Comparison of Cellulolytic Activities in Clostridium thermocellum and Three Thermophilic, Cellulolytic Anaerobes

Avicelase, carboxymethyl cellulase (CMCase), and β-glucosidase activities have been compared between Clostridium thermocellum and three extremely thermophilic, cellulolytic anaerobes, isolates TP8, TP11, and KT8. The three isolates were all small, gram-negative staining, oval-ended rods which occurred singly and, at exponential phase, in long chains. They were nonflagellated and no spores were visible. The KT8 and TP11 isolates caused clumping of the cellulose during growth. In all four organisms the CMCase activity paralleled cell growth; however, in C. thermocellum and TP8 the avicelase activity did not increase until early stationary phase. Total CMCase activity in C. thermocellum was significantly higher than in the three isolates; however, avicelase activities were much more comparable among the four organisms. C. thermocellum produced higher levels of ethanol, and all four organisms produced similar concentrations of acetate. The amounts of free and bound CMCase and avicelase activities were investigated. In C. thermocellum and TP8 most of the CMCase and avicelase activities were bound to the cellulose in the medium. In contrast, most of the CMCase activity in TP11 and KT8 was free in the culture supernatant; a significant percentage of avicelase activity was also free. The TP8 isolate was also grown on a defined medium with urea as sole nitrogen source and cellulose serving as the carbon source. Under these conditions the pattern of enzyme production was the same as that in the enriched medium, although the level of that production was considerably reduced.     

Assimilation of Cellooligosaccharides by a Cell Surface-Engineered Yeast Expressing β-Glucosidase and Carboxymethylcellulase from Aspergillus aculeatus

Since Saccharomyces cerevisiae lacks the cellulase complexes that hydrolyze cellulosic materials, which are abundant in the world, two types of hydrolytic enzymes involved in the degradation of cellulosic materials to glucose were genetically co-immobilized on its cell surface for direct utilization of cellulosic materials, one of the final goals of our studies. The genes encoding FI-carboxymethylcellulase (CMCase) and β-glucosidase from the fungus Aspergillus aculeatus were individually fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin and introduced into S. cerevisiae. The delivery of CMCase and β-glucosidase to the cell surface was carried out by the secretion signal sequence of the native signal sequence of CMCase and by the secretion signal sequence of glucoamylase from Rhizopus oryzae for β-glucosidase, respectively. The genes were expressed by the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase and β-glucosidase activities were detected in the cell pellet fraction, not in the culture supernatant. The display of CMCase and β-glucosidase proteins on the cell surface was confirmed by immunofluorescence microscopy. The cells displaying these cellulases could grow on cellobiose or water-soluble cellooligosaccharides as the sole carbon source. The degradation and assimilation of cellooligosaccharides were confirmed by thin-layer chromatography. This result showed that the cell surface-engineered yeast with these enzymes can be endowed with the ability to assimilate cellooligosaccharides. This is the first step in the assimilation of cellulosic materials by S. cerevisiae expressing heterologous cellulase genes.     

Processive Endoglucanase Active in Crystalline Cellulose Hydrolysis by the Brown Rot Basidiomycete Gloeophyllum trabeum

Brown rot basidiomycetes have long been thought to lack the processive cellulases that release soluble sugars from crystalline cellulose. On the other hand, these fungi remove all of the cellulose, both crystalline and amorphous, from wood when they degrade it. To resolve this discrepancy, we grew Gloeophyllum trabeum on microcrystalline cellulose (Avicel) and purified the major glycosylhydrolases it produced. The most abundant extracellular enzymes in these cultures were a 42-kDa endoglucanase (Cel5A), a 39-kDa xylanase (Xyn10A), and a 28-kDa endoglucanase (Cel12A). Cel5A had significant Avicelase activity—4.5 nmol glucose equivalents released/min/mg protein. It is a processive endoglucanase, because it hydrolyzed Avicel to cellobiose as the major product while introducing only a small proportion of reducing sugars into the remaining, insoluble substrate. Therefore, since G. trabeum is already known to produce a β-glucosidase, it is now clear that this brown rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.     

