora fusca and Persea americana (avocado) are also members. Endoglucanases from Clostridium thermocellum and Pseudomonas fluorescens form subgroup 2.     

Analysis of Molecular Size Distributions of Cellulose Molecules during Hydrolysis of Cellulose by Recombinant Cellulomonas fimi β-1,4-Glucanases

Four β-1,4-glucanases (cellulases) of the cellulolytic bacterium Cellulomonas fimi were purified from Escherichia coli cells transformed with recombinant plasmids. Previous analyses using soluble substrates had suggested that CenA and CenC were endoglucanases while CbhA and CbhB resembled the exo-acting cellobiohydrolases produced by cellulolytic fungi. Analysis of molecular size distributions during cellulose hydrolysis by the individual enzymes confirmed these preliminary findings and provided further evidence that endoglucanase CenC has a more processive hydrolytic activity than CenA. The significant differences between the size distributions obtained during hydrolysis of bacterial microcrystalline cellulose and acid-swollen cellulose can be explained in terms of the accessibility of β-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.     

Endoglucanase activity and relative expression of glycoside hydrolase genes of Fibrobacter succinogenes S85 grown on different substrates

The endoglucanase activity of cells and extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, soluble polysaccharides (beta-glucan, lichenan) and intact plant polysaccharides, was compared. The specific activity of cells grown on cellulose or forages was 6- to 20-fold higher than that of cells grown on soluble substrates, suggesting an induction of endoglucanases by the insoluble substrates. The ratios of cells to extracellular culture fluid endoglucanase activities measured in cultures grown on sugars or insoluble polysaccharides suggested that the endoglucanases induced by the insoluble polysaccharides remained attached to the cells. The mRNA of all the F. succinogenes glycoside hydrolase genes sequenced so far were then quantified in cells grown on glucose, cellobiose or cellulose. The results show that all these genes were transcribed in growing cells, and that they are all overexpressed in cultures grown on cellulose. Endoglucanase-encoding endB and endA(FS) genes, and xylanase-encoding xynC gene appeared the most expressed genes in growing cells. EGB and ENDA are thus likely to play a major role in cellulose degradation in F. succinogenes.     

Assessment of the endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1.

The extracellular endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1 were analyzed by high-performance liquid chromatography (HPLC) by using DEAE ion-exchange, hydroxylapatite, and gel filtration chromatography and polyacrylamide gel electrophoresis (PAGE). Two endo-1,4-beta-glucanase peaks were resolved by DEAE-HPLC and termed endoglucanases A and B. Carboxymethyl cellulose (CMC) zymograms were achieved by enzyme separation using nondenaturing PAGE followed by incubation of the gel on top of a CMC-agarose gel. This revealed no less than 13 and 5 endo-1,4-beta-glucanase components present in endoglucanases A and B, respectively. Hydroxylapatite chromatography of endoglucanases A and B revealed one activity peak for each preparation, which contained 4 and 5 endo-1,4-beta-glucanase components, respectively. Gel filtration chromatography of endoglucanase A following hydroxylapatite chromatography resolved the most active carboxymethylcellulase (CMCase) component from other endo-1,4-beta-glucanase activities. Gel filtration of endoglucanase B following hydroxylapatite chromatography showed one CMCase activity peak. Protein stains of sodium dodecyl sulfate-PAGE and nondenaturing PAGE gels of endoglucanases A and B from hydroxylapatite and gel filtration chromatography revealed multiple protein components. When xylan was substituted for CMC in zymograms, identical separation patterns for CMCase and xylanase activities were observed for both endoglucanases A and B. These data suggest that both 1,4-beta linkage-hydrolyzing activities reside on the same polypeptide or protein complex. The highest endo-1,4-beta-glucanase-specific activities were observed following DEAE-HPLC chromatography, with 16.2 and 7.5 mumol of glucose equivalents per min per mg of protein for endoglucanases A and B, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new type of Clostridium thermocellum endoglucanase produced by the recombinant strain of E. coli. Some properties and identification in donor cells

