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At least 253 records · Page 14Linked to original sources
PubMed1986

Synthesis and processing of cellulase from ripening avocado fruit.

The biosynthesis and processing of cellulase from ripening avocado fruit was studied. The mature protein is a glycoprotein, as judged by concanavalin A binding, with a molecular weight of 54,200. Upon complete deglycosylation by treatment with trifluoromethane sulfonic acid the mature protein has a molecular weight of 52,800 whereas the immunoprecipitated in vitro translation product has a molecular weight of 54,000. This result indicates that cellulase is synthesized as a large molecular weight precursor, which presumably possesses a short-lived signal peptide. A membrane-associated and heavily glycosylated form of the protein was also identified. This putative secretory precursor was enzymically active and the carbohydrate side chains were sensitive to endoglycosidase H cleavage. Results of partial endoglycosidase H digestion suggest that this precursor form of the mature glycoprotein possesses two high-mannose oligosaccharide side chains. The oligosaccharide chains of the mature protein were insensitive to endoglycosidase H cleavage, indicating that transport of the membrane-associated cellulase to the cell wall was accompanied by modification of the oligosaccharide side chains. The presence of a large pool of endoglycosidase H-sensitive membrane-associated cellulase (relative to an endoglycosidase H-insensitive form) suggest that transit of this protein through the Golgi is rapid relative to transit through the endoplasmic reticulum.

Journal Article↗
PubMed1991

Suppression of cellulase and polygalacturonase and induction of alcohol dehydrogenase isoenzymes in avocado fruit mesocarp subjected to low oxygen stress.

Expression of polygalacturonase and cellulase, two hydrolytic enzymes of avocado (Persea americana, cv Hass) fruit which are synthesized de novo during ripening, and alcohol dehydrogenase, a known anaerobic protein, were studied under different O(2) regimes. Low O(2) concentrations (2.5-5.5%) diminished the accumulation of polygalacturonase and cellulase proteins and the expression of their isoenzymes. This pattern of change in cellulase protein was also reflected in the steady-state amount of its mRNA. In contrast, 7.5 and 10% O(2) did not alter the changes observed in fruits ripened in air. On the other hand, alcohol dehydrogenase was induced in 2.5, 3.5, and 5.5% O(2) but not in 7.5 or 10% O(2). The recovery from the hypoxic stress upon returning the fruits back to air for 24 hours, was also a function of O(2) tensions under which the fruits were kept. Thus, the synthesis of polygalacturonase and cellulase was directly related to O(2) levels, while the activity of the isoenzymes of alcohol dehydrogenase was inversely related to O(2) levels. The results indicate that hypoxia exerts both negative and positive effects on the expression of certain genes and that these effects are initiated at the same levels of O(2).

Journal Article↗
PubMed1977

Semiquantitative Plate Assay for Determination of Cellulase Production by Trichoderma viride.

A plate clearing assay was devised to screen for high-producing cellulase mutants of Trichoderma viride. The method employs (i) the use of either rose bengal or oxgall to limit colony size and (ii) Phosfon D (tributyl-2, 4-dichloroben-zylphosphonium chloride) to enhance cellulase detection, in combination with acid-swollen cellulose on agar plates. The method was used to isolate constitutive cellulase mutants of T. viride and should prove useful for isolating high-producing mutants from a range of organisms. This technique has been also used to determine the concentration at which glucose and glycerol inhibit cellulase synthesis by catabolite repression in the wild-type strains.

Journal Article↗
PubMed1972

Cellulolytic activity of Thermomonospora curvata: nutritional requirments for cellulase production.

The use of a minimal medium for cellulase (C(1) and C(x)) production by Thermomonospora curvata increased extracellular C(1) activity (measured by rate of cotton fiber hydrolysis) 11-fold compared with the previously used yeast extract medium. Ground cotton fibers supported the highest cellulase production when compared to other soluble and insoluble carbohydrate sources. Maximal cellulase production occurred at 45 C, slightly less at 55 C, and was insignificant at 65 C (the highest temperature at which cellulase activity appeared stable). At a temperature of 55 C, an optimal pH of 8.0, and a cotton fiber concentration of 8 mg/ml, shake cultures of T. curvata degraded about 75% of the cellulose during the 10-day period.