Biochemical Characterization of a Protease Involved in the Processing of a Streptomyces reticuli Cellulase (Avicelase)

A 36-kDa protease from Streptomyces reticuli had recently been shown to be responsible for the in vivo and in vitro processing of the 82-kDa cellulase (Avicelase) Cel-1 from S. reticuli to a 42-kDa truncated enzyme. It was induced only in the presence of Avicel, hydroxyethylcellulose, and xylan. The addition of the nonionic detergent Tween 80 to the culture medium containing Avicel as the carbon source led to a 10-fold increase in extracellular proteolytic activity. The protease, which has an isoelectric point of 3.9, was purified to homogeneity from the culture filtrate by a combination of anion-exchange and hydrophobic-interaction chromatographies and was characterized biochemically. The enzyme hydrolyzed gelatin and the chromogenic substrates Azocoll, Azocasein, and Azoalbumin. Its highest activity was determined between pH 7.0 and 7.7 and at 55°C. The proteolytic activity was inhibited by 1,10-phenanthroline and EDTA; however, no metal ions were detected to be associated with the protein. The protease was stable in the presence of 1 M urea and 0.01 M sodium dodecyl sulfate. The inhibitory effect of alpha-2-macroglobulin indicated an endo-mode of proteolytic cleavage. Studies with lectins and sugar analysis by mass spectroscopy indicated that the cellulase (Avicelase) Cel-1 was neither N nor O glycosylated. Its processing by the protease occurred at temperatures ranging from 30 to 55°C, pH 7.5, in the presence of 2 mM dithiothreitol.     

Lignocellulolytic Enzymes Produced by Volvariella volvacea, the Edible Straw Mushroom

Volvariella volvacea, commonly known as the straw or paddy mushroom, had the following growth characteristics: minimum temperature, 25°C; optimal temperature, 37°C; maximal temperature, 40°C; pH optimum 6.0. Optimal pH for cellulase production was 5.5. The optimal initial pH for cellulase production and mycelial growth was found to be 6.0. The pH and temperature optima for cellulolytic activity were 5.0 and 50°C, respectively. Maximal cellulolytic activity was obtained within 5 days in shake-flask culture. The cellulases were found to be partly cell free and partly cell bound during growth on microcrystalline cellulose. The endoglucanase activity was primarily extracellular, and β-glucosidase activity was found exclusively extracellularly. Weak cellulase activity was detected when cells were grown on cellobiose and lactose. V. volvacea could not digest the lignin portion of newspaper in shake-flask cultivation. Phenol oxidase, an important enzyme in lignin biodegradation, also was lacking in the cell-free filtrate. However, the organism oxidized phenolic compounds when it was cultured on agar plates containing commercial lignin.     

Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as “transceptors” with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.     

Subunit Composition and Glycosidic Activities of the Cellulase Complex from Clostridium thermocellum JW20

The subunit composition of the extracellular complex from Clostridium thermocellum was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Twenty-six bands, representing proteins with apparent molecular sizes ranging from 37,500 to 185,000 Da, could be detected by silver staining. Cultivation of the bacteria with the substrate Avicel, Sigma cellulose, Solka floc, or cellobiose as the carbon source had no influence on the number of detectable protein bands. By activity staining with the substrate carboxymethyl cellulose or xylan added to the SDS-polyacrylamide gels, 15 of the 26 bands exhibited endoglucanase activity and 13 showed xylanase activity. In 8 of the 26 bands, both activities could be found. As minor activities, β-glucosidase, β-xylosidase, β-galactosidase, and β-mannosidase activities could be demonstrated in the cellulase complex. Upon measuring the release of para-nitrophenol (PNP) from PNP-cellobioside and determining the amount of glucose formed, the presence of exoglucanase activity was indicated. Upon glycoprotein staining of SDS-polyacrylamide gels, 14 of the 26 bands reacted positive, indicating the glycoprotein nature of the respective proteins. Four proteins (apparent molecular sizes, 58,000, 72,500, 94,000, and 110,000 Da) could be enriched from the originally bound cellulase complex by preparative SDS-PAGE. The two smaller proteins exhibited xylanase activity, whereas the 94,000-Da protein had endo- and exoglucanase activity, and the 110,000-Da protein degraded PNP-pyranosides.     

