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PubMed1995

Ultrastructural diversity of the cellulase complexes of Clostridium papyrosolvens C7.

Transmission electron microscopy was used to investigate the ultrastructural features of diverse cellulase and cellulase-xylanase multiprotein complexes that are components of the cellulase-xylanase system of Clostridium papyrosolvens C7. The multiprotein complexes were separated by anion-exchange chromatography into seven biochemically distinguishable fractions (F1 to F7). Most individual F fractions contained, in relatively large numbers, an ultrastructurally recognizable type of particle that occurred only in smaller numbers, or not at all, in the other F fractions. It is suggested that these ultrastructurally distinct particles represent the biochemically distinct multiprotein complexes that constitute the cellulase-xylanase system of C. papyrosolvens C7. Some of the particles consisted of tightly packed globular components that appeared to be arranged in the shape of a ring with conical structures pointing out along its axis. Other particles had triangular, polyhedral, or star shapes. The major protein fraction (F4) almost exclusively contained particles consisting of loosely aggregated components, many of which appeared to be arranged along filamentous structures. The ultrastructural observations reported here support our previous conclusion that the cellulase-xylanase system of C. papyrosolvens C7 comprises at least seven different high-molecular-weight multiprotein complexes. Furthermore, results of this and earlier studies indicate that the interactions between C. papyrosolvens C7 and cellulose are different from those that have been described for Clostridium thermocellum.

Cellulase↗
PubMed1976

Cellulase induction and the use of cellulose as a preferred growth substrate by Cellvibrio gilvus.

Cellvibrio gilvus produced cellulase when grown in the presence of cellulose or carboxymethyl cellulose (CMC) but not when grown in the presence of glucose or cellobiose. This was so whether or not these compounds were the sole carbon sources present. Repeated addition of small amounts of glucose prevented cellulase formation in the presence of cellulose. It is concluded that cellulose and CMC induce cellulase formation and glucose and cellobiose repressit. Cellulose stimulated growth when added to organic media. Physical contact between cells and cellulose fibres was necessary for both cellulase induction and growth stimulation. If cellulase induction was prevented by glucose, cellulose did not stimulate growth.

Bacterial Proteins↗
PubMed2005

Extracellular cellulase production by tropical isolates of Aureobasidium pullulans.

Cellulase production by Aureobasidium pullulans from the temperate regions has remained speculative, with most studies reporting no activity at all. In the current study, tropical isolates from diverse sources were screened for cellulase production. Isolates were grown on a synthetic medium containing cell walls of Msasa tree (Brachystegia sp.) as the sole carbon source, and their cellulolytic activities were measured using carboxymethyl cellulose and alpha-cellulose as substrates. All isolates studied produced carboxymethyl cellulase (endoglucanase) and alpha-cellulase (exoglucanase) activity. Endoglucanase-specific activities of ten selected isolates ranged from 2.375 to 12.884 micromol glucose.(mg protein)-1.h-1, while activities on alpha-cellulose (exoglucanase activity) ranged from 0.293 to 22.442 micromol glucose.(mg protein)-1.day-1. Carboxymethyl cellulose induced the highest cellulase activity in the selected isolates, while the isolates showed variable responses to nitrogen sources. The current study indicates that some isolates of A. pullulans of tropical origin produce significant extracellular cellulolytic activity and that crude cell walls may be good inducers of cellulolytic activity in A. pullulans.

Ascomycota↗
PubMed2006

Pathogenic cellulase assay of pine wilt disease and immunological localization.

