Electrophoretic and enzymatic studies on the crude extracellular enzyme system of the cellulolytic bacterium Cellulomonas sp. ATCC 21399

Cellulomonas sp. ATCC 21399 produced extracellular enzyme activities against Avicel, H(3)PO(4)-swollen Avicel, carboxymethylcellulose, (1-3, 1-4)-beta-D-heteroglucan, xylan, galactomannan, and amylose drying growth on microcrystalline cellulose. No extracellular cellobiase activity was produced. Crossed immunoelectrophoresis of the crude extracellular enzyme system revealed 15 immunologically distinct immunoprecipitates. The immunoprecipitates of endoglucanase A, endoglucanase B and the xylanase appeared heterogeneous with several optima, whereas the immunoprecipitates of endoglucanase C and the amylase appeared homogeneous. The heterogeneity of endoglucanase A, endoglucanase B and xylanase was also visualized using electrofocusing-immunoelectrophoresis. Electro-focusing could resolve the activity against carboxymethylcellulose into six peaks, whereas only one peak of activity against Avicel was observed. The later peak coincided with the major peak of activity against carboxymethylcellulose with isoelectric point between pH 4.0-5.0.     

Degradation of microcrystalline cellulose: synergism between different endoglucanases of Cellulomonas sp. ATCC 21399

Three immunologically and enzymatically distinct endoglucanases of Cellulomonas sp. ATCC 21399 were purified previously. Endoglucanase A and endoglucanase B acted synergistically on microcrystalline cellulose (Avicel), whereas no synergistic action was observed between endoglucanase B or endoglucanase C. Only endoglucanase A was capable of hydrolyzing Avicel when acting alone and this enzyme resulted in "short fiber formation" when acting on Avicel. The end product of hydrolysis of acid swollen Avicel produced by the three endoglucanases was in all cases dominated by cellobiose and showed lower content of glucose and cellotriose. Higher cellodextrins appeared as transient end products. The results indicate that the function of endoglucanase A in the cellulase system of Cellulomonas might be very similar to the function of the cellobiohydrolases of Trichoderma reesei.     

Purification of an extracellular cellulose-binding endoglucanase of Cellulomonas sp. ATCC 21399 by affinity chromatography on H3PO4-swollen cellulose

A cellulose-binding endoglucanase (endoglucanase A) of Cellulomonas sp. ATCC 21399 was purified to immunological homogeneity by affinity chromatography ob H(3)PO(4)-swollen cellulose. This method of purification turned out to be an easy and very gentle method for obtaining a high yield of cellulose-binding endoglucanase. The purified enzyme was immunologically homogeneous but appeared heterogeneous when analyzed by denaturing polyacrylamide gel electrophoresis. In addition to the cellulose-binding of endoglucanase A, the enzyme also had a strong affinity for Concanavaline A, indicating that the enzyme was glycosylated. Purified endoglucanase A showed an endo mode of action on carboxymethylcellulose. The enzyme could hydrolyze microcrystalline cellulose when acting alone, and the enzyme had a high specific activity on H(3)PO(4)-swollen cellulose.     

Purification and characterization of endoglucanase and exoglucanase components from Trichoderma viride