The properties of endoglucanase produced by the recombinant strain of E. coli carrying plasmid pCU 104 with a 2.9 kb insert of chromosomal DNA of C. thermocellum encoding the multiple forms of the 35.5 kD polypeptide (pI 4.3-4.7) were studied. The enzyme has a broad pH optimum of activity (6.0-7.5). The half-inactivation time for different forms of the enzyme at 65 degrees C is similar and is equal to 25-30 minutes. The enzyme is related to endoglucanases weakly adsorbed on cellulose (Kp = 0.065 1/g). Hydrolysis of microcrystalline cellulose is completed within 7 days (7-9%) and is accompanied by the formation of cellobiose and cellotriose. The enzyme splits dyed lichenan (mixed 1,3-1,4-beta-glucane) at a higher rate than the dyed CM-cellulose. A guinea pig antiserum to enzyme isoforms with a pI of 4.46-4.54 was obtained. Using direct solid phase immunoenzymatic analysis, it was demonstrated that all the enzyme isoforms under study (pI 4.3-4.7) are immunologically related (serum titers for different enzyme isoforms vary from 1:20,000 to 1:50,000). In the original culture fluid of C. thermocellum, the antigen related to the enzyme isolated from the recombinant strain was unobserved. However, SDS-PAAG electrophoresis of SDS- and mercaptoethanol-treated culture fluids revealed among 11 protein bands at least 4 antigens interacting with antibodies (Mr = 107, 76, 67 and 37 kD), although their antibody titers were far lower and did not exceed 1:300-1:500. The cumulative data suggest that the endoglucanase under study is not identical to the earlier described enzymes encoded by the cel A- and ceI B-genes of C. thermocellum.     

Assessment of the endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1

The extracellular endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1 were analyzed by high-performance liquid chromatography (HPLC) by using DEAE ion-exchange, hydroxylapatite, and gel filtration chromatography and polyacrylamide gel electrophoresis (PAGE). Two endo-1,4-beta-glucanase peaks were resolved by DEAE-HPLC and termed endoglucanases A and B. Carboxymethyl cellulose (CMC) zymograms were achieved by enzyme separation using nondenaturing PAGE followed by incubation of the gel on top of a CMC-agarose gel. This revealed no less than 13 and 5 endo-1,4-beta-glucanase components present in endoglucanases A and B, respectively. Hydroxylapatite chromatography of endoglucanases A and B revealed one activity peak for each preparation, which contained 4 and 5 endo-1,4-beta-glucanase components, respectively. Gel filtration chromatography of endoglucanase A following hydroxylapatite chromatography resolved the most active carboxymethylcellulase (CMCase) component from other endo-1,4-beta-glucanase activities. Gel filtration of endoglucanase B following hydroxylapatite chromatography showed one CMCase activity peak. Protein stains of sodium dodecyl sulfate-PAGE and nondenaturing PAGE gels of endoglucanases A and B from hydroxylapatite and gel filtration chromatography revealed multiple protein components. When xylan was substituted for CMC in zymograms, identical separation patterns for CMCase and xylanase activities were observed for both endoglucanases A and B. These data suggest that both 1,4-beta linkage-hydrolyzing activities reside on the same polypeptide or protein complex. The highest endo-1,4-beta-glucanase-specific activities were observed following DEAE-HPLC chromatography, with 16.2 and 7.5 mumol of glucose equivalents per min per mg of protein for endoglucanases A and B, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple forms of endo-1,4-beta-glucanases in the endosperm of Euphorbia heterophylla L

Germinating seeds of Euphorbia heterophylla L. contain endo-1,4-beta-glucanases which degrade carboxymethylcellulose (CMC). The activity decreased approximately 66% in extracts of endosperm containing isopropanol or ethanol. The endoglucanases were isolated from endosperm extracts using ammonium sulphate fractionation followed by Sephacryl S-100-HR chromatography resulting in two main peaks: I and II. Peak I endoglucanase was further purified about 15-fold on DEAE-Sephadex A50 and then by affinity chromatography (CF11-cellulose). Peak II endoglucanases were further purified 10-fold on CM-cellulose chromatography. The results indicated the occurrence of a 66 kDa endoglucanase (fractionated by SDS-PAGE and visualized by activity staining using Congo Red). Several acidic (pI 3.0 to 5.7) and basic (pI 8.5 to 10.0) forms from both peaks which differed in their capacities for degrading CMC or xyloglucans from Copaifera langsdorffii or Hymenaea courbaril were detected.     