Isolation of Cellulolytic Anaerobic Extreme Thermophiles from New Zealand Thermal Sites

Avicel enrichment cultures from 47 thermal-pool sites in the New Zealand Rotorua-Taupo region were screened for growth and carboxymethyl cellulase activity at 75°C. Eight anaerobic cellulolytic cultures were obtained. The effect of temperature on carboxymethyl cellulase activity was measured, and bacteria were isolated from the five best cultures. Bacteria from two sources designated TP8 and TP10 grew at 75°C, accumulated reducing sugar in the growth medium and gave free cellulases with avicelase activity. Bacteria from sources designated Tok4, Tok8, and Wai21 grew at 75°C, accumulated no free sugars in the medium, and gave free carboxymethyl cellulases with virtually no avicelase activity. All were obligate anaerobic nonsporeforming rods which stained gram negative, grew on pentoses as well as hexoses, and gave ethanol and acetate as major fermentation end products. The isolated strain which produced the most active and stable cellulases (trivially designated TP8.T) had lower rates of free endocellulase accumulation at 75°C than did Clostridium thermocellum at 60°C, but its cellulase activity against avicel and filter paper in culture supernatants was comparable. Tested at 85°C, TP8.T carboxymethyl cellulases included components which were very stable, whereas C. thermocellum carboxymethyl cellulases were all rapidly inactivated. The TP8.T avicelase activity was relatively unaffected by Triton X-100, EDTA, and dithiothreitol. Evidence was obtained for the existence of unisolated, cellulolytic extreme thermophiles producing cellulases which were more stable and active than those from TP8.T.     

Production and Purification of Thermostable Cellulases from Humicola insolens YH-8

Humicola insolens YH-8, a thermophilic fungus isolated from manure and compost heaps, produced a significant amount of thermostable cellulases in cultures on wheat bran medium (50°C, 4 days). The mold bran extract hydrolyzed Avicel, CMC and newsprint at 90%, 45% and 35%, respectively, to glucose. Then, Avicelase and CMCase were purified from the culture extract by adsorption onto Avicel, heat and acid treatment and consecutive column chromatographies to a homogeneous state on polyacrylamide gel disc electrophoresis. The purified cellulases, especially CMCase, was found highly thermostable. The optimal temperature of both enzymes was 50°C. Avicelase was stable after heating at 65°C for 5 mm and CMCase retained 45% of the original activity after heating at 95°C for 5 min.     

Saccharification of Complex Cellulosic Substrates by the Cellulase System from Clostridium thermocellum

True cellulase activity has been demonstrated in cell-free preparations from the thermophilic anaerobe Clostridium thermocellum. Such activity depends upon the presence of Ca²⁺ and a thiol-reducing agent of which dithiothreitol is the most promising. Under these conditions, native (cotton) and derived forms of cellulose (Avicel and filter paper) were all extensively solubilized at rates comparable with cellulase from Trichoderma reesei. Maximum activity of the Clostridium cellulase was displayed at 70°C and at pH 5.7 and 6.1 on Avicel and carboxymethylcellulose, respectively. In the absence of substrate at temperatures up to 70°C, carboxymethylcellulase was much more unstable than the Avicel-hydrolyzing activity.     

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.     

Studies on Cellulose Hydrolysis by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus extracellular cellulase extensively hydrolyzed crystalline celluloses such as Avicel (FMC Corp., Food and Pharmaceutical Products Div., Philadelphia, Pa.) but only if it was desalted and supplemented with Ca²⁺. The Ca²⁺ effect was one of increased enzyme stability in the presence of the ion. Although preincubation of the cellulase complex at 40°C for 5 h without added Ca²⁺ had a negligible effect on endoglucanase activity or on the subseqent hydrolysis of amorphous cellulose, the capacity of the enzyme to hydrolyze crystalline cellulose was almost completely lost. Adsorption studies showed that 90% of the Avicel-solubilizing component of the total enzyme preparation bound to 2% Avicel at 40°C. Under these conditions, only 15% of the endoglucanase and 25% of the protein present in the enzyme preparation adsorbed to the substrate. The protein profile of the bound enzyme, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was complex and distinctly different from the profile observed for total cellulase preparations. The specific activity of A. cellulolyticus cellulase with respect to Avicel hydrolysis was compared with that of commercially available Trichoderma reesei cellulase.     

