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At least 613 records · Page 34Linked to original sources
PubMed2000

Treatment of recycled kraft pulps with Trichoderma reesei hemicellulases and cellulases.

Effects of recycling ECF-bleached softwood kraft pulp on pulp properties were evaluated in the laboratory. The tensile strength, fiber flexibility and WRV lost during drying of the pulp were recovered by refining between the cycles which, however, resulted in deteriorated drainage properties. The recycled pulps were treated with purified Trichoderma reesei cellulases and hemicellulases and the changes in fiber properties due to enzymatic treatments were characterized. The endoglucanases (EG I and EG II) significantly improved pulp drainage already at low dosage levels, and EG II was found to be more effective at a given level of carbohydrate solubilization. Combining hemicellulases with the endoglucanase treatments increased the positive effects of the endoglucanases on pulp drainage. However, as a result of the endoglucanase treatments a slight loss in strength was observed. Combining mannanase with endoglucanase treatments appeared to increase this negative effect, whereas the impact of xylanase was not significant. Although the drainage properties of the pulps could be improved by selected enzymes, the water retention capacity of the dried hornified fibers could not be recovered by any of the enzymes tested.

Biotechnology↗
PubMed2003

Efficient synthesis of glyceroyl beta-lactoside and its derivatives through a condensation reaction by cellulase.

Condensation reaction between lactose and glycerol was effectively catalyzed by utilizing a commercially available cellulase preparation from Trichoderma reesei. The enzyme induced the formation of 1-O-beta-lactosyl-(R,S)-glycerol (1) and 2-O-beta-lactosyl glycerol (2) in a molar ratio of 7:3 and in a 20% yield based on lactose added. The enzyme also induced the condensation of lactose with 1,3-propanediol to produce O-beta-lactosyl propanediol (3) in a yield of 15%. When various alkanols (N: 2-8) and allyl alcohol were used in the condensation reaction, the corresponding alkyl and allyl beta-lactoside were obtained in the yields of 0.9-3.8% of the desired compounds.

Cellulase↗
PubMed2003

A novel cold-tolerant Clostridium strain PXYL1 isolated from a psychrophilic cattle manure digester that secretes thermolabile xylanase and cellulase.

A Clostridium strain PXYL1 was isolated from a cold-adapted cattle manure biogas digester at 15 degrees C. It could grow at temperatures as low as 5 degrees C up to 50 degrees C with highest specific growth rate at 20 degrees C and is a psychrotroph. It produced extracellular hydrolytic enzymes namely xylanase, endoglucanase, beta-xylosidase, beta-glucosidase and filter paper cellulase, all of which had maximal activity at 20 degrees C. The induction of xylanase was highest on birch wood xylan (37 IU(mg protein)(-1)) compared with xylose (1.11 IU(mg protein)(-1)), cellobiose (1.43 IU(mg protein)(-1)) and glucose (no activity). The xylanase was thermolabile with a half-life of 30 min at 40 degrees C and 8 min at 50 degrees C but stable for over 2 h at 20 degrees C. The crude enzyme released reducing sugars (1.25 g l(-1)) from finger millet flour at 20 degrees C, while commercial food-grade xylanases showed no hydrolysis at this temperature. This is the first report of a Clostridium strain growing at 20 degrees C and producing an array of xylanolytic and cellulolytic enzymes, possessing low temperature optima of 20 degrees C, which may facilitate degradation of plant fibre under low-temperature conditions.

Animals↗
PubMed2003

New integration vector using a cellulase gene as a screening marker for Lactobacillus.

The new integration vector for Lactobacillus, pJC4, was developed using the extracellular endoglucanase A gene (celA) of Clostridium thermocellum as a screening marker. pJC4 was transformed into four Lactobacillus species, Lb. johnsonii, Lb. gasseri, Lb. bulgaricus, and Lb. plantarum. In each species, the pJC4 integrants were easily and accurately detected by the appearance of a clear halo on a cellulase screening plate without any false transformants. Polymerase chain reaction and Southern hybridization indicated that all transformants with clear halos contained pJC4 in their chromosomal DNAs. The celA gene could be a useful screening marker for other lactic acid bacteria.