Endoglucanase sensitivity for substituents in methyl cellulose hydrolysis studied using MALDI-TOFMS for oligosaccharide analysis and structural analysis of enzyme active sites

The properties of modified cellulose polymers, such as methylcellulose, are significantly influenced by the distribution of substituents along the polymer backbone. This distribution is difficult to determine due to the lack of suitable analytical methods. One approach is to use cellulose-degrading enzymes to gain information from the capability of the enzymes to cleave the bonds between glucose units. Endoglucanases are cellulase enzymes that can break internal glycosidic linkages and degrade low substituted regions of modified cellulose where the substituents do not interfere with the enzyme active site. In this work methyl cellulose was degraded using five endoglucanases from glycosyl hydrolase families 5 and 7 from three different species. The products were analyzed with reducing end analysis, chromatography (SEC-MALS-RI), and MALDI-TOFMS. The results were correlated with available determined enzyme structures and using structural alignment for unknown enzyme structures. This was performed in order to elucidate the relationship between active site structures and sensitivity for substituents on derivatized cellulose. The evaluation of endoglucanase hydrolysis of methyl cellulose showed that differences in sensitivity could be related to differences in steric hindrance of substituents in the active site, which could explain differences within family 5 and 7 enzymes, as well as the generally higher substituent tolerance for family 5 enzymes. This information is important for use of endoglucanases as tools for characterization of substituent distribution. The results are also valuable since soluble cellulose derivatives are generally used as substrates during enzyme characterization and in endoglucanase activity assays.     

Enzymic activities of endo-1,4-beta-D-glucanases purified from Trichoderma viride

Endoglucanases II, III and IV (EC 3.2.1.4) from Trichoderma viride are highly active in degrading CM-cellulose or phosphoric acid swollen cellulose, and only slightly active on Avicel. The specific activities of the endoglucanases increase with the length of the cellooligosaccharide substrates. By rate and product analyses using high pressure liquid chromatography the mode of action of Endoglucanase III was differentiated from that of Endoglucanases II and IV. Endoglucanase III has a low affinity for cellobiose, reacts rapidly with cellotriose, and gradually increases in reactivity with cellooligosaccharides as degree of polymerization increases from four to six. In addition to cleaving internal glycosidic bonds of polymeric substrates, it preferentially cleaves cellobiosyl units from the non-reducing end of oligosaccharides. The cellobiosyl units are often, under initial reaction conditions, transferred to the substrate-acceptor. Endoglucanases II and IV show a preference for internal glycosidic bonds of cellooligosaccharides. The soluble products from the initial action of Endoglucanases II and IV on swollen cellulose are glucose, cellobiose, and cellotriose, which are slowly converted to glucose and some cellobiose.     

Purification and characterization of major extracellular proteinases from Trichophyton rubrum.

Two extracellular proteinases that probably play a central role in the metabolism and pathogenesis of the most common dermatophyte of man, Trichophyton rubrum, were purified to homogeneity. Size-exclusion chromatography and Chromatofocusing were used to purify the major proteinases 42-fold from crude fungal culture filtrate. The major enzyme has pI 7.8 and subunit Mr 44 000, but forms a dimer of Mr approx. 90 000 in the absence of reducing agents. A second enzyme with pI 6.5 and subunit Mr 36 000, was also purified. It is very similar in substrate specificity to the major enzyme but has lower specific activity, and may be an autoproteolysis product. The major proteinase has pH optimum 8, a Ca2+-dependence maximum of 1 mM, and was inhibited by serine-proteinase inhibitors, especially tetrapeptidyl chloromethane derivatives with hydrophobic residues at the P-1 site. Kinetic studies also showed that tetrapeptides containing aromatic or hydrophobic residues at P-1 were the best substrates. A kcat./Km of 27 000 M-1 X S-1 was calculated for the peptide 3-carboxypropionyl-Ala-Ala-Pro-Phe-p-nitroanilide. The enzyme has significant activity against keratin, elastin and denatured type I collagen (Azocoll).     

