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PubMed1999

Disintegration of uncooked rice by carboxymethyl cellulase from Sporotrichum sp. HG-I.

A thermostable carboxymethyl cellulase (CMCase) was purified to homogeneity from a wheat bran culture of the thermophilic fungus Sporotrichum sp. HG-1, an isolate from a compost heap. The enzyme had a molecular weight (M(r)) of 33,000 by SDS-PAGE. The optimum temperature and pH for enzyme activity were 70 degrees C and 4.5-5.0, and the enzyme was heat stable. Uncooked Thai rice was digested so as to cause its disintegration by the addition of purified CMCase, but not by the addition of xylanase purified from strain HG-1. Ferulic acid conjugated to oligosaccharide was released significantly by the combined action of CMCase and xylanase, but the free form of ferulic acid was not detectable.

Journal Article↗
PubMed1991

An active single-chain antibody containing a cellulase linker domain is secreted by Escherichia coli.

Single-chain antibodies consist of the variable, antigen-binding domains of antibodies joined to a continuous polypeptide by genetically engineered peptide linkers. We have used the flexible interdomain linker region of a fungal cellulase to link together the variable domains of an anti-2-phenyloxazolone IgG1 and show here that the resulting single-chain antibody is efficiently secreted and released to the culture medium of Escherichia coli. The yield of affinity-purified single-chain antibody is 1-2 mg/l of culture medium and its affinity and stability are comparable to those of the corresponding native IgG.

Amino Acid Sequence↗
PubMed1974

An Apparent Cellulase Complex in Tomato (Lycopersicon esculentum L.) Fruit.

Four enzyme-containing fractions were separated by ammonium sulfate fractionation of 2-day, postbreaker tomato (Lycopersicon esculentum L. cv. Manhattan). The pH optima and substrate specificities are reported. The enzymes were identified as a nonspecific beta-glucosidase, an exo-beta-1,4-glucanase, and two endocellulases. Both endocellulase fractions were able to catalyze the hydrolysis of various insoluble cellulose materials.Together, the enzymes studies are capable of completely degrading insoluble cellulose to short chain cellodextrins and glucose. Ripening tomato fruit apears to contain a complex of cellulases similar to that of cellulolytic bacteria and fungi.

Journal Article↗
PubMed1990

Regulation of a sesquiterpene cyclase in cellulase-treated tobacco cell suspension cultures.

The regulation of an elicitor-inducible sesquiterpene cyclase in tobacco (Nicotiana tabacum) cell suspension cultures was investigated. Sesquiterpene cyclase activity was absent from control cell cultures but induced to a maximum within 15 hours of cellulase addition to the cell cultures. The induction of the cyclase activity was correlated with an absolute amount of the cyclase protein as measured in immunoblots. Both the in vivo synthesis rate, measured as the incorporation of [(35)S]methionine by cell cultures into immunoprecipitable cyclase protein, and the cyclase mRNA translational activity, measured as the incorporation of [(35)S]methionine into immunoprecipitable cyclase protein synthesized by in vitro translation of isolated RNA, were maximal at that time when the increase in cyclase enzyme activity was maximal. Using thiouridine to selectively label and isolate de novo synthesized mRNA, the in vitro translation products encoded by the newly synthesized RNA from elicitor-treated, but not control, cell cultures contained immunoprecipitable cyclase protein. These results suggest that the induction of the sesquiterpene cyclase in elicitor-treated cell cultures is primarily regulated by transcriptional control of the cyclase gene.

Journal Article↗
PubMed1980

Use of a cellulase-derepressed mutant of cellulomonas in the production of a single-cell protein product from cellulose.

A cellulase-derepressed mutant of a Cellulomonas species was used to produce single-cell protein from crystalline cellulose. In preliminary tests, maximum yield of single-cell protein was obtained at 30 degrees C (pH 7.0) with urea as the nitrogen source. A continuous-flow foam flotation procedure was developed for rapid and efficient separation of bacteria from the culture liquid and cellulose residue. A pH of 4.5 was optimum for foam flotation of this organism. In preliminary trials, recovery was 85% of the cells with the flotation procedure. Cellulomonas was 68% true protein and had an essential amino acid profile featuring a high lysine content (6.5% of protein). The Cellulomonas product was evaluated nutritionally with weanling rats. The net protein utilization value for the protein supplemented with methionine was 50.4% Weight gain of rats on the Cellulomonas diet was similar to that of rats fed a casein diet.