Actinomycetales↗
PubMed1999

Preserving the activity of cellulase in a batch foam fractionation process.

Foam fractionation is one of the low operating-cost techniques for removing proteins from a dilute solution. The initial bulk solution pH and air superficial velocity play an important role in the foam-fractionation process. Denaturation of proteins (enzymes) can occur, however, during the foam-fractionation process from the shear forces resulting from bursting air bubbles. At the extreme bulk solution pHs (lower than 3.0 and higher than 10.0), the enzymatic activity of cellulase in the foamate phase drops significantly. Within these two pH bounds an increase in the air superficial velocity, V0, and a decrease in the bulk solution pH leads to a decrease in the separation ratio (SR), defined as the ratio of the protein concentration in the foamate to the protein concentration in the residue. On the other hand, an increase in V0 provides a higher foamate-protein recovery. The process efficiency is defined as the product of foamate-protein recovery times the SR times the cellulase activity. The optimal operating condition of the cellulase foam-fractionation process is taken into account at the maximum value of the process efficiency. In this study, that optimal condition is at an air superficial velocity of 32 cm/min and a bulk-solution pH of 10.0. At this condition, the recovered foamate is about 80% of the original protein mass, the SR is about 12, and the enzymatic activity is about 60% of the original cellulase activity.

Journal Article↗
PubMed2026

The novel transcriptional activator Bhr1 combining NTPase and Zn(II)2Cys6 DNA-binding domains controls (hemi-)cellulase response to mannose-rich substrates in the white-rot fungus Dichomitus squalens.

The regulatory landscape responsible for lignocellulose degradation in white-rot basidiomycete fungi remains largely unexplored. In this study, we characterize a novel transcriptional activator, Bhr1, in the white-rot fungus Dichomitus squalens. Bhr1 exhibits an unusual domain architecture that combines a septin-like P-loop NTPase fold with Zn(II)2Cys6 DNA-binding domains and plays a critical role in activating (hemi-)cellulase enzyme production when D. squalens is exposed to mannose-rich substrates. Using CRISPR/Cas9-mediated gene editing, we generated a bhr1 disruption mutant that displayed distinct phenotypes and enzyme activity profiles on mannose and guar gum compared to the wild type. RNA sequencing data indicate that Bhr1 induces specific (hemi-)cellulase-encoding genes without altering the expression of genes encoding sugar transporters or sugar metabolic enzymes. Phylogenetic analyses show that Bhr1 is basidiomycete specific and largely restricted to saprotrophic and plant-associated Agaricomycetes fungi. Based on the domain architecture of Bhr1 and the effects of its disruption in D. squalens, our findings reveal a lineage-specific regulatory innovation in basidiomycetes that is distinct from those described in ascomycetes. Elucidating the function and evolutionary conservation of Bhr1 advances our understanding of lignocellulose degradation at the molecular level in basidiomycete fungi and may inform studies of their ecological adaptation and the development of biotechnological applications.IMPORTANCEUnderstanding the transcriptional regulatory mechanisms in white-rot fungi, such as Dichomitus squalens, is crucial for advancing our knowledge of lignocellulose degradation. This study identifies D. squalens Bhr1 as a key regulator of (hemi-)cellulase production on mannose-rich substrates and further distinguishes basidiomycete transcription factors involved in plant biomass degradation from their ascomycete counterparts. Our findings highlight the significance of lineage-specific regulators in facilitating adaptive enzyme production for efficient biomass utilization, which is critical to carbon cycling in terrestrial ecosystems. This work establishes a foundation for exploring novel regulatory strategies among wood-degrading fungi, potentially enabling targeted strain engineering in biotechnological applications.

Mannose↗
PubMed2002

Chemoenzymatic synthesis of a bifunctionalized cellohexaoside as a specific substrate for the sensitive assay of cellulase by fluorescence quenching.