Cellulase↗
PubMed2000

Isolation and analysis of two cellulase cDNAs from Orpinomyces joyonii.

Two cellulase cDNAs, celB29 and celB2, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Orpinomyces joyonii strain SG4. The nucleotide sequences of celB2 and celB29 and the primary structures of the proteins encoded by these cDNAs were determined. The larger celB29 cDNA was 1966bp long and encoded a 477 amino acid polypeptide with a molecular weight of 54kDa. Analysis of the 1451bp celB2 cDNA revealed an 1164bp open reading frame coding for a 44kDa protein consisting of 388 amino acids. Both deduced proteins had a high sequence similarity in central regions containing putative catalytic domains. Primary structure analysis revealed that CelB29 contained a Thr/Pro-rich sequence that separated the N-terminal catalytic domain from a C-terminal reiterated region of unknown function. Homology analysis showed that both enzymes belong to glycosyl hydrolase family 5 and were most closely related to endoglucanases from the anaerobic fungi Neocallimastic patriciarum, Neocallimastix frontalis and Orpinomyces sp. The classification of CelB29 and CelB2 as endoglucanases was supported by enzyme assays. The cloned enzymes had high activities towards barley beta-glucan, lichenan and carboxymethylcellulose (CMC), but not Avicel, laminarin, pachyman, xylan and pullulan. In addition, CelB29 and CelB2 showed activity against p-nitrophenyl-beta-D-cellobioside (pNP-G(2)) to p-nitrophenyl-beta-D-cellopentaoside (pNP-G(5)) but not p-nitrophenyl-beta-D-glucopyranoside (pNP-G(1)) with preferential activity against p-nitrophenyl-beta-D-cellotrioside (pNP-G(3)). Based on these results, we proposed that CelB29 and CelB2 are endoglucanases with broad substrate specificities for short- and long-chain beta-1,4-glucans.

Amino Acid Sequence↗
PubMedDate not supplied

Growth and cellulase production of Micromonospora chalcae and Pseudonocardia thermophila.

Beta(1,4)glucosidases and carboxymethylcellulases were demonstrated in both strains when using carboxymethylcellulose as a carbon source for growth. Beta(1,4)Glucosidases appeared mainly as cell-bound activities, whereas carboxymethylcellulases were evenly distributed between the incubation fluids and the cellular fractions. In both microorganisms, glucose appeared to repress biosynthesis of the enzymes, and cellobiose and carboxymethylcellulose acted as inducers of the cellulase complex.

Actinomycetales↗
PubMed2001

Studies on carboxymethyl cellulase produced by an alkalothermophilic actinomycete.

A novel alkalothermophilic actinomycete having optimum growth at pH 9 and 50 degrees C was isolated from self-heating compost from the Barabanki district of Uttar Pradesh, India. Based on its morphology, susceptibility of spores to heat and novobiocin, guaninecytosine content of chromosomal DNA and cell wall composition, the organism was classified under Thermomonospora. The alkalothermophilic actinomycete produced 23 IU/ml carboxymethyl cellulase (CMCase). The CMCase was purified by fractional ammonium sulphate precipitation followed by cellulose affinity chromatography and Sephacryl S-200 gel filtration. The CMCase had a molecular weight of 38 KD and pI of 4.1. The enzyme exhibited optimum activity at pH 5 and temperature 50 degrees C. The CMCase showed pH stability in the range 7-10. The enzyme retained 100% activity at 50 degrees C for 72 h and had half-lives of 7 and 3 h at 60 degrees C and 70 degrees C, respectively. The CMCase was stable in the presence of commercial detergents such as Ariel, Henko and Surf Excel, indicating its potential as an additive to laundry detergents.