Journal Article↗
PubMed1984

Production and Characterization of Cellulase and beta-Glucosidase from a Mutant of Alternaria alternata.

A mutant of Alternaria alternata excreted enhanced levels of carboxymethylcellulase and particularly beta-glucosidase when grown in cellulose liquid media. Both enzymes were purified two- to four-fold by ammonium sulfate precipitation and gel filtration, and the kinetic data showed K(m) values of 16.64 mg/ml of culture fluid for carboxymethylcellulase and 0.14 mM p-nitrophenyl-beta-d-glucoside and 0.81 mM cellobiose for beta-glucosidase at pH 5. Carboxymethylcellulase and extracellular beta-glucosidase functioned optimally at pH 5 to 6 and 4.5 to 5 and at temperatures of 55 to 60 and 70 to 75 degrees C, respectively. Both temperature optima and thermostability of beta-glucosidase were among the highest ever reported for the same enzyme excreted from cellulase and beta-glucosidase hyperproducing microorganisms.

Journal Article↗
PubMed1985

Effect of Colloidal Materials on Cellulase Production by Trichoderma reesei Rut-C30.

The addition of positively charged colloidal materials to the growth medium markedly increased the concentration of cellulase enzymes produced by Trichoderma reesei Rut-C30. Filter paper activities of up to 4 and 13 IU/ml have been achieved by the addition of colloidal materials, using 3% lactose and 3% cellulose, respectively, as a substrate. The particles exert their effect by binding soluble sugars and slowing their uptake by the organism.

Journal Article↗
PubMed1987

Calorimetric versus Growth Microbial Analysis of Cellulase Enzymes Acting on Cellulose.

ASSAY OF CELLULASE ENZYMOLOGY ON CELLULOSE WAS INVESTIGATED BY TWO METHODS: (i) plate colony counting to determine microbial growth and (ii) microbial calorimetry. These methods were chosen because they accept raw samples and have the potential to be far more specific than spectrophotometric reducing sugar assays. Microbial calorimetry requires ca. 0.5 to 1 h and 10 to 100 muM concentrations of cellulolytic lower sugars (glucose and cellobiose). Growth assay (liquid culture, plating, colony counting) requires 15 to 20 h and ca. 0.5 mM sugars. Microbial calorimetry requires simply aerobic metabolism, whereas growth assay requires completion of the cell cycle. A stripping technique is described for use in conjunction with the calorimetric method to enable separate analysis of the two sugars. Mixtures of glucose and cellobiose are equilibrated with Escherichia coli and spun out to remove glucose. The supernatant is calorimetrically combusted with Klebsiella sp. to quantitate cellobiose, and the same organism combusting the nonstripped mixture gives heat proportional to the sum of the two sugars. Calorimetry of cellulolysis products from individual exo- and endocellulases, and from their reconstituted mixture, was carried out to develop a microbial calorimetric means for demonstrating enzyme synergism.

Journal Article↗
PubMed1992

Biochemical and Electron Microscopic Studies of the Streptomyces reticuli Cellulase (Avicelase) in Its Mycelium-Associated and Extracellular Forms.

Streptomyces reticuli is able to grow efficiently with crystalline cellulose (Avicel) as the sole carbon source. Cultivation in the presence of the nonionic detergent Tween 80 at a concentration of 0.1% led to a 10-fold increase in extracellular cellulolytic activity. Under these conditions, one single 82-kDa cellulase (Avicelase) capable of degrading crystalline and soluble cellulose as well as cellodextrins and p-nitrophenylcellobioside was purified to apparent homogeneity by a procedure which consisted of two consecutive anion-exchange chromatographies followed by chromatofocusing. Aggregation, which was a major problem during protein purification, could be avoided by including Triton X-100 at a concentration of 0.1% in every chromatographic step. The Avicelase was identified in extracellular and mycelium-associated forms, the latter of which could be released efficiently by nonionic detergents. In addition, a 42-kDa truncated form retaining cellulolytic activity was identified which had been generated from the 82-kDa enzyme by a protease. Antibodies raised against the mycelium-associated Avicelase reacted with the 42-kDa derivative and the extracellular form. The mycelial association of the enzyme was confirmed by immunofluorescence and immunoelectron microscopies.