A new bifunctionalized cellohexaose derivative was synthesized as a specific substrate for continuous assay of cellulases by resonance energy transfer. This cellohexaoside has a naphthalene moiety (EDANS) as a fluorescent energy donor at the reducing end and a 4-(4'-dimethylaminobenzeneazo)-benzene derivative as an acceptor chromophore at the non-reducing end. The key steps for the preparation of the target molecule involved transglycosylation reactions of cellobiosyl and cellotetraosyl fluoride donors onto cellobiosyl acceptors catalysed by the E197A mutant of cellulase Cel7B from Humicola insolens. Upon digestion with various cellulases, the energy transfer was disrupted and an increase of fluorescence was observed.

Cellulase↗
PubMed2004

Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems.

Information pertaining to enzymatic hydrolysis of cellulose by noncomplexed cellulase enzyme systems is reviewed with a particular emphasis on development of aggregated understanding incorporating substrate features in addition to concentration and multiple cellulase components. Topics considered include properties of cellulose, adsorption, cellulose hydrolysis, and quantitative models. A classification scheme is proposed for quantitative models for enzymatic hydrolysis of cellulose based on the number of solubilizing activities and substrate state variables included. We suggest that it is timely to revisit and reinvigorate functional modeling of cellulose hydrolysis, and that this would be highly beneficial if not necessary in order to bring to bear the large volume of information available on cellulase components on the primary applications that motivate interest in the subject.

Cellulase↗
PubMed2004

Microplate-based filter paper assay to measure total cellulase activity.

The standard filter paper assay (FPA) published by the International Union of Pure and Applied Chemistry (IUPAC) is widely used to determine total cellulase activity. However, the IUPAC method is not suitable for the parallel analyses of large sample numbers. We describe here a microplate-based method for assaying large sample numbers. To achieve this, we reduced the enzymatic reaction volume to 60 microl from the 1.5 ml used in the IUPAC method. The modified 60-microl format FPA can be carried out in 96-well assay plates. Statistical analyses showed that the cellulase activities of commercial cellulases from Trichoderma reesei and Aspergillus species determined with our 60-microl format FPA were not significantly different from the activities measured with the standard FPA. Our results also indicate that the 60-microl format FPA is quantitative and highly reproducible. Moreover, the addition of excess beta-glucosidase increased the sensitivity of the assay by up to 60%.

Cellulase↗
PubMed1979

Cellulase production and ammonia metabolism in Trichoderma reesei on high levels of cellulose.

Trichoderma can be cultured in stirred-tank fermentors on high (8%) cellulose concentrations without increasing the salt concentration of the medium when NH4OH is used to control pH and as a nitrogen source. Approximately 90% of the ammonia consumed by the organism can be added as NH4OH. The advantage of using high concentrations of cellulose is that culture filtrates with greater cellulase activity are obtained. The advantage of a low salts medium is that unwanted solutes in the final enzyme preparation are reduced. The appearance of cellulase in the medium occurs later than net ammonia uptake so that only 20% of the final amount of cellulase has appeared when 80% of the maximum amount of ammonia has been consumed.

Ammonia↗
PubMed1998

Aryl thioglycoside-based affinity purification of exo-acting cellulases.

The influence of ligand-coupling chemistry and mobile-phase composition on the interaction of exo-acting cellulases with an immobilized complementary ligand was investigated. p-Aminophenyl 1-thio-beta-D-cellobioside (APTC) was used as a representative affinity ligand to which exo-acting cellulases (cellobiohydrolases, CBHs) preferentially bind. A "crude" cellulase preparation from the fungus Trichoderma reesei served as an enzyme source. The adsorption properties of the two principal exo-acting CBHs in this preparation, CBH I and CBH II, are shown to be distinctly different under several scenarios. Their relative affinities, based on column elution behavior and partition equilibrium experiments, are shown to be highly dependent on the functional groups employed for ligand coupling, the extent of functional group hydrolysis, the composition of the mobile phase, and the inherent nature of the enzymes. The dependency on the chemistry of the supporting matrix was illustrated using agarose supports containing cyanate ester, N-hydroxy-succinimide, and epoxy functional groups. When compared under apparent optimal conditions, the affinity of CBH II for immobilized APTC was approximately 10-fold that of CBH I. However, selective adsorption of CBH I or CBH II can be achieved by adjusting experimental parameters.

Basidiomycota↗
PubMed1995

Palladium--a new inhibitor of cellulase activities.