The pine wilt disease caused by Bursaphelenchus xylophilus (BX), also known as the pine wood nematode (PWN), is the most devastating disease of pine trees. In this work, a high molecular weight B. xylophilus cellulase antigen (BXCa) was purified from total homogenates of nematodes. BXCa was found to be able to hydrolyze carboxymethyl cellulose (CMC) efficiently (155.65 U/mg) and to have an approximate molecular mass of 58.9 kDa. We harvested anti-BXCa antibodies and performed immunocytochemical assays, which revealed the localization of cellulase pools in the esophageal gland cells of the PWN. It was also discovered that cellulase was secreted from the stylet and was used to hydrolyze cellulose to facilitate the PWN entering host cells. These results are consistent with other plant parasitical nematodes. Interestingly, strong fluorescence signals from cellulase staining were observed in tracheid cells in naturally infected pine wood, in addition to ray cells and the resin canal zone. These results strongly suggest that the cellulase released by the PWN is one of the pathogenic substances of pine wilt disease and is responsible for the development of the early symptoms of the disease.

Animals↗
PubMed2004

Dynamics of cellulase production by glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose.

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily fermentable low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity products with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources-including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucose-grown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka-floc (delignified pine pulp), and the response was monitored in terms of CO2 evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO2 evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO2 evolution are analyzed and discussed herein.

Biomass↗
PubMed2005

Weak lignin-binding enzymes: a novel approach to improve activity of cellulases for hydrolysis of lignocellulosics.

Economic barriers preventing commercialization of lignocellulose-to-ethanol bioconversion processes include the high cost of hydrolytic enzymes. One strategy for cost reduction is to improve the specific activities of cellulases by genetic engineering. However, screening for improved activity typically uses "ideal" cellulosic substrates, and results are not necessarily applicable to more realistic substrates such as pretreated hardwoods and softwoods. For lignocellulosic substrates, nonproductive binding and inactivation of enzymes by the lignin component appear to be important factors limiting catalytic efficiency. A better understanding of these factors could allow engineering of cellulases with improved activity based on reduced enzyme-lignin interaction ("weak lignin-binding cellulases"). To prove this concept, we have shown that naturally occurring cellulases with similar catalytic activity on a model cellulosic substrate can differ significantly in their affinities for lignin. Moreover, although cellulose-binding domains (CBDs) are hydrophobic and probably participate in lignin binding, we show that cellulases lacking CBDs also have a high affinity for lignin, indicating the presence of lignin-binding sites on the catalytic domain.

Cellulases↗
PubMed1989

Regulatory aspects of cellulase biosynthesis and secretion.

The cellulase enzyme system consists of cellobiohydrolase, endoglucanase, and beta-glucosidase and has been extensively studied with respect to its biosynthesis, properties, mode of action, application, and, most recently, secretion mechanisms. A knowledge of the factors governing the biosynthesis and secretion of these enzymes at the molecular level will be useful in maximizing enzyme productivity in extracellular fluid. Among other topics, the regulatory effects of sorbose (a noninducing sugar which is not a product of cellulose hydrolysis) on cellulase synthesis and release are described. Cellulase genes have recently been cloned into a number of microorganisms with a view to understanding the gene structure and expression and to obtaining the enzyme components in pure form. The factors governing biosynthesis and secretion of cellulases in recombinant cells are also discussed. Cellulases are known to be glycoproteins, therefore, the role of O- and N-linked glycosylation on enzyme stability and secretion is also detailed.

Amino Acid Sequence↗
PubMed1999

A modular cinnamoyl ester hydrolase from the anaerobic fungus Piromyces equi acts synergistically with xylanase and is part of a multiprotein cellulose-binding cellulase-hemicellulase complex.