Actinomycetales↗
PubMed2001

Regulation of cellulases and xylanases from a derepressed mutant of Cellulomonas flavigena growing on sugar-cane bagasse in continuous culture.

When the wild type Cellulomonas flavigena was grown on glycerol, xylose or cellobiose, it produced basal levels of carboxymethyl-cellulase (CMCase), filter-paperase (FPase) and xylanase activities. By comparison, a catabolic derepressed mutant strain of the same organism produced markedly higher levels of these enzymes when grown on the same carbon sources. Sugar-cane bagasse induced both the wild type and the mutant strain to produce three- to eight-time higher levels of FPase and xylanase than was observed with xylose or cellobiose. Continuous culture was used to determine the minimal cellobiose or glucose concentrations that repress the enzyme synthesis in both strains. 2.5 g l(-1) glucose repressed FPase and xylanases from wild type, while 1.6 times more glucose was needed to repress the same activities in the PN-120 strain. In the same way, twofold more cellobiose was needed to reduce by 75% the CMCase and xylanase activities in the mutant compared to the wild type. The FPase in the presence of 4 g l(-1) cellobiose did not change in the same strain. Therefore, its derepressed and feedback resistant characters of PN-120 mutant are evident. On the other hand, isoelectrofocused crude extracts of mutant and wild strains induced by sugar-cane bagasse, did not show differences in protein patterns, however, the Schiffs staining was more intense in the PN-120 than in the wild strain. These results point out that the mutational treatment did not apparently change the extracellular proteins from mutant PN-120 and this could affect their regulation sites, since derepressed and feed-back resistant enzymes may be produced.

Actinomycetales↗
PubMed2001

Hydrolysis of rice hull by crosslinked Aspergillus niger cellulase.

A. niger cellulase was crosslinked by glutaraldehyde to obtain a heat-stable enzyme preparation for rice hull cellulose hydrolysis. Under optimized crosslinking conditions of 0.12 M glutaraldehyde, pH 7.0, temperature 40 degrees C and at 45 min of crosslinking, a preparation having 15% more activity than free enzyme was obtained which also had considerable improvement in heat stability at 65 degrees C and 70 degrees C. Whereas the free enzyme lost 80% of its activity in 4 h at 65 degrees C, the crosslinked preparation lost only 30% activity. The crosslinked preparation hydrolyzed cellulosic biomass more effectively giving 2.2 mg/ml glucose and 52% corresponding saccharification in 4 h at 65 degrees C as compared to 14% saccharification by free enzyme under similar conditions.

Animal Feed↗
PubMed1995

Mitochondrial functions mediate cellulase gene expression in Trichoderma reesei.

We examined the effects of inhibition of mitochondrial functions on the expression of two nuclear genes encoding the extracellular cellobiohydrolase I (cbh1) and endoglucanase I (egl1) of the cellulase system of the filamentous fungus Trichoderma reesei. The cbh1 and egl1 transcripts are repressed at a low oxygen tension, and by glucose at a concentration known to repress mitochondrial respiration. The transcripts are also down-regulated by chemical agents known to dissipate the proton electrochemical gradient of the inner mitochondrial membrane and blocking of the electron-transport chain, such as DNP and KCN, respectively. These results suggest that expression of those transcripts is influenced by the physiological state of the mitochondria. In addition, heterologous gene fusion shows that the sensitivity of the expression of those transcripts to the functional state of the mitochondria is transcriptionally controlled through the 5'-flanking DNA sequence of those genes.

Cellulase↗
PubMed2000

Specificity and affinity of substrate binding by a family 17 carbohydrate-binding module from Clostridium cellulovorans cellulase 5A.