Palladium complexes have been shown to strongly inhibit cellobiohydrolase I (CBH I) and endoglucanase II (EG II), two cellulases produced by Trichoderma reesei. Also inhibited were total cellulase (Avicelase) and beta-glucosidase (cellobiase) activities. The catalytic domain of CBH II, the second most abundant component of this cellulase, appeared less susceptible to inhibition by palladium. The inhibition was irreversible and could be prevented if histidine, cysteine or cystine was added to the enzyme reaction mixture simultaneously with the inhibitor. The binding of CBH I to microcrystalline cellulose (Avicel) was unaffected by palladium.

Binding Sites↗
PubMed1995

Informational suppression to investigate structural functional and evolutionary aspects of the Erwinia chrysanthemi cellulase EGZ.

The cellulase EGZ produced by the plant pathogen Erwinia chrysanthemi belongs to family 5 of the beta-glycohydrolases (also referred to as cellulase family A), which contains over 40 members from Gram-negative and Gram-positive bacteria and fungi. Amber mutations were introduced into 16 codons of the celZ gene encoding EGZ. Targeted residues included: (1) two Glu, two His and one Arg residue, strictly conserved throughout family 5; (2) one Arg and one His residue conserved in sub-family 5-2; and (3) one His and six Arg residues not conserved at all. Each amber allele was introduced into 13 Escherichia coli strains each carrying a different suppressor tRNA that inserts an amino acid at the mutated position. In vivo stability of the mutated forms of EGZ and their cellulase activity were analysed as well as suppression efficiency. For some positions of particular interest, missense mutations were introduced into the celZ gene either to confirm the effect of the suppressor-mediated amino acid substitution or to broaden the spectrum of mutations available. The substitution patterns of the two Glu positions were interpretable in the light of the stereospecificity of the reaction catalysed by EGZ: Glu133 and Glu220 are proposed to act as a proton donor and as a nucleophile, respectively, forming the glycosyl-enzyme intermediate. Substitution at His-occupied positions, including two non-conserved positions, yielded proteins affected in their catalytic activity but not their in vivo stability. In particular, evidence was obtained for His at position 98 to be involved in interactions with the substrate. The view that Arg residues are important in stabilizing proteins was supported by the identification of three Arg residues, whose substitution yielded thermosensitive forms of EGZ. In addition, Pro substitutions of any of the six Arg residues altered protein stability in vivo but the substitutions scored almost neutral for activity. Five positions, predicted to be within alpha-helices, were found to be susceptible to Pro substitutions (but not to Ala) with respect to stability in vivo. Overall, the systematic alteration of all His and Arg residues coupled with the simultaneous analysis of activity and in vivo stability allowed us to demonstrate that substitution matrices vary at each position and for each biological property considered. Ideally, therefore, substitution matrices used in sequence alignment procedures should be reconsidered as position-specific and as property-specific.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

Direct isolation of functional genes encoding cellulases from the microbial consortia in a thermophilic, anaerobic digester maintained on lignocellulose.

Gene libraries ("zoolibraries") were constructed in Escherichia coli using DNA isolated from the mixed liquor of thermophilic, anaerobic digesters, which were in continuous operation with lignocellulosic feedstocks for over 10 years. Clones expressing cellulase and xylosidase were readily recovered from these libraries. Four clones that hydrolyzed carboxymethylcellulose and methylumbelliferyl-beta-D-cellobiopyranoside were characterized. All four cellulases exhibited temperature optima (60-65 degrees C) and pH optima (pH 6-7) in accordance with conditions of the enrichment. The DNA sequence of the insert in one clone (plasmid pFGH1) was determined. This plasmid encoded an endoglucanase (celA) and part of a putative beta-glucosidase (celB), both of which were distinctly different from all previously reported homologues. CelA protein shared limited homology with members of the A3 subfamily of cellulases, being similar to endoglucanase C from Clostridium thermocellum (40% identity). The N-terminal part of CelB protein was most similar to beta-glucosidase from Pseudomonas fluorescens subsp. cellulosa (28% homology). The use of zoolibraries constructed from natural or laboratory enrichment cultures offers the potential to discover many new enzymes for biotechnological applications.