Major cellulase components—four endoglucanases (Endo I, II, III and IV) and one exoglucanase (Exo II)—were isolated from a commercial cellulase preparation derived from Trichoderma viride by a series of chromatographic procedures. The average molecular weights were determined by SDS-polyacrylamide gel electrophoresis. Endos I, III and IV, with M_rs of 52,000, 42,000 and 38,000, respectively, exhibited a more random hydrolytic mode on carboxymethylcellulose (CMC) than Endo II, which has an M_r of 60,000. Endo II showed low activity towards CMC, but out of the four purified endoglucanases this enzyme had the highest specific activity against Avicel. In the hydrolysis of H₃PO₄-swollen cellulose by Endos I, III and IV, cellobiose was the major product, but equimolar amounts of glucose and cellobiose were formed by Endo II. Exo II, with an M_r of 62,000, released cellobiose as the main product in the hydrolysis of H₃PO₄-swollen cellulose, but glucose was negligible. The combination of Endo I, II, III or IV with Exo II resulted in a synergistic effect in the degradation of Avicel at various combination ratios of these enzymes; the specific optimum ratio of endoglucanase to exoglucanase was largely dependent upon the random hydrolytic mode of the endoglucanase. On the other hand, adsorption of cellulase components was found apparently to obey the Langmuir isotherm, and the thermodynamic parameter (ΔH) was calculated from the adsorption equilibrium constant (K). The enthalpies of adsorption of the endoglucanases were in the range of −2.6–−7.2 KJmol⁻¹, much smaller than that of Exo II (−19.4 KJmol⁻¹). This suggest that Exo II shows stronger preferential adsorption than endoglucanases, and that the enthalpy of adsorption will be effective in distinguishing endoglucanase from exoglucanase.     

Characterization of an unglycosylated low molecular weight 1,4-β-glucan-glucanohydrolase of Trichoderma reesei

A low molecular weight endoglucanase (1,4-beta-glucan glucanohydrolase E.C.3.2.1.4) was purified to homogeneity by a two-step procedure from 7 day old culture filtrates of Trichoderma reesei. The endoglucanase was obtained by BioGel A 0.5 m gel chromatography followed by preparative PAGIF. The purified endoglucanase was homogeneous upon titration curve separation. Enzyme characteristics were: Mr 25 kDa, pI 7.5. The amino acid composition is predominantly neutral (mainly glycine). The N-terminus is arginine. The pH-optimum for this endoglucanase was 5.8 and its optimal temperature was at 52 degrees C. The activity of this endoglucanase gave a strong increase in CMC-fluidity with only a small release of reducing sugars. The endoglucanase was 0.2% of total culture medium protein content. The reducing sugars upon CMC digestion were G1-G4. The enzyme had no specificity towards crystalline cellulose (Avicel) or xylan. The endoglucanase is not a glycoprotein.     

A comparative analysis of two cDNA clones of the cellulase gene family from anaerobic fungus Piromyces rhizinflata

Cellulase family and some other glycosyl hydrolases of anaerobic fungi inhabiting the digestive tract of ruminants are believed to form an enzyme complex called cellulosome. Study of the individual component of cellulosome may shed light on understanding the organization of this complex and its functional mechanism. We have analysed the primary sequences of two cellulase clones, cel5B and cel6A, isolated from the cDNA library of ruminal fungus, Piromyces rhizinflata strain 2301. The deduced amino acid sequences of the catalytic domain of Cel5B, encoded by cel5B, showed homology with the subfamily 4 of the family 5 (subfamily 5(4)) of glycosyl hydrolases, while cel6A encoded Cel6A belonged to family 6 of glycosyl hydrolases. Phylogenetic tree analysis suggested that the genes of subfamily 5(4) glycosyl hydrolases of P. rhizinflata might have been acquired from rumen bacteria. Cel5B and Cel6A were modular enzymes consisting of a catalytic domain and dockerin domain(s), but not a cellulose binding domain. The occurrence of dockerin domains indicated that both enzymes were cellulosome components. The catalytic domain of the Cel5B (Cel5B') and Cel6A (Cel6A') recombinant proteins were purified. The optimal activity conditions with carboxymethyl cellulose (CMC) as the substrate were pH 6.0 and 50 degrees C for Cel5B', and pH 6.0 and 37-45 degrees C for Cel6A'. Both Cel5B' and Cel6A' exhibited activity against CMC, barley beta-glucan, Lichenan, and oat spelt xylan. Cel5B' could also hydrolyse p-nitrophenyl-beta-d-cellobioside, Avicel and filter paper while Cel6A' did not show any activity on these substrates. It is apparent that Cel6A' acted as an endoglucanase and Cel5B' possessed both endoglucanase and exoglucanase activities. No synergic effect was observed for these recombinant enzymes in vitro on Avicel and CMC.     