Journal Article↗
PubMed1996

Physiological Studies of Cellulase (Avicelase) Synthesis in Streptomyces reticuli.

Cellulase (Avicelase, Cel1) from Streptomyces reticuli efficiently hydrolyzes crystalline cellulose (Avicel) to cellobiose. The synthesis of the enzyme was found to be dependent on the presence of insoluble Avicel but not on either soluble hydroxyethylcellulose, cellooligomers, or cellobiose. Glycerol and various metabolizable mono- and disaccharides repress Avicelase synthesis, whereas yeast extract has no inducing or repressing effect. Glucose kinase is not required for the repression effect. In the course of cultivation, S. reticuli secretes significant quantities of acid, predominantly pyruvate and succinate, which reduce the pH to 4 in commonly used media with low buffering capacity. Comparative studies with media with low and high buffering capacities revealed that Avicelase synthesis is strongly repressed at a low pH.

Journal Article↗
PubMed1996

The Cellulases Endoglucanase I and Cellobiohydrolase II of Trichoderma reesei Act Synergistically To Solubilize Native Cotton Cellulose but Not To Decrease Its Molecular Size.

Degradation of cotton cellulose by Trichoderma reesei endoglucanase I (EGI) and cellobiohydrolase II (CBHII) was investigated by analyzing the insoluble cellulose fragments remaining after enzymatic hydrolysis. Changes in the molecular-size distribution of cellulose after attack by EGI, alone and in combination with CBHII, were determined by size exclusion chromatography of the tricarbanilate derivatives. Cotton cellulose incubated with EGI exhibited a single major peak, which with time shifted to progressively lower degrees of polymerization (DP; number of glucosyl residues per cellulose chain). In the later stages of degradation (8 days), this peak was eventually centered over a DP of 200 to 300 and was accompanied by a second peak (DP, (apprx=)15); a final weight loss of 34% was observed. Although CBHII solubilized approximately 40% of bacterial microcrystalline cellulose, the cellobiohydrolase did not depolymerize or significantly hydrolyze native cotton cellulose. Furthermore, molecular-size distributions of cellulose incubated with EGI together with CBHII did not differ from those attacked solely by EGI. However, a synergistic effect was observed in the reducing-sugar production by the cellulase mixture. From these results we conclude that EGI of T. reesei degrades cotton cellulose by selectively cleaving through the microfibrils at the amorphous sites, whereas CBHII releases soluble sugars from the EGI-degraded cotton cellulose and from the more crystalline bacterial microcrystalline cellulose.

Journal Article↗
PubMed1974

Cellulase Production by Trichoderma viride on Feedlot Waste.

Feedlot waste contains essentially all the necessary nutrients for batch fermentation with the fungus Trichoderma viride. The organism utilizes two-thirds of the carbohydrate in feedlot waste while elaborating cellulase in quantities comparable to commercial preparations. Essentially odor-free, the fermented waste contains all of the original nitrogen but has 24% less organic matter.

Journal Article↗
PubMed2006

High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell.

BACKGROUND: The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins. RESULTS: We used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved CelF mutants and corresponding optimized clones in expression-ready plasmids. CONCLUSION: The multiplex method using an integrated automated platform for high-throughput screening in a functional proteomic assay allows rapid identification of plasmids containing optimized clones ready for use in subsequent applications including transformations to produce improved strains or cell lines.

Journal Article↗
PubMed2001

Reduction in operator supervision time using a modified malfunction detection system with fuzzy inference in an industrial-scale fermentation of alkaline cellulase.

A malfunction detection system based on fuzzy inference, in which a fermentation process is categorized into three states (normal, intermediate and abnormal) according to the computation of malfunction degree, was modified to reduce the supervision tasks required for an industrial-scale production of alkaline cellulase. The time lag of the culture, tau, was determined from the difference in the cumulated amount of CO2 evolved between the actual and standard values. By employing tau, the deviations of the actual values of process data (pH of broth and the amount of antifoaming agent supplied) from the standard values could be compensated for in terms of a delayed or forward effect in fermentation time. In the detection tests conducted for a series of 200 runs using the modified method, two runs and a single run were categorized as belonging to the abnormal and intermediate states, respectively, and all of the other runs were recognized to be in a normal state. As a result, the total supervision time for operators was reduced by the modification.