Amino Acid Sequence↗
PubMed1990

Quantification and identification of the main components of the Trichoderma cellulase complex with monoclonal antibodies using an enzyme-linked immunosorbent assay (ELISA).

An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies has been developed to measure the concentration of three main cellulase components from Trichoderma reesei, cellobiohydrolase I (CBH I), cellobiohydrolase II (CBH II) and endoglucanase I (EG I), in both commercial enzyme preparations as well as in samples from laboratory fermentations. The sensitivity of the assay is 1-10 ng protein, depending on the type of cellulase. The coefficient of variability is between 10% and 20%. By a combination of two different domain-specific monoclonals against CBH I or II it is also possible to quantify the concentration of intact and truncated forms of these two enzymes, respectively. The use of the ELISA to quantify the formation of the three cellulase components under different cultivation conditions is described.

Antibodies, Monoclonal↗
PubMed1995

Studies on the capacity of the cellulase of the anaerobic rumen fungus Piromonas communis P to degrade hydrogen bond-ordered cellulose.

The anaerobic rumen fungus Piromonas communis, when cultured on cotton fibre as the carbon source, produces an extracellular cellulase that is capable of solubilizing "crystalline" hydrogen-bond-ordered cellulose, in the form of the cotton fibre, at a rate that is greater than that of any other cellulases reported in the literature hitherto. The cell-free culture fluid is also very rich in xylan-degrading enzymes. The activity towards crystalline cellulose resides in a high-molecular-mass (approximately 700-1000 kDa) component (so-called crystalline-cellulose-solubilizing component, CCSC) that comprises endo (1-->4)-beta-D-glucanase (carboxymethylcellulase), beta-D-glucosidase and another enzyme that appears to be important for the breakdown of hydrogen-bond-ordered cellulose. The CCSC is associated with only a small amount of the endo(1-->4)-beta-D-glucanase (1.9%), beta-D-glucosidase (0.7%) and protein (0.5%) found in the crude cell-free cellulase preparation. The CCSC, unlike the bulk of the endo(1-->4)-beta-D-glucanase and beta-D-glucosidase, is very strongly absorbed on the microcrystalline cellulose, Avicel.

Anaerobiosis↗
PubMed1993

High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes.

Four cellulase genes of Trichoderma reesei, cbh1, cbh2, egl1 and egl2, have been replaced by the amdS marker gene. When linear DNA fragments and flanking regions of the corresponding cellulase locus of more than 1 kb were used, the replacement frequencies were high, ranging from 32 to 52%. Deletion of the major cellobiohydrolase 1 gene led to a 2-fold increase in the production of cellobiohydrolase II; however, replacement of the cbh2 gene did not affect the final cellulase levels and deletion of egl1 or egl2 slightly increased production of both cellobiohydrolases. Based on our results, endoglucanase II accounts for most of the endoglucanase activity produced by the hypercellulolytic host strain. Furthermore, loss of the egl2 gene causes a significant drop in the filter paper-hydrolysing activity, indicating that endoglucanase II has an important role in the total hydrolysis of cellulose.

Cellulase↗
PubMed1993

Molecular analysis of the major cellulase (CelV) of Erwinia carotovora: evidence for an evolutionary "mix-and-match" of enzyme domains.

The structural gene for the major cellulase of Erwinia carotovora subspecies carotovora (Ecc) was isolated and expressed in Escherichia coli. Sequencing of the gene (celV) revealed a typical signal sequence and two functional domains in the enzyme; a catalytic domain linked by a short proline/threonine-rich linker to a cellulose-binding domain (CBD). The deduced amino acid sequence of the catalytic domain showed homology with cellulases of Family A, including enzymes from Bacillus spp. and Erwinia chrysanthemi CelZ, whereas the CBD showed homology with cellulases from several diverse families, supporting a "mix-and-match" hypothesis for evolution of this domain. Analysis of the substrate specificity of CelV showed it to be an endoglucanase with some exoglucanase activity. The pH optimum is about 7.0 and the temperature optimum about 42 degrees C. CelV is secreted by Ecc and by the taxonomically related Erwinia carotovora subspecies atroseptica (Eca) but not by E. coli. Overproduction of the enzyme from multicopy plasmids in Ecc appears to overload the secretory mechanism.

Amino Acid Sequence↗