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PubMed2004

Cellulase retention and sugar removal by membrane ultrafiltration during lignocellulosic biomass hydrolysis.

Technologies suitable for the separation and reuse of cellulase enzymes during the enzymatic saccharification of pretreated corn stover are investigated to examine the economic and technical viability of processes that promote cellulase reuse while removing inhibitory reaction products such as glucose and cellobiose. The simplest and most suitable separation is a filter with relatively large pores on the order of 20-25 mm that retains residual corn stover solids while passing reaction products such as glucose and cellobiose to form a sugar stream for a variety of end uses. Such a simple separation is effective because cellulase remains bound to the residual solids. Ultrafiltration using 50-kDa polyethersulfone membranes to recover cellulase enzymes in solution was shown not to enhance further the saccharification rate or overall conversion. Instead, it appears that the necessary cellulase enzymes, including beta-glucosidase, are tightly bound to the substrate; when fresh corn stover is contacted with highly washed residual solids, without the