Occurrence and Some Properties of Cellulase in the Filtrates of Conidiospores and Mycelia of Pyricularia oryzae Cavara

Occurrence of cellulase activity was demonstrated in the filtrates of germinating conidiospores and growing mycelia of P. oryzae. Activity and some properties of cellulase in the filtrate of mycelia grown on rice plant powder as carbon source were compared among various strains.

Cellulase activity (C₁ and C_x enzymes; cellulose and carboxymethylcellulose as substrates, respectively) in the filtrate of germinating conidiospores was detected in the pathogenic T–l (Ken 53–33) strain as well as nonpathogenic 0 (THU 3 × 1) strain of P. oryzae. The activity was higher in the former than the latter strains. Cellulase activity (C_x enzyme) in the filtrate of growing mycelia was detected in the four strains used, T–l (Ken 53–33), C–3 (N 87), N–1 (H373), and 0 (THU 3 × 1). Cellulase activity (C_x enzyme) in the filtrate of mycelia was optimal at pH 5.0 and 40°C, and stable up to 40°C. Their properties did not differ significantly except for the pH-activity curve at alkaline side among various strains; but cellulase activity (C₁ enzyme) was found to be correlated with their pathogenicity except for the case of C–3 strain.     

Production of Cellulase(s) byMyricoccum albomyces

The ability ofMyricoccum albomyces to produce extracellular cellulase(s) has been studied in a stationary liquid medium. Different cellulosic carbon sources were used. The organism was able to produce cellulose 1,4-β-cellobiosidase (C₁) and cellulase (C_x) activities. The optimum temperature for C₁ and C_x activity was 45 °C. The optimum pH for C₁ activity was pH 6 while that for C_x was pH 5.     

Partial Purification of Intra- and Extra-cellular Cellulases from Pyricularia oryzae: with Reference to Optimum pH at Alkaline Side

In order to elucidate the relation between the difference in cellulase activity among various strains of P. oryzae and the optimum pH at alkaline side, and also to know the relation between the intra- and extra-cellulases, the elution patterns of the enzymes from the Sephadex G–100 column were compared and the occurrence of the enzyme fractions showing the optimum pH at alkaline side was investigated.

The elution patterns of the intracellular cellulases were shown to be relatively constant, but those of the extracellular enzymes did not. The peak e appeared comparatively constant, but the peak c was considered to undergo some change during the excretion into the medium.

The optimum pH at alkaline side was shown to occur in the peak e among five peaks on Sephadex G–100 of the partially purified intra- and extra-cellular cellulases. The peak seems to be significant for P. oryzae.     

Cellulase production by a thermophilic clostridium species

Strain M7, a thermophilic, anaerobic, terminally sporing bacterium (0.6 by 4.0 μm) was isolated from manure. It degraded filter paper in 1 to 2 days at 60 C in a minimal cellulose medium but was stimulated by yeast extract. It fermented a wide variety of sugars but produced cellulase only in cellulose or carboxymethyl-cellulose media. Cellulase synthesis not only was probably repressed by 0.4% glucose and 0.3% cellobiose, but also cellulase activity appeared to be inhibited by these sugars at these concentrations. Both C₁ cellulase (degrades native cellulose) and C_x cellulase (β-1,4-glucanase) activities in strain M7 cultures were assayed by measuring the liberation of reducing sugars with dinitrosalicylic acid. Both activities had optima at pH 6.5 and 67 C. One milliliter of a 48-h culture of strain M7 hydrolyzed 0.044-meq of glucose per min from cotton fibers. The cellulase(s) from strain M7 was extracellular, produced during exponential growth, but was not free in the growth medium until approximately 30% of the cellulose was hydrolyzed. Glucose and cellobiose were the major soluble products liberated from cellulose by the cellulase. ZnCl₂ precipitation appeared initially to be a good method for the concentration of cellulase activity, but subsequent purification was not successful. Isoelectric focusing indicated the presence of four C_x cellulases (pI 4.5, 6.3, 6.8, and 8.7). The rapid production and high activity of cellulases from this organism strongly support the basic premise that increased hydrolysis of native cellulose is possible at elevated temperature.     