Subcellulosome preparation with high cellulase activity from Clostridium thermocellum

We have prepared a much simpler cellulase preparation than that of cellulosomes from the extracellular broth of Clostridium thermocellum. This "subcellulosome" preparation from C. thermocellum was obtained by column chromatography on CM-Bio-Gel A and then on a lectin-affinity material (Jacalin). The subcellulosome preparation is a macromolecular complex, composed of six main protein subunits (molecular weight, 210,000 to 58,000) revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activities of carboxymethylcellulase (CMCase) and Avicelase are 15- and 8-fold-higher, respectively, than those of crude extracellular cellulase. We could not further fractionate this preparation without denaturing it. The optimum pH and temperature of the subcellulosome preparation are 5.5 to 7.0 and 70 degrees C for CMCase and 5.5 to 7.0 and 65 degrees C for Avicelase. The subcellulosome preparation acted on various types of carboxymethyl cellulose, cellulose, and p-nitrophenyl-beta-D-cellobioside but not on p-nitrophenyl-beta-D-glucoside. Sulfhydryl reagents and N-bromosuccinimide inhibited both CMCase and Avicelase activities, whereas EDTA and o-phenanthroline inhibited Avicelase activity only.     

液态发酵条件下拟康宁木霉8985产纤维素酶能力初探

液态发酵条件下,以微晶纤维素为唯一碳源,比较了拟康宁木霉Trichoderma koningiopsis 8985和里氏木霉T. reesei QM9414产纤维素酶的能力。8985发酵12 h开始产生纤维素酶,36 h时酶活达到产酶峰值的50%,此时QM9414尚未诱导产酶。测定8985发酵84 h时上清液中滤纸纤维素酶、羧甲基纤维素酶、β-葡萄糖苷酶和木聚糖酶的酶活分别为1.06、3.62、1.80和6.67 IU/mL,分别是QM9414上述酶活的1.72、1.70、6.35和1.12倍。8985滤纸纤维素酶酶活的最适反应条件为pH 4.5,反应温度50 ℃,在Fe³⁺ (≤ 4 mmol/L)和Cu²⁺ (0-10 mmol/L)存在条件下酶活稳定。     

Subcellulosome preparation with high cellulase activity from Clostridium thermocellum.

We have prepared a much simpler cellulase preparation than that of cellulosomes from the extracellular broth of Clostridium thermocellum. This "subcellulosome" preparation from C. thermocellum was obtained by column chromatography on CM-Bio-Gel A and then on a lectin-affinity material (Jacalin). The subcellulosome preparation is a macromolecular complex, composed of six main protein subunits (molecular weight, 210,000 to 58,000) revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activities of carboxymethylcellulase (CMCase) and Avicelase are 15- and 8-fold-higher, respectively, than those of crude extracellular cellulase. We could not further fractionate this preparation without denaturing it. The optimum pH and temperature of the subcellulosome preparation are 5.5 to 7.0 and 70 degrees C for CMCase and 5.5 to 7.0 and 65 degrees C for Avicelase. The subcellulosome preparation acted on various types of carboxymethyl cellulose, cellulose, and p-nitrophenyl-beta-D-cellobioside but not on p-nitrophenyl-beta-D-glucoside. Sulfhydryl reagents and N-bromosuccinimide inhibited both CMCase and Avicelase activities, whereas EDTA and o-phenanthroline inhibited Avicelase activity only.     

Cellulolytic Enzyme System of Thermoactinomyces sp. Grown on Microcrystalline Cellulose

The carboxymethyl-cellulase and Avicelase activities of Thermoactinomyces sp., strain YX, were produced simultaneously with cell growth. Throughout the growth phase these activities were primarily extracellular, with up to 50% adsorbed to residual cellulosic substrate at any one time. On the other hand, the β-glucosidase activity was associated with the culture solids throughout the entire fermentation and appears to be intracellular. Preparative isoelectric focusing of the culture filtrate, in the pH range 3 to 5, separated three major fractions with cellulolytic activities towards both carboxymethyl-cellulose and Avicel. Based on the carboxymethyl-cellulase and Avicelase activities of these separated fractions, it was not possible to discriminate between endo- and exoglucanases produced by Thermoactinomyces sp. Analytical isoelectric focusing of culture filtrates obtained throughout the growth phase of Thermoactinomyces indicated that all the extracellular cellulolytic enzymes are produced and released into the culture filtrate simultaneously, with no evidence of a sequential appearance of the various enzymes or isoenzymes.     