Purification and properties of the endoglucanase C of Clostridium thermocellum produced in Escherichia coli

The celC gene, which codes for a new endoglucanase of Clostridium thermocellum, termed endoglucanase C, was found to be expressed when cloned in Escherichia coli. The enzyme was purified to electrophoretic homogeneneity from E. coli and its biochemical properties were studied. It differs from the previously studied endoglucanases A and B. In particular, endoglucanase C displays features common to endo- and exoglucanases, since it had a high activity on carboxymethylcellulose and on p-nitrophenyl-beta-D-cellobioside where only the agluconic bond was split. In addition, the enzyme was able to release cellobiose units from G3, G4 and G5 cellodextrins. Endoglucanase C was characterized by Western blot in a culture supernatant from C. thermocellum grown on cellulose, using an antiserum raised against the enzyme produced by E. coli.     

Detection and characterization of the specific and nonspecific endoglucanases of Trichoderma reesei: Evidence demonstrating endoglucanase activity by cellobiohydrolase II

The cellulase enzyme system of Trichoderma reesei RUT C-30 has been separated by DEAE ion exchange chromatography into four fractions. Their specificity towards substituted cellulose and cellooligosaccharides was revealed by analytical IEF and activity stains. Fraction EGI (26% of the total protein) exhibited mainly endoglucanase activity on carboxymethylcellulose (CMC) whereas endoglucanases EGII and EGIII (15% of the total protein) showed high activity towards CMC as well as xylan, 4-methylumbelliferyl cellobioside [MeUmb(Glc)₂] and p-nitrophenyl lactoside (pNPL). A subfraction of EGI (pI 5.9) which has been described in the literature as a cellobiohydrolase (CBHII) was isolated by preparative isoelectric focusing, and was shown to have only 3 U CMCase activity per milligram. Turbidimetric measurements and phase contrast microscopy demonstrated differences between endoglucanase and cellobiohydrolase behaviour during the hydrolysis of purified cellulose (Solka Floc BW-40). Treatment of the purified cellulose with endoglucanases resulted in fibre breakdown into small particles. This was contrasted with no morphological change to the fibres when contacted with the cellobiohydrolase. By this technique it was revealed that the EGI subfraction (pI 5.9) behaves as an endoglucanase and not as a cellobiohydrolase. Incubation of this enzyme with acid-swollen cellulose resulted in cellotriose production, as it did with other endoglucanases which exhibited CMCase activities >; 100 U mg⁻¹. Cellotriose was not present during the hydrolysis of acid-swollen cellulose with the CBHI fraction.     

Production and Characteristics of Avicel-Disintegrating Endoglucanase from a Protease-Negative Humicola grisea var. thermoidea Mutant

Mutational experiments were performed to decrease the protease productivity of Humicola grisea var. thermoidea YH-78 using UV light and N-methyl-N′-nitro-N-nitrosoguanidine. A protease-negative mutant, no. 140, exhibited higher endoglucanase activity than the parent strain in mold bran culture at 50°C for 4 days. The culture extract rapidly disintegrated filter paper but produced a small amount of reducing sugar. About 30% of total endoglucanase activity in the extract was adsorbed onto Avicel. The electrophoretically homogeneous preparation of Avicel-adsorbable endoglucanase (molecular weight, 128,000) showed intensive filter-paper-disintegrating activity but did not release reducing sugar. The preparation also exhibited a highly synergistic effect with the cellulase preparation from Trichoderma reesei in the hydrolysis of microcrystalline cellulose. This endoglucanase was observed via scanning electron microscopy to disintegrate Avicel fibrils layer by layer from the surface, yielding thin sections with exposed chain ends. A mutant, no. 191, producing higher protease activity and an Avicel-unadsorbable, Avicel-nondisintegrating endoglucanase was isolated. The purified enzyme (molecular weight, 63,000) showed no disintegrating activity on filter paper and Avicel and a less synergistic effect with the T. reesei cellulase in hydrolyzing microcrystalline cellulose than did the former enzyme. Endoglucanase was therefore divided into two types, Avicel disintegrating and Avicel nondisintegrating.     