A collection of clones, isolated from a Piromyces equi cDNA expression library by immunoscreening with antibodies raised against affinity purified multienzyme fungal cellulase-hemicellulase complex, included one which expressed cinnamoyl ester hydrolase activity. The P. equi cinnamoyl ester hydrolase gene (estA) comprised an open reading frame of 1608 nt encoding a protein (EstA) of 536 amino acids and 55540 Da. EstA was modular in structure and comprised three distinct domains. The N-terminal domain was closely similar to a highly conserved non-catalytic 40-residue docking domain which is prevalent in cellulases and hemicellulases from three species of anaerobic fungi and binds to a putative scaffolding protein during assembly of the fungal cellulase complex. The second domain was also not required for esterase activity and appeared to be an atypically large linker comprising multiple tandem repeats of a 13-residue motif. The C-terminal 270 residues of EstA contained an esterase catalytic domain that exhibited overall homology with a small family of esterases, including acetylxylan esterase D (XYLD) from Pseudomonas fluorescens subsp. cellulosa and acetylxylan esterase from Aspergillus niger. This region also contained several smaller blocks of residues that displayed homology with domains tentatively identified as containing the essential catalytic residues of a larger group of serine hydrolases. A truncated variant of EstA, comprising the catalytic domain alone (EstA'), was expressed in Escherichia coli as a thioredoxin fusion protein and was purified to homogeneity. EstA' was active against synthetic and plant cell-wall-derived substrates, showed a marked preference for cleaving 1-->5 ester linkages between ferulic acid and arabinose in feruloylated arabino-xylo-oligosaccharides and was inhibited by the serine-specific protease inhibitor aminoethylbenzene-sulphonylfluoride. EstA' acted synergistically with xylanase to release more than 60% of the esterified ferulic acid from the arabinoxylan component of plant cell walls. Western analysis confirmed that EstA is produced by P. equi and is a component of the aggregated multienzyme cellulase-hemicellulase complex. Hybrid proteins, harbouring one, two or three iterations of the conserved 40-residue fungal docking domain fused to the reporter protein glutathione S-transferase, were produced. Western blot analysis of immobilized P. equi cellulase-hemicellulase complex demonstrated that each of the hybrid proteins bound to a 97 kDa polypeptide in the extracellular complex.

Amino Acid Sequence↗
PubMed1998

[The intergeneric compatibility of heredity and expression for cellulase genomes between Aspergillus niger and Trichoderma reesei].

By using the developed display techniques of cellulase isozymes and the RAPD-PCR analysis guided by a deduced universal sequence of cellulase genes, the polymorphisms of genomic DNA fingerprints and cellulase isozymes were compared among three typical stable recombinants (3a, 3b, A7-1) and their two parents (Aspergillus niger AMS11, Trichoderma reesei QM9414) in order to provide the molecular evidence of gene recombination, to demonstrate the compatibility of heredity and expression of intergeneric genomes, and to assay on the molecular fundamentals of hybridization dominance. The results showed that in these recombinant strains the recombinantal fingerprints of genomic DNA could be stablly hereditary and the expression of recombinantal CMCase (carboxymethylcellulase) and beta GLase (beta-glucosidase) could be compatibly enhanced. The diversity of molecular fundamentals of cellulase hybridization dominance were (1) the compatible co-existence and enhanced expression of some hereditary beta GLase-coding genes from two parents in recombinant 3b; and (2) the compatibly enhancement of expression between the hereditary genes encoding beta GLase and CMCase from two parents, resulting in the dramatic increase of proteins of corresponding isozymes in recombinants 3a and A7-1. Based on these, a proposal model for the double synergism on cellulase activity in vitro and on its biosynthesis in vivo mediated by beta GLase was suggested. A practical method for assaying on the molecular fundamentals and the stability of hybridization dominance of recombinants was thereby established in this study.

Aspergillus niger↗
PubMed2003

Isolation of a multi-functional endogenous cellulase gene from mollusc, Ampullaria crossean.

The cellulase genes of some animals, most coding for endo-beta-1,4-glucanases, were found and cloned. There has been no reports about genes encoding exo-beta-1,4-glucanase or endo- -1,4-xylanase from animal. Here we cloned the cDNA of a cellulase designated as EGX from mollusc, Ampullaria crossean, and expressed it in Pichia pastoris for the first time. The cellulase EGX is a multi-functional beta cellulase with the activities of exo-beta-1,4-glucanase, endo-beta-1,4-glucanase and endo-beta-1,4-xylanase. The opening reading frame of EGX cDNA is 1185 bp and encodes 395 amino acids. The EGX gene can also be amplificated from the genomic DNA by PCR, which verified the endogenous origin of this gene. This EGX gene was the first multi-functional cellulase gene that was directly isolated from animals.