Inhibitory evaluation of Curculigo latifolia on α-glucosidase,DPP (IV) and in vitro studies in antidiabetic with molecular docking relevance to type 2 diabetes mellitus

The inhibition of α-glucosidase and DPP enzymes capable of effectively reducing blood glucose level in the management of type 2 diabetes. The purpose of the present study is to evaluate the inhibitory potential of α-glucosidase and DPP (IV) activity including with the 2-NBDG uptake assay and insulin secretion activities through in vitro studies. The selected of active compounds obtained from the screening of compounds by LC-MS were docked with the targeted enzyme that involved in the mechanism of T2DM. From the results, root extracts displayed a better promising outcome in α-glucosidase (IC₅₀ 2.72 ± 0.32) as compared with the fruit extracts (IC₅₀ 3.87 ± 0.32). Besides, root extracts also displayed a better activity in the inhibition of DPP (IV), enhance insulin secretion and glucose uptake activity. Molecular docking results revealing that phlorizin binds strongly with α-glucosidase, DPP (IV) and Insulin receptor (IR) enzymes with achieving the lowest binding energy value. The present work suggests several of the compounds have the potential that contribute towards inhibiting α-glucosidase and DPP (IV) and thus effective in lowering post-prandial hyperglycaemia.     

Binding of Extracellular Carboxymethylcellulase Activity from the Marine Shipworm Bacterium to Insoluble Cellulosic Substrates

The binding of extracellular endoglucanase, a carboxymethylcellulase (CMCase), produced by the marine shipworm bacterium to insoluble cellulose substrates was investigated. Up to 70% of CMCase activity bound to cellulosic substrates, and less than 10% bound to noncellulosic substrates. CMCase binding to cellulose was enhanced in basal salt medium or sodium phosphate buffer containing 0.5 M NaCl. Increased cellulose particle size correlated with decreased CMCase binding. Also, cellulose treated with either 5 N NaOH or commercial cellulase reduced the CMCase binding to these surfaces. Pretreatment of CMCase preparations with 0.01% sodium dodecyl sulfate, 5% β-mercaptoethanol, and 5 mM EDTA or ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) had little effect on binding to cellulose. While pretreatment of CMCase with trypsin, chymotrypsin, and pronase had little effect on CMCase enzymatic activity, the ability to bind to cellulose was greatly diminished by these treatments.     

Comparison of Extracellular Cellulase Activities of Clostridium thermocellum LQRI and Trichoderma reesei QM9414

The crude extracellular cellulase of Clostridium thermocellum LQRI (virgin strain) was very active and solubilized microcrystalline cellulose at one-half the rate observed for the extracellular cellulase of Trichoderma reesei QM9414 (mutant strain). C. thermocellum cellulase activity differed considerably from that of T. reesei as follows: higher endoglucanase/exoglucanase activity ratio; absence of extracellular cellobiase or β-xylosidase activity; long-chain oligosaccharides instead of short-chain oligosaccharides as initial (15-min) hydrolytic products on microcrystalline cellulose; mainly cellobiose or xylobiose as long-term (24-h) hydrolysis products of Avicel and MN300 or xylan; and high activity and stability at 60 to 70°C. Under optimized reaction conditions, the kinetic properties (V_max, 0.4 μmol/min per mg of protein; energy of activation, 33 kJ; temperature coefficient, 1.8) of C. thermocellum cellulose-solubilizing activity were comparable to those reported for T. reesei, except that the dyed Avicel concentration at half-maximal velocity was twofold higher (182 μM). The cellulose-solubilizing activity of the two crude cellulases differed considerably in response to various enzyme inhibitors. Most notably, Ag²⁺ and Hg²⁺ effectively inhibited C. thermocellum but not T. reesei cellulase at <20 μM, whereas Ca²⁺, Mg²⁺, and Mn²⁺ inhibited T. reesei but not C. thermocellum cellulase at >10 mM. Both enzymes were inhibited by Cu²⁺ (>20 mM), Zn²⁺ (>1.0 mM), and ethylene glycol-bis(β-aminoethyl ether)- N,N-tetraacetic acid (>10 mM). T. reesei but not C. thermocellum cellulose-solubilizing activity was 20% inhibited by glucose (73 mM) and cellobiose (29 mM). Both cellulases preferentially cleaved the internal glycosidic bonds of cellooligosaccharides. The overall rates of cellooligosaccharide degradation were higher for T. reesei than for C. thermocellum cellulase, except that the rates of conversion of cellohexaose to cellotriose were equivalent.     

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.