Improvement of Aspergillus oryzae NRRL 3484 by mutagenesis and optimization of culture conditions in solid-state fermentation for the hyper-production of extracellular cellulase

Spore suspensions of Aspergillus oryzae NRRL 3484 were subjected to mutagenesis using ultraviolet-irradiation followed by chemical treatments to improve the biosynthesis of cellulase. Ten mutant strains namely UEAC7, UEAR5, UNAC4, UNAC16, UNAR19, UNBC7, UNBR3, UNBR10, UNBR23 and UNBR25 were selected and their extracellular cellulase activities were assayed. Mutant UNAC4 gave the highest cellulase production [2,455 ± 28 U/g-dry substrate (ds) for filter paper-ase (FP-ase)] in a yield 4-fold exceeding that of the wild type strain (578 ± 5.0 U/g-ds for FP-ase). Rice straw (RS) was used as a sole carbon source for the enzyme production at a concentration of 10 % (w/v). Maximum cellulase production was achieved at initial medium pH 5.5, initial moisture content 77 % and an incubation temperature 28 °C on the fifth day of growth. NH₄Cl proved to be the suitable added nitrogen source for maximum enzyme production followed by peptone. These results clearly indicate the cost-effectiveness of solid state fermentation technology in the economic production of extracellular cellulase. The hyper-production of cellulase by mutant strain UNAC4 has potential for industrial processes that convert lignocellulosic material (e.g. RS) into products of commercial value such as glucose and biofuels.     

Penicillium verruculosum WA 30 a new source of cellulase

Summary Of fungi 110 strains were screened for extracellular cellulase production in shake flask experiments. Twelve strains produced the enzyme in significant quantity. Since the enzyme activity was assayed by different methods (liberation of reducing sugar from cotton, filter paper, carboxymethylcellulose and cellobiose), the estimation of the productivity of the strains differed according to the substrate used. The best cotton degrading activity per fermentation volume as well as per mg of secreted soluble protein was achieved by Penicillium verruculosum WA 30, a wild-type strain, for which the cellulase productivity has not yet been published. The cotton degrading (so-called C₁) activity was successfully enhanced nearly threefold in medium experiments. Analyses of saccharification digests showed that glucose was the predominant product, with negligible amounts of cellobiose. The pH and temperature optima for WA 30 cellulase complex were pH 4.2 and 60°C.     

Induction and regulation of cellulase synthesis in Trichoderma pseudokoningii mutants EA3-867 and N2-78

Two mutants, EA₃-867 and N₂-78, with high cellulase yields were obtained from wild strains of Trichoderma pseudokoningii Rifai, 1096 and Mo₃, respectively, by mutagenic treatments with a linear accelerator, ⁶⁰Co, u.v., nitrosoguanidine (NTG) and diethylsulphate (DTS). The mutants grew slowly to produce small colonies on agar plates with synthetic medium. On agar plates of peptone-yeast extract, the small colonies were as large as those of wild strains. The cellulase activities of these mutants in Koji extracts, shake flask culture filtrates, and enzyme preparations were markedly higher than those of their parents. The mutant N₂-78 reached quite high cellulase activity level when cultured for 60 h in shake flasks in a simple medium containing milled straw, wheat bran, mineral salts plus waste glucose molasses. The cellulase saccharifying activities on CMC, filter paper and cotton, were 255, 8.2 and 13.4 mg glucose/ml enzyme, respectively, or 11, 4.3 and 6 times more than those of its parent Mo₃.The cellulase synthesis of EA₃-867 and N₂-78 was strongly induced by sophorose, isolated from pods of Sophora japonica L., and was inhibited by glucose, sugar phosphates, glycerol and organic acids. We conclude that cellulase synthesis of the mutants is regulated by catabolite repression as well as by induction. The increase in cellulase production by both mutants results from changes in the regulatory systems for cellulase synthesis, i.e. the mutants showed higher sensitivity to inducer and lower susceptibility to catabolite repression than did the wild types.A cellulase preparation of Trichoderma pseudokoningii Rifai N₂-78 induced by sophorose was fractionated by DEAE-Sephadex A-50 and Sephadex G-100 column chromatography, selective inactivation and polyacrylamide gel electrophoresis. The components C₁(exo-β1,4-glucanase), C_x(endo-β1,4-glucanase) and β-glucosidase were separated, and their molecular weights were estimated to be 67 000, 62 000 and 42 000 respectively. The homogeneity of C₁ was verified by polyacrylamide gel electrophoresis, immunoelectrophoresis and ultracentrifugal analysis. It is a glycoprotein and is rich in glycine, aspartic acid, threonine, serine and glutamic acid. The C₁ showed a strong synergistic action with C_x in the degradation of cotton, Avicel and Walseth cellulose.A poly(A)-RNA, induced by sophorose in N₂-78 mycelium, was isolated by oligo(dT)-cellulose affinity chromatography.     