Purification and biochemical characterization of endoglucanase from Penicillium pinophilum MS 20

Cellulases find increasing prominence in sustainable production of fuel and feedstock from lignocellulosic biomass. The purification and biochemical characterization of individual components of cellulase complex is important to understand the mechanism of their action for the solubilization of crystalline cellulose. In this study, an extra-cellular endoglucanase isolated from culture filtrate of Penicillium pinophilum MS 20 was purified to homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and gel filtration. The purified endoglucanase (specific activity 69 U/mg) was a monomeric protein with molecular mass of 42 kDa, as determined by SDS-PAGE. The endoglucanase was active over a broad range of pH (4-7) with maximum activity at pH 5 and showed optimum temperature of 50 degrees C. It retained 100% activity at 50 degrees C for 6 h and half- lives of 4 h and 3 h at 60 degrees C and 70 degrees C, respectively. The kinetic constants for the endoglucanase determined with carboxymethyl cellulose as substrate were V(max) of 72.5 U/mg and apparent K(m) of 4.8 mg/ml. The enzyme also showed moderate activity towards H3PO4 swollen cellulose and p-nitrophenyl beta-D-glucoside, but no activity towards filter paper, Avicel and oat spelt xylan. The activity was positively modulated by 47, 32 and 25% in the presence of Co2+, Zn2+ and Mg2+, respectively to the reaction mixture. The wide pH stability (4-7) and temperature stability up to 50 degrees C of endoglucanase makes the enzyme suitable for use in cellulose saccharification at moderate temperature and pH.     

The cellulase of Trichoderma koningii. Purification and properties of some endoglucanase components with special reference to their action on cellulose when acting alone and in synergism with the cellobiohydrolase.

1. Four principal endoglucanase components of Trichoderma koningii cellulase were separated and purified by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE- and sulphoethyl-Sephadex and isoelectric focusing. 2. All four endoglucanases hydrolysed CM-cellulose, H3PO4-swollen cellulose, cellotetraose and cellopentaose, but differed in the rate and mode of attack. 3. Attack on cotton fibre by the endoglucanases was minimal, but resulted in changes that were manifested by an increased capacity for the uptake of alkali, and a decrease in tensile strength. 4. All four endoglucanases acted synergistically with the exoglucanase [cellobiohydrolase; Wood & McCrae (1972) Biochem. J. 128, 1183-1192] of T. koningii during the early stages of the breakdown of cotton fibre, but only two could produce extensive solubilization of cotton cellulose when acting in admixture with the exoglucanase component. 5. The mode of action of the enzymes is discussed in relation to these synergistic effects. It is suggested that the results are compatible with the interpretation that the 'crystalline' areas of cotton cellulose are hydrolysed only by those endoglucanases capable of forming of forming an enzyme-enzyme complex with the cellobiohydrolase on the surface of the cellulose chains.     

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)     

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)     

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.     

The brown-rot basidiomycete Fomitopsis palustris has the endo-glucanases capable of degrading microcrystalline cellulose

Two endoglucanases with processive cellulase activities, produced from Fomitopsis palustris grown on 2% microcrystalline cellulose (Avicel), were purified to homogeneity by anion-exchange and gel filtration column chromatography systems. SDS-PAGE analysis indicated that the molecular masses of the purified enzymes were 47 kDa and 35 kDa, respectively. The amino acid sequence analysis of the 47-kDa protein (EG47) showed a sequence similarity with fungal glycoside hydrolase family 5 endoglucanase from the white-rot fungus Phanerochaete chrysosporium. N-terminal and internal amino acid sequences of the 35-kDa protein (EG35), however, had no homology with any other glycosylhydrolases, although the enzyme had high specific activity against carboxymethyl cellulose, which is a typical substrate for endoglucanases. The initial rate of Avicel hydrolysis by EG35 was relatively fast for 48 h, and the amount of soluble reducing sugar released after 96 h was 100 microg/ml. Although EG47 also hydrolyzed Avicel, the hydrolysis rate was lower than that of EG35. Thin layer chromatography analysis of the hydrolysis products released from Avicel indicated that the main product was cellobiose, suggesting that the brown-rot fungus possesses processive EGs capable of degrading crystalline cellulose.     