Amino Acid Sequence↗
PubMed1980

[Relationship between the kinetics of hydrolysis of soluble and native celluloses under the action of cellulase multi-enzyme complexes].

The kinetics of cellulase multi-enzyme complex under steady- and non-steady-state conditions have been studied. To verify the kinetic regularities obtained the action of eight different cellulases from Trichoderma, Geotrichum and Aspergillus has been studied with respect to ball milled cotton linters. On the other hand, the activities of the individual components of cellulase complexes, i.e. endoglucanase, exoglucosidase and cellobiase have been determined with respect to soluble cellulose derivatives. Theoretical and experimental evidence for the existence of a linear correlation between the rate of hydrolysis of native cellulose under certain experimental conditons, on the one hand, and that of its soluble polymeric compounds (particularly, carboxymethyl cellulose) on the other, have been obtained. The kinetics of the cellulase multi-enzyme system suggest that this correlation is a substantiation of the fact that endoglucanse is a cellulolytic component which is the first to attack native cellulose. The data obtained contradict the hypothesis being discussed in literature for the last 30 years about the presence in cellulase complexes of some "pre-hydrolytic C1-enzyme", which presumably attacks native cellulose. Evidently, a hypothetical "C1-enzyme" is the endoglucanase of random action.

Aspergillus↗
PubMed1977

Kinetic properties and substrate specificities of two cellulases from auxin-treated pea epicotyls.

Two cellulases purified from growing regions of auxin-treated peas (buffer-soluble and buffer-insoluble) hydrolyze cellulose powder, partially substituted carboxymethylcellulose (CM-cellulose), higher cellodextrins, and certain mixed linkage glucans (e.g. barley beta-glucan), at rates comparable to these reported for the most active fungal cellulases, and with kinetics and product formation characteristic of endohydrolase action. They are unable to cleave 1,3-linkages in beta-glucans, or 1,4-linkages in dextrins containing excessive substitution at C6, alpha configuration, alternating beta-1,3- and 1,4-linkages, or residues other than anhydroglucose. They are not active towards cellobiose or the 1,4-linkage adjacent to the reducing end of cellodextrin chains. It is concluded that buffer-soluble and buffer-insoluble cellulases are true beta-1,4-glucan 4-glucanohydrolases (EC 3.2.1.4). On a molar basis, Vmax values for buffer-insoluble are higher than buffer-soluble cellulase acting towards any of the substrates tested, but Km values towards CM-cellulose and cellohexaose are essentially identical. Both cellulases were inhibited by C12+, Hg2+, and sulfhydryl-binding reagents. Buffer-insoluble, but not buffer-soluble, cellulose was inactivated by reagents that bind serine and threonine, which reflects differences in their amino acid composition. No major qualitative differences have been detected in the mode of action of the two enzymes. Despite marked differences in their physical and immunological properties, close similarities between buffer-soluble and buffer-insoluble enzymic properties suggest that their active sites are the same.

Cellulase↗
PubMed1991

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains.

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and beta-octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but - unlike in extracellular culture fluids from Trichoderma - CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.

Antibodies, Monoclonal↗
PubMed1992

Cellobiose oxidase of Phanerochaete chrysosporium enhances crystalline cellulose degradation by cellulases.

The effect of Phanerochaete chrysosporium cellobiose oxidase (CBO) on microcrystalline cellulose hydrolysis by Trichoderma cellulases was determined. Addition of 10 micrograms.ml-1 CBO to a reaction mixture containing T. viride cellulase increased glucose and cellobiose production by 10% and 48%, respectively. Cellulose weight loss was also enhanced by 19%. At higher concentrations (20-80 micrograms.ml-1), CBO decreased glucose and cellobiose production. Cellulose weight loss at 60 micrograms.ml-1 CBO was 76% compared to control cellulase reactions. This decrease appears to be due to inactivation of cellulase by H2O2 produced via CBO reaction, because addition of catalase enhances sugar production and cellulose weight loss. These findings suggest that at low, perhaps physiologically relevant concentrations, CBO enhances crystalline cellulose degradation by cellulases.