The C-terminal carbohydrate-binding module (CBM17) from Clostridium cellulovorans cellulase 5A is a beta-1,4-glucan binding module with a preference for soluble chains. CBM17 binds to phosphoric acid swollen Avicel (PASA) and Avicel with association constants of 2.9 (+/-0.2) x 10(5) and 1.6 (+/-0.2) x 10(5) M(-1), respectively. The capacity values for PASA and Avicel were 11.9 and 0.4 micromol/g of cellulose, respectively. CBM17 did not bind to crystalline cellulose. CBM17 bound tightly to soluble barley beta-glucan and the derivatized celluloses HEC, EHEC, and CMC. The association constants for binding to barley beta-glucan, HEC, and EHEC were approximately 2.0 x 10(5) M(-1). Significant binding affinities were found for cello-oligosaccharides greater than three glucose units in length. The affinities for cellotriose, cellotetraose, cellopentaose, and cellohexaose were 1.2 (+/-0.3) x 10(3), 4.3 (+/-0.4) x 10(3), 3.8 (+/-0.1) x 10(4), and 1.5 (+/-0.0) x 10(5) M(-1), respectively. Fluorescence quenching studies and N-bromosuccinimide modification indicate the participation of tryptophan residues in ligand binding. The possible architecture of the ligand-binding site is discussed in terms of its binding specificity, affinity, and the participation of tryptophan residues.

Amino Acid Motifs↗
PubMedDate not supplied

Surfactant-induced unfolding of cellulase: kinetic studies.

Surfactant-induced unfolding is a significant degradation pathway for detergent enzymes. This study examines the kinetics of surfactant-induced unfolding for endoglucanase III, a detergent cellulase, under conditions of varying pH, temperature, ionic strength, surfactant type, and surfactant concentration. Interactions between protein and surfactant monomer are shown to play a key role in determining the kinetics of the unfolding process. We demonstrate that the unfolding rate can be slowed by (1) modifying protein charge and/or pH conditions to create electrostatic repulsion of ionic surfactants and (2) reducing the amount of monomeric ionic surfactant available for interaction with the enzyme (i.e., by lowering the critical micelle concentration). Additionally, our results illustrate that there is a poor correlation between thermodynamic stability in buffer (DeltaG(unfolding)) and resistance to surfactant-induced unfolding.

Bacteria↗
PubMedDate not supplied

Efficiency of new fungal cellulase systems in boosting enzymatic degradation of barley straw lignocellulose.

This study examined the cellulytic effects on steam-pretreated barley straw of cellulose-degrading enzyme systems from the five thermophilic fungi Chaetomium thermophilum, Thielavia terrestris, Thermoascus aurantiacus, Corynascus thermophilus, and Myceliophthora thermophila and from the mesophile Penicillum funiculosum. The catalytic glucose release was compared after treatments with each of the crude enzyme systems when added to a benchmark blend of a commercial cellulase product, Celluclast, derived from Trichoderma reesei and a beta-glucosidase, Novozym 188, from Aspergillus niger. The enzymatic treatments were evaluated in an experimental design template comprising a span of pH (3.5-6.5) and temperature (35-65 degrees C) reaction combinations. The addition to Celluclast + Novozym 188 of low dosages of the crude enzyme systems, corresponding to 10 wt % of the total enzyme protein load, increased the catalytic glucose yields significantly as compared to those obtained with the benchmark Celluclast + Novozyme 188 blend. A comparison of glucose yields obtained on steam-pretreated barley straw and microcrystalline cellulose, Avicel, indicated that the yield improvements were mainly due to the presence of highly active endoglucanase activity/activities in the experimental enzyme preparations. The data demonstrated the feasibility of boosting the widely studied T. reeseicellulase enzyme system with additional enzymatic activity to achieve faster lignocellulose degradation. We conclude that this supplementation strategy appears feasible as a first step in identifying truly promising fungal enzyme sources for fast development of improved, commercially viable, enzyme preparations for lignocellulose degradation.

Ascomycota↗
PubMedDate not supplied

Enhancement of cellulase activity by clones selected from the combinatorial library of the cellulose-binding domain by cell surface engineering.