Screening of highly cellulolytic fungi and the action of their cellulase enzyme systems

Over 100 strains of wood-rotting fungi were compared for their ability to degrade wood blocks. Some of these strains were then assayed for extracellular cellulase [1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] activity using a variety of different solid media containing carboxymethyl cellulose or acid swollen cellulose. The diameter of clearing on these plates gave an approximate indication of the order of cellulase activities obtained from culture filtrates of these strains. Trichoderma strains grown on Vogels medium gave the highest cellulase yields. The cellulase enzyme production of T. reesei C30 and QM9414 was compared with that of eight other Trichoderma strains. Trichoderma strain E58 had comparable endoglucanase and filter paper activities with the mutant strains while the β- -glucosidase [β- -glucoside glucohydrolase, EC 3.2.1.21] activity was approximately six to nine times greater.     

Carboxymethylcellulase and avicelase activities from a cellulolytic Clostridium strain A11

The extracellular cellulase enzyme system of Clostridium A11 was fractionated by affinity chromatography on Avicel: 80% of the initial carboxymethylcellulase (CMCase) activity was adhered. This cellulase system was a multicomponent aggregate. Several CMCase activities were detected, but the major protein P1 had no detectable activity. Adhered and unadhered cellulases showed CMCase activity with the highest specific activity in Avicel-adhered fraction. However, only afhered fractions could degrade Avicel. Thus, efficiency of the enzymatic hydrolysis of Avicel was related to the cellulase-adhesion capacity. Carboxymethylcellulase and Avicelase activities were studied with the extracellular enzyme system and cloned cellulases. Genomic libraries from Clostridium A11 were constructed with DNA from this Clostridium, and a new gene cel1 was isolated. The gene(s) product(s) from cel1 exhibited CMCase and p-nitrophenylcellobiosidase (pNPCbase) activities. This cloned cellulase adhered to cellulose. Synergism between adhered enzyme system and cloned endoglucanases was observed on Avicel degradation. Conversely, no synergism was observed on CMC hydrolysis. Addition of cloned endoglucanase to cellulase complex led to increase of the V_max without significant K _m variation. Cloned endoglucanases can be added to cellulase complexes to efficiently hydrolyze cellulose.     

Partial characterization of a crude cold-active lipase from Rhodococcus cercidiphylli BZ22

Cold-active lipase production by the psychrophilic strain Rhodococcus cercidiphylli BZ22 isolated from hydrocarbon-contaminated alpine soil was investigated. Depending on the medium composition, high cell densities were observed at a temperature range of 1–10 °C in Luria–Bertani (LB) broth or 1–30 °C in Reasoner’s 2A (R2A). Maximum enzyme production was achieved at a cultivation temperature of 1–10 °C in LB medium. About 70–80 % of the secreted enzyme was bound to the cell and was highly active as a cell-immobilized lipase which exhibited good reusability; more than 60 % of the initial lipase activity was retained after five-fold reuse. The properties of the lipase produced by the investigated strain were compared with those of a mesophilic porcine pancreatic lipase (PPL). The thermal stability of the cell-immobilized bacterial lipase was higher than that of the extracellular enzyme. Highest activity was detected at 30 °C for the cell-immobilized enzyme and for PPL, while the extracellular enzyme displayed highest activity at 10–20 °C. The bacterial lipase hydrolyzed p-nitrophenyl (p-NP) esters with different acyl chain lengths (C₂–C₁₈). The highest hydrolytic activity was obtained with p-NP-butyrate (C₄) as substrate, while the highest substrate affinity was obtained with p-NP-dodecanoate (C₁₂) as substrate, indicating a clear preference of the enzyme for medium acyl chain lengths.     