Basidiomycota↗
PubMed1966

Cellulases of a marine mollusc, Dolabella sp.

1. A crude cellulase extract was prepared from the hepatopancreas of a marine mollusc, Dolabella sp., and partially purified by ammonium sulphate fractionation. 2. The optimum pH values of the partially purified preparation were 6.5 and 8.0 for Walseth cellulose and CM-cellulose respectively. It was most stable at pH6.0 and showed moderate thermostability. 3. The partially purified preparation was subjected to starch-zone electrophoresis, and incompletely resolved into several fractions that contained one or more cellulase components of different substrate specificity. 4. Some of these cellulase fractions showed practically no aryl beta-glucosidase activity and hydrolysed aryl beta-cellobioside with difficulty. From substrates such as higher cello-oligosaccharides, cellodextrin, CM-cellulose, Walseth cellulose and cotton fibre, they produced cellobiose as the major and cellotriose as the minor end products, both of which were resistant to further attack by cellulase. 5. From the slope of the curves of viscosity-reducing power for CM-cellulose, the cellulase components from Dolabella were presumed to be of a ;more-random' or a ;less-random' type in the mode of action. 6. In the hepatopancreas of this mollusc, beta-glucosidases were also present, which hydrolysed cellobiose as well as aryl beta-glucosides. The optimum pH values of these enzymes were about 5.5.

Animals↗
PubMed1969

Activity of pectin esterase and cellulase in the abscission zone of citrus leaf explants.

The activity of pectin esterase and cellulase in abscission of citrus explants was studied. No relation was established between pectin esterase and abscission, while cellulase activity was markedly increased before abscission and for a certain period after excision. IAA and cycloheximide delay abscission and cellulase activity, while ethylene and, to a lesser extent, GA(3) accelerate them. Application of cycloheximide during the lag period and before cellulase activity can be measured, inhibits to a certain extent the formation of cellulase. An escape from the inhibitory effect of cycloheximide is detected when inhibitor is supplied at the end of the lag period.

Journal Article↗
PubMed1970

Synthesis of Cellulase during Abscission of Phaseolus vulgaris Leaf Explants.

When abscission in leaf explants from Phaseolus vulgaris, cultivar Red Kidney, was allowed to proceed while the explants were in (2)H(2)O, a 1.25% increase in the buoyant density of cellulase in a cesium chloride gradient was observed. These data indicate that the increase in cellulase activity during abscission is a result of the synthesis of new protein. Two differentially soluble forms of cellulase are present in the abscission zone. The form which is soluble only in a high salt buffer seems more closely related to the abscission process than the form which is soluble in dilute buffer. The correlation between changes in pull force and increase in cellulase activity and the effects of several hormones on cellulase activity are discussed.

Journal Article↗
PubMed1973

Hormonally Induced Changes in the Stem and Petiole Anatomy and Cellulase Enzyme Patterns in Phaseolus vulgaris L.

Time course changes were observed in petiole and stem anatomy and cellulase enzyme patterns in bean (Phaseolus vulgaris L.) explants when 10(-5) or 10(-2)m indoleacetic acid in lanolin paste was applied to acropetal cut surfaces in the presence or absence of ethylene. Auxin (10(-2)m) in the presence of ethylene stimulated rapid ordered cell division and dedifferentiation, with ensuing lateral root formation. Auxin (10(-5)m) caused moderate cortical swelling, pit formation in pith parenchyma, and chloroplast development in certain cortical cells. Exogenous ethylene reduced cell division activity and caused cortical cell swelling and separation. Removal of endogenously generated ethylene by mercuric perchlorate resulted in less ordered cell division patterns and no lateral root formation. Auxin treatments enhanced formation of an active acidic pI cellulase, exogenous ethylene-stimulated formation of an active basic pI cellulase. The absence of basic pI cellulase activity by the removal of endogenously generated ethylene suggests a close dependence of basic pI cellulase activity on ethylene.

Journal Article↗