Relative molecular mass determination of a major, highest relative molecular mass extracellular amelogenin of developing bovine enamel

Proteins of developing bovine enamel were fractionated by molecular sieving and ion-exchange chromatography. The major fraction corresponding to the highest Mr amelogenin of Mr approximately 26 000-30 000 was isolated and its Mr determined by SDS-PAGE, molecular sieving on G-100 resin and high performance liquid chromatography and by sedimentation-equilibrium ultracentrifugation, the latter three procedures in guanidine hydrochloride. SDS-PAGE and HPLC molecular sieving, employing commonly used Mr standards, gave Mr values of approximately 22 000-26 000. SDS-PAGE and HPLC molecular sieving, using proline-rich CNBr peptides of collagen as standards, and sedimentation-equilibrium ultracentrifugation, gave Mr values of approximately 15 000-18 000 and approximately 17 385, respectively. These latter values correspond well with those reported earlier and with the Mr of the major amelogenin computed from recent amino acid sequence data (approximately 19 000). It is concluded that the recently described, highest Mr amelogenin of Mr = 26 000-30 000 is not a new component but is identical to the proline-rich components having relative molecular masses ranging from 15 000 to 18 000 described much earlier by several groups of workers.     

The endo-(1→4)-β- -glucanase system of Penicillium pinophilum cellulase: Isolation, purification, and characterization of five major endoglucanase components

Five major endo-(1→4)-β- -glucanases (I–V) have been isolated from a cellulase preparation of P. pinophilum. The pI values for I–V were 7.4, 4.8, 4.1, 3.7, and 4.0, respectively, and the respective molecular weights were 25,000, 39,000, 62,500, 54,000, and 44,500, when determined by SDS-gel electrophoresis. Endoglucanase V was optimally active at 65–70° and I–IV were most active at 50–60°. The pH optima of I and III–V were in the range 4.0–5.0. Antiserum prepared to I reacted only with I; II antiserum reacted only with II. Endoglucanases I and V were more random in their attack on CM-cellulose and H₃PO₄-swollen cotton cellulose, and showed no activity against cello-oligosaccharides containing less than five -glucose residues, whereas III and IV were active against all the cello-oligosaccharides tested and acted in a less random manner, and II was intermediate in its catalytic action. III was adsorbed completely on both Avicel PH101 and H₃PO₄-swollen cellulose, whereas IV was not adsorbed. The endoglucanases I–V have distinct roles in the digestion of cellulose.     

Liquid chromatography combined with mass spectrometry for the investigation of endoglucanase selectivity on carboxymethyl cellulose

Endoglucanases are useful tools in the chemical structure analysis of cellulose derivatives. However, knowledge on the endoglucanase selectivity, which is of central importance for data interpretation, is still limited. In this study, new reverse-phase liquid chromatography mass spectrometry (LC–MS) methods were developed to investigate the selectivity of the endoglucanases Cel5A, Cel7B, Cel45A, and Cel74A from the filamentous fungus Trichoderma reesei. The aim was to improve the identification of the regioisomers in the complex mixtures that are obtained after enzymatic hydrolysis. Reduction followed by per-O-methylation was performed in order to improve the separation in reverse-phase LC, increase MS sensitivity, and to facilitate structure analysis by MS/MS of O-carboxymethyl glucose and cellooligosaccharides. The cellulose selective enzymes that were investigated displayed interesting differences in enzyme selectivity on CMC substrates.     

A model for the Escherichia coli FtsB/FtsL/FtsQ cell division complex

Background

Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy) database.

Results

Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion.

Conclusions

Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient.     

Cellulase complex from Chaetomium cellulolyticum: isolation and properties of major components.

Four major components of the cellulase complex of Chaetomium cellulolyticum have been isolated by gel-filtration, ion-exchange chromatography on DEAE-Toyopearl and Macro Prep Q, and chromatofocusing on Mono P. These components include three endoglucanases (19, 35, and 40 kD) and a cellobiohydrolase (45 kD). The isoelectric points of the enzymes vary from 3.8 to 4.2. The optimal pH values for catalytic activity are in the range 4.5-6.0, and the optimal temperatures are in the range 60-70 degrees C. Of these enzymes the 19 kD endoglucanase is the most stable; it retained high activity within a broad pH range (from 5.0 to 9.6) at 50 degrees C for 3 h. This enzyme also had the highest topolytic activity determined by the efficiency of removal of indigo from the surface of cotton.