Hydrolytic enzyme(s) production in Micrococcus roseus growing on different cellulosic substrates

Micrococcus roseus (G12) isolated from higher termite Odontotermes obesus gut exhibited cellulose digesting properties. A lignocellulosic substrate, rice husk induced endoglucanase, β-glucosidase, β-xylanase and β-xylosidase production. Besides rice husk, CMC also induced endoglucanase production. β-Glucosidase activity was quite pronounced when rice husk was supplemented with CMC or cellobiose. Both β-xylanase and β-xylosidase activities could be induced by xylan as well as xylobiose, whereas CMC induced partial activity. Endoglucanase and β-xylanase enzymes were secreted into the culture medium, whereas β-glucosidase and β-xylosidase activities were intracellular in nature. Enzyme production was subject to end product inhibition. The extracellular enzyme(s) possessed the potential to saccharify rice husk, xylan and CMC to reducing sugars.     

The cellulase of Trichoderma viride. Purification, characterization and comparison of all detectable endoglucanases, exoglucanases and beta-glucosidases

Six endoglucanases (Endo I; II; III; IV; V; VI), three exoglucanases (Exo I; II; III) and a beta-glucosidase (beta-gluc I) were isolated from a commercial cellulase preparation derived from Trichoderma viride, using gel filtration on Bio-Gel, anion exchange on DEAE-Bio-Gel A, cation exchange on SE-Sephadex and affinity chromatography on crystalline cellulose. Molecular masses were determined by polyacrylamide gel electrophoresis. One group of endoglucanases (Endo I, Endo II and Endo IV) with Mr of 50 000, 45 000 and 23 500 were more random in their attack on carboxymethylcellulose than another group (Endo III, Endo V and Endo VI) showing Mr of 58 000, 57 000 and 53 000 respectively. Endo III was identified as a new type of endoglucanase with relatively high activity on crystalline cellulose and moderate activity on carboxymethylcellulose. Exo II and Exo III with Mr of 60 500 and 62 000 respectively showed distinct adsorption affinities on a column of crystalline cellulose and could be eluted by a pH gradient to alkaline regions. These enzymes were cellobiohydrolases as judged by high-pressure liquid chromatography of the products obtained from incubation with H3PO4-swollen cellulose. It was concluded that these exoglucanases are primarily active on newly generated chain ends. Exo I was essentially another type of exoglucanase which in the first instance was able to split off a cellobiose molecule from a chain end and then hydrolyse this molecule in a second step to two glucose units beta-Gluc I was a new type of aryl-beta-D-glucosidase which had no activity on cellobiose. The enzyme had a Mr of 76 000 and was moderately active on CM-cellulose, crystalline cellulose and xylan and highly active on p-nitrophenyl-beta-D-glucose and p-nitrophenyl-beta-D-xylose.     

Characterization and substrate specificity of an endo-beta-1,4-D-glucanase I (Avicelase I) from an extracellular multienzyme complex of Bacillus circulans.