Journal Article↗
PubMed2003

Isolation and characterization of solventogenic, cellulase-free xylanolytic Clostridia from cow rumen.

Hemicellulolytic obligate anaerobes capable of converting a range of agricultural substrates and monomeric sugars of hemicellulose to solvents and acids were isolated from cow rumen fluid. The strains were identified as 5 strains of C. beijerinckii (3 different genotypes), 3 strains of C. acetobutylicum (2 different genotypes), 1 strain of C. butyricum and 3 strains of C. bifermentans (2 different genotypes). All of them produced cellulase-free xylanase activity. C. acetobutylicum LU1 produced solvent, a high of 5.1 gl(-1) and C.bifermentans LU1 produced xylanase activity a high of 4.2 IU ml(-1).

Animals↗
PubMed2005

[Cloning and expressing of cellulase gene (cbh2) from thermophilic fungi Chaetomium thermophilum CT2].

Chaetomium thermophilum CT2 can produce extracellular cellulase with industrial value. We designed two degenerate primers to amplify catalytic domain sequence of cellobiohydrolase II ( CBH II). Full length of cDNA was obtained by rapid amplification of cDNA ends technologies. DNA sequencing revealed that cbh2 has an open reading frame of 1428bp, which encodes a putative polypeptide of 476 amino acids. The deduced amino acid sequence shows that the predicted molecular mass is 53 kD and the cbh2 consists of a fungal-type carbohydrate binding domain (CBD) separated from a catalytic domain by a linker region rich in proline/serine/threonine. PCR product consisting of the entire CBH II coding region without its signal sequences was cloned into the yeast secretive plasmid pPIC9K, which was then transformed into Pichia pastoris GS115. Highly efficient production of the cellobiohydrolase II was achieved in P. pastoris under the control of the AOX1 promoter, and the expressing level was 1.2 mg/mL by small-scale culturing. The recombinant cellobiohydrolase II was purified by using ammonium sulfate fraction, DEAE-Sepharose Fast flow chromatography. A molecular mass of the purified enzyme is 67 kD determined by SDS-PAGE and this is similar to the native cellobiohydrolase II purified from C. thermophilum CT2. The recombinant enzyme exhibited optimum catalytic activity at pH 4.0 and 50 degrees C respectively. It was thermostable at 50 degrees C and retained 50% of its original activity after 30 min at 70 d degrees C . The high level of fully active recombinant cellobiohydrolase II got from P. pastoris makes this expression system attractive for fermentor and industrial applications.

Amino Acid Sequence↗
PubMed2006

[Changes in glycosidases and cellulase activities, and cell wall composition in strawberry fruits during development and ripening].

Experiments were carried out with two strawberry (Fragaria x nanassa Duch.) cultivars Fengxiang and Hongfeng, with different softening characteristics during growth, ripening and postharvest storage. The fruits were harvested at different stages of growth and ripening, as assessed by size and the coloration of the surface of the fruits. We selected the following stages: small and green (S1), large and green (S2), white (S3), reddish (S4), and fully red (S5). The main results were as follows. Both alpha- and beta-galactosidase activities were changed with ripening of strawberry fruits (Fig.1A, B). Of the strawberry cultivars tested, no correlation was found between glucosidase activity and fruit ripening (Fig.1C, D). Alpha-mannosidase is an enzyme being ionically bound with cell wall and its activities is correlated with the softening of strawberry fruits (Fig.1E, F). No beta-mannosidase has been detected in strawberry. The activities of cellulase increased as the strawberry fruits developed from stage of small and green to stage of overripe (Fig.2A). The activities of PME increased during the development of strawberry fruits (Fig.2B). Endo-PG was not detected in strawberry, and exo-PG was not related to fruit ripening (Fig.2C). Changes in cell wall component contents were clearly related to the changes in the firmness of strawberry fruits. The increase in soluble pectin, together with reduction of ionically bound pectin content, covalently bound pectin content (Fig.3A, B) and cellulose (Fig.3A, B) resulted in softening of strawberry fruits.

Cell Wall↗