Mutants of the white-rot fungus Sporotrichum pulverulentum with increased cellulase and β-d-glucosidase production

This paper reports the isolation of mutants of the white-rot fungus Sporotrichum pulverulentum and the results of a survey of enzymic activity among these mutants. The strains were screened for extracellular cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) production in shake flask experiments. Apart from strain 63-2, strains 6, 63, 9, L5, E-1 and UV-18 showed equal or higher endo-1,4-β-d-glucanase (cellulase), filter paper-degrading and β-d-glucosidase activities than S. pulverulentum. The cellulase activity obtained, measured as filter paper activity, was comparable to that reported for Trichoderma reesei QM9414. However, the β-d-glucosidase activity was about six times higher than for the QM9414 strain. The pH and temperature-activity profiles of crude β-d-glucosidase preparations from the various strains were determined and were found to be identical. The thermal stability at pH 4.5 and 40°C was 5 days for all these preparations.     

Enumeration of gut associated extracellular enzyme‐producing yeasts in some freshwater fishes

Extracellular enzyme‐producing yeasts might be involved in the supplementation of enzymes within the gastrointestinal tract of fish. The present study was intended to detect yeasts in the intestine of three Indian major carps (Labeo rohita, Catla catla, Cirrhinus mrigala), three exotic carps (Hypophthalmichthys molitrix, Ctenopharyngodon idella, Cyprinus carpio), as well as Nile tilapia (Oreochromis niloticus), and to identify the most promising extracellular enzyme‐producing (e.g. amylase, protease, lipase, cellulase, xylanase and phytase) yeast strains by 18S rDNA sequence analysis. Selected for qualitative enzyme assay were 121 yeast strains, from which 28 were further studied for quantitative enzyme assay. The strain CMH6A isolated from C. mrigala exhibited the best extracellular enzyme activities except for amylase and cellulase. The strain ONF19B isolated from O. niloticus was noted as the best extracellular enzyme producer among the strains that produced all of the extracellular enzymes studied. Sequencing of the 18S rDNA fragment followed by nucleotide blast in the National Centre for Biotechnology Information (NCBI) GenBank revealed that strains CMH6A and ONF19B were similar to Pichia kudriavzevii (Accession no. KF479403 ) and Candida rugosa (Accession no. KF479404 ), respectively. The test of antagonism (in vitro) revealed that the isolated yeasts could not affect the growth of the autochthonous gut bacteria. This might indicate likely co‐existence of autochthonous yeasts and bacteria in the fish gut. Further research is necessary to explore the possibilities of utilizing the extracellular enzyme‐producing yeasts detected in the present study for commercial aquaculture.     

The production of highly effective enzyme complexes of cellulases and hemicellulases based on the Penicillium verruculosum strain for the hydrolysis of plant raw materials

Methods for the production and analysis of cellulase and hemicellulase enzyme preparations of various compositions based on the Penicillium verruculosum carbohydrase complex and intended for the effective hydrolysis of different types of cellulose-containing materials (CCMs) have been developed. New recombinant strains of P. verruculosum producing multienzyme carbohydrase complexes with increased activities of cellulases (due to the expression of endo-β-1,4-glucanases I and IV and cellobiohydrolase II from Trichoderma reesei) and hemicellulases (due to the expression of endo-β-1,4-xylanases from P. canescens and T. reesei and endo-β-1,4-mannanase from T. reesei) were constructed. The hydrolytic efficiency of the enzyme preparations (EPs) produced by the new recombinant strains during continuous hydrolysis of three CCM types (milled aspen, depitched pine wood, and milled bagasse) was studied. It was shown that new EPs containing recombinant proteins and retaining their own basic cellulase complex are characterized by the highest hydrolytic ability, exceeding that of the EP based on the original P. verruculosum strain. The recombinant enzyme preparations were highly stable; the optimal pH and temperature values for cellulase, xylanase and mannanase activities were in the range of 3.5–5.5 and 50–80°C, respectively.     