To improve the cellulolytic activity of a yeast strain displaying endoglucanase IotaIota (EG II) from Trichoderma reesei, a combinatorial library of the cellulose-binding domain (CBD) of EG II was constructed by using cell surface engineering. When EG II degrades celluloses, CBD binds to cellulose, and its catalytic domain cleaves the glycosidic bonds of cellulose. CBD had a flat face, composed of five amino acids for binding. It was supposed that the three hydrophobic amino acid residues of the five amino acid residues were essential for binding to cellulose. Therefore, by improving the two remaining amino acid residues, construction of mutants with a combinatorial library of the two amino acids in CBD was carried out and binding ability and hydrolysis activity were measured. In the first screening by halo assay using the Congo Red staining method, about 200 of the 2000 colonies formed clear halos, and then five colonies with the clearest halos were finally selected. In the second screening, the binding ability of the five mutants to phosphoric acid-swollen Avicel was measured. In addition, the measurement of hydrolysis activity toward carboxymethylcellulose (CMC) using the screened mutants was carried out. As a result, the mutated EG II exhibiting higher binding ability (1.5-fold) had higher hydrolysis activity (1.3-fold) compared to the parent EG II-displaying yeast cell, demonstrating that CBD has confirmatively some effect on the cellulase activity through its binding ability of the enzyme to cellulose.

Binding Sites↗
PubMed1999

Fractionation of cellulase and beta-glucosidase in a Trichoderma reesei culture liquid by use of two-phase partitioning.

An aqueous two-phase system based on the two polymers poly(ethylene glycol) and dextran has been used for the fractionation of cellulase enzymes present in culture liquid obtained by fermentation with Trichoderma reesei. The activities of beta-glucosidase and glucanases were separated to high degree by using the two-phase systems for a counter-current distribution process in nine transfer steps. While the glucanases had high affinity to the poly(ethylene glycol) rich top phase the beta-glucosidase was enriched in the dextran-containing bottom phase. Multiple counter-current distribution performed indicates the heterogeneity of beta-glucosidase activities assuming at least four isoenzyme forms. One step concentration of beta-glucosidase by using system with 46:1 phase volume ratio resulted in 16 times higher enzyme activity.

Cellulase↗
PubMed2003

Expression and import of an active cellulase from a thermophilic bacterium into the chloroplast both in vitro and in vivo.

A bacterial thermostable cellulase, the endo-1,4-beta-D-glucanase E1 from Acidothermus cellulolyticus, was imported into chloroplasts, and an active enzyme was recovered both in vitro and in vivo. Precursor fusion proteins were synthesized with E1 or its catalytic domain, CD, fused to the transit peptide of ferredoxin or ribulose-bisphosphate carboxylase activase for stromal targeting. A spacer region of 1, 5 or 15 amino acids was included carboxy to the transit peptide. The efficiency of import and processing by the stromal processing peptidase depended on the nature of the transit peptide and the passenger protein, and increased with the length of the spacer between them. Besides finding E1 or CD in the stroma, protein was arrested in the envelope during import showing that structural features of E1 and CD, along with their proximity to the transit peptide, influence translocation. The cellulose binding domain and/or serine/proline/threoline-rich linker of E1 may impede efficient import. Significantly, most precursors for E1 and CD synthesized by in vitro translation possessed endoglucanse activity that was temperature-dependent, and required the residues AGGGY at the N-terminus of E1 and CD. Furthermore, activity was detected upon import into chloroplasts. Based on the in vitro analyses, five precursor fusion proteins were selected to determine if E1 and CD would be successfully targeted to chloroplasts in vivo. In transgenic tobacco plants, E1 and CD accumulated in both the stromal and membrane fractions and, importantly, chloroplast extracts showed endoglucanase activity.

Actinomycetales↗
PubMed1978

Affinity chromatography of the cellulase system of Trichoderma koningii.

A procedure involving affinity chromatography on cellulose was developed for separating enzymic components of the cellulase complex. Cellobiase, carboxymethylcellulase, component C2 and cellobiohydrolase are adsorbed with increasing tenacity, and released, as highly purified components, as the ionic strength of the eluent is decreased.

Cellulase↗