An endo-1,4-beta-D-glucanase I (Avicelase I; EC 3.2.1.4) was purified to homogeneity from an extracellular celluloxylanosome of Bacillus circulans F-2. The purification in the presence of 6 M urea yielded homogeneous enzyme. The enzyme had a monomeric structure, its relative molecular mass being 75 kDa as determined by gel filtration and 82 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI was 5.4, and the N-terminal amino acid sequence was ASNIGGWVGGNESGFEFG. The optimal pH was 4.5, and the enzyme was stable at pH 4 to 10. The enzyme has a temperature optimum of 50 degrees C, it was stable at 55 degrees C for 46 h, and it retains approximately 20% of its activity after 30 min at 80 degrees C. It showed high-level activity towards carboxymethyl cellulose (CMC) as well as p-nitrophenyl-beta-D-cellobioside, 4-methylumbelliferyl cellobioside, xylan, Avicel, filter paper, and some cello-oligosaccharides. Km values for birch xylan, CMC, and Avicel were 4.8, 7.2, and 87.0 mg/ml, respectively, while Vmax values were 256, 210, and 8.6 mumol x min-1 x mg-1, respectively. Cellotetraose was preferentially cleaved into cellobiose (G2) plus G2, and cellopentaose was cleaved into G2 plus cellotriose (G3), while cellohexaose was cleaved into cellotetraose plus G2 and to a lesser extent G3 plus G3. G3 was not cleaved at all. G2 was the main product of Avicel hydrolysis. Xylotetraose (X4) and xylobiose (X2) were mainly produced by the enzyme hydrolysis of xylan. G2 inhibited the activity of carboxymethyl cellulase and Avicelase, whereas Mg2+ stimulated it. The enzyme was completely inactivated by Hg2+, and it was inhibited by a thiol-blocking reagent. Hydrolysis of CMC took place, with a rapid decrease in viscosity but a slow liberation of reducing sugars. On the basis of these results, it appeared that the cellulase should be regarded as endo-type cellulase, although it hydrolyzed Avicel.     

The N-terminal region of an endoglucanase from Pseudomonas fluorescens subspecies cellulosa constitutes a cellulose-binding domain that is distinct from the catalytic centre

The substrate specificity of an endoglucanase (EGB) from Pseudomonas fluorescens subspecies cellulosa was determined. The enzyme was most active against barley beta-glucan, but showed significant activity against amorphous and crystalline cellulose. EGB was purified to homogeneity by affinity chromatography with crystalline cellulose (Avicel). The Mr of the purified enzyme was 50,000, which is in good agreement with the size of EGB deduced from the nucleotide sequence of the celB gene, coding for EGB. The N-terminal region of the mature form of EGB showed strong homology to another endoglucanase and to a xylanase expressed by the same organism; homologous sequences included highly conserved serine-rich regions. Truncated forms of celB, in which the gene sequence encoding the conserved domain had been deleted, directed the synthesis of a functional endoglucanase that did not bind to crystalline cellulose. This indicates that the conserved region of endoglucanases and xylanases expressed by P. fluorescens subsp. cellulosa constitutes a cellulose-binding domain, which is distinct from the active centre. The possible role of this substrate-binding region is discussed.     

Purification and characterization of five cellulases and one xylanase from Penicillium brasilianum IBT 20888

The filamentous fungus Penicillium brasilianum IBT 20888 was cultivated on a mixture of 30 g l⁻¹ cellulose and 10 g l⁻¹ xylan for 111 h and the resulting culture filtrate was used for protein purification. From the cultivation broth, five cellulases and one xylanase were purified. Hydrolysis studies revealed that two of the cellulases were acting as cellobiohydrolases by being active on only microcrystalline cellulose (Avicel). Three of the cellulases were active on both Avicel and carboxymethyl cellulose indicating endoglucanase activity. Two of these showed furthermore mannanase activity by being able to hydrolyze galactomannan (locust bean gum). Adsorption studies revealed that the smaller of the two enzymes was not able to bind to cellulose. Similarity in molecular mass, pI and hydrolytic properties suggested that these two enzymes were identical, but the smaller one was lacking the cellulose-binding domain or an essential part of it. The basic xylanase (pI>9) was only active towards xylan. Two of the purified cellulases with endoglucanase activity were partly sequenced and based on sequence homology with known enzymes they were classified as belonging to families 5 and 12 of the glycosyl hydrolases.