Cellulase from Fusarium solani: Purification and properties of the C1 component

The C₁ component from Fusarium solani cellulase was purified extensively by molecular-sieve chromatography on Ultrogel AcA-54 and ion-exchange chromatography on DEAE-Sephadex. The purified component showed little capacity for hydrolysing highly ordered substrates (e.g., cotton fibre), but poorly ordered substrates (e.g., H₃PO₄-swollen cellulose), and the soluble cello-oligosaccharides cellotetraose and cellohexaose, were readily hydrolysed; cellobiose was the principal product in each case. Attack on O-(carboxymethyl)cellulose, a substrate widely used for measuring the activity of the randomly acting enzymes (C_x enzymes) of the cellulase complex, was minimal, and ceased after the removal of a few unsubstituted residues from the end of the chain. These observations, and the fact that the rate of change of degree of polymerisation of H₃PO₄-swollen cellulose was very slow compared with that effected by the randomly acting endoglucanases (C_x, CM-cellulases), indicate that C₁ is a cellobiohydrolase. Fractionation by a variety of methods gave no evidence for the non-identity of the cellobiohydrolase and the component that acted in synergism with the randomly acting C_x enzyme when solubilizing cotton fibre.     

Milk Clotting Enzyme from Microorganisms

The purification of the milk clotting enzyme from Mucor pusillus Lindt could be achieved by column chromatography on Amberlite IRC-50 by raising pH from 3.5 to 4.5 and about 70% of activity was recovered after this treatment. After the treatment through the column of DEAE-Sephadex A-25, the trace cellulase activity could be eliminated.

The homogeneity of the purified preparation was proved by ultracentrifugal analysis and electrophoretic patterns at various pH values.

Isoelectric point of this enzyme is considered to lie between pH 3.5 and 3.8.

The enzyme activity was inhibited by Hg⁺⁺ or Fe⁺⁺⁺.

Trypsinogenkinase activity was not contained in this enzyme.

The antiserum against the milk clotting enzyme from Mucor pusillus reacted with the purified and crude enzyme preparations in precipitin test and inhibited their enzyme activities, but did not react with other enzymes such as rennin, pepsin, acid proteases from Aspergillus saitoi and Aspergillus oryzae, or the culture filtrates of some strains of Mucor and Rhizopus.

The antigen-antibody reaction was so specific that it might be possible with this antibody to identify this enzyme and also the strain itself.

Normal sera from some mammals inhibited this enzyme activity too, but the degree was less than that with rennin.     

Production of pectolytic and cellulolytic enzymes byfusarium oxysporum andF. moniliforme under different cultivation conditions

Six-day incubation was most suitable for production of pectolytic and cellulolytic enzymes byFusarium on different culture media. Czapek’s medium favoured maximum production of polygalacturonase (PG) and cellulase (C_x), peptone dextrose gave highest yields of pectin methyl galacturonase (PMG) withF. oxysporum. Cole’s medium was found to be poor for the enzyme production by both organisms. A positive correlation was observed between the growth rate of the pathogenic forms and their enzyme production. InF. oxysporum the PG secretion was maximum at pH 4.5 and inF. moniliforme at pH 5.0. PMG production optimum was at pH 5.5. No PG and PMG were produced above pH 7. InF. oxysporum the C_x activity was highest at pH 5.5 and inF. moniliforme at pH 4.5. Maximum PG and PMG activities were recorded at 35 °C in both pathogens. The C_x activity of both organisms was maximum at 45 °C but some carboxymethyl cellulose hydrolysis was found even at 60 °C.     

Response surface optimization of cellulase production from Aneurinibacillus aneurinilyticus BKT-9: An isolate of urban Himalayan freshwater

Due to their vast industrial potential, cellulases have been regarded as the potential biocatalysts by both the academicians and the industrial research groups. In the present study, culturable bacterial strains of Himalayan Urban freshwater lake were investigated for cellulose degrading activities. Initially, a total of 140 bacterial strains were isolated and only 45 isolates were found to possess cellulose degrading property. On the basis of preliminary screening involving cellulase activity assay on CMC agar (with clear zone of hydrolysis) and biosafety assessment testing, only single isolate named as BKT-9 was selected for the cellulase production studies. Strain BKT-9 was characterized at the molecular level using rRNA gene sequencing and its sequence homology analysis revealed its identity as Aneurinibacillus aneurinilyticus. Further, various physico-chemical parameters and culture conditions were optimized using one factor approach to enhance cellulase production levels in the strain BKT-9. Subsequently, RSM based statistical optimization led to formulation of cellulase production medium, wherein the bacterial strain exhibited ~60 folds increase in enzyme activity as compared to un-optimized culture medium. Further studies are being suggested to scale up cellulase production in A. aneurinilyticus strain BKT-9 so that it can be utilized for biomass saccharification at an industrial level.     

Mutants of the white-rot fungus Sporotrichum pulverulentum with increased cellulase and β- -glucosidase production

This paper reports the isolation of mutants of the white-rot fungus Sporotrichum pulverulentum and the results of a survey of enzymic activity among these mutants. The strains were screened for extracellular cellulase [see 1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] and β- -glucosidase (β- -glucoside glucohydrolase, EC 3.2.1.21) production in shake flask experiments. Apart from strain 63-2, strains 6, 63, 9, L5, E-1 and UV-18 showed equal or higher endo-1,4-β- -glucanase (cellulase), filter paper-degrading and β- -glucosidase activities than S. pulverulentum. The cellulase activity obtained, measured as filter paper activity, was comparable to that reported for Trichoderma reesei QM9414. However, the β- -glucosidase activity was about six times higher than for the QM9414 strain. The pH and temperature-activity profiles of crude β- -glucosidase preparations from the various strains were determined and were found to be identical. The thermal stability at pH 4.5 and 40°C was 5 days for all these preparations.     

Optimization of the composition of the nutrient medium for cellulase and protein biosynthesis by thermophilic Aspergillus fumigatus NRC 272

An active strain of Aspergillus spp. has been selected for the production of cellulolytic enzymes and proteins when grown on peracetic acid-treated wheat straw. This strain produced a considerable amount of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] in the extracellular supernatant and exhibited good overall cellulolytic activity, as measured using filter paper and Avicel as substrates. Also, under the same conditions the strain showed high activities of β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) and β-d-xylosidase (1,4-β-d-xylan xylohydrolase, EC 3.2.1.37). The maximum enzyme yields (carboxymethylcellulose activity 26.4 units ml^?1, filter paper activity 2.26 units ml^?1 and Avicel activity 16.82 units ml^?1; β-d-glucosidase 9.09 units ml^?1 and β-d-xylosidase 1.92 units ml^?1) were obtained after 96 h incubation at 45°C.     

Variation of bagasse crystallinity and cellulase activity during the fermentation of Cellulomonas bacteria

A characteristic behavior of the fermentation process was observed during the growth of Cellulomonas on sugarcane bagasse. At the early stage of the fermentation the crystallinity index of bagasse increased, suggesting that the major metabolized fraction corresponded to the hemicellulose during this stage. Some time later the crystallinity achieved a steady state and then deceased, which indicated that the most complex structure of bagasse was being attacked. The analysis of the cellulolytic activity of extracellular enzyme in the medium showed a sharp increase followed by an abrupt leveling off and decline in activity. These results along with the reduction of crystallinity index and bagasse utilization (70%) justify the assumption that the C₁ component was present in the cellulase complex synthesized by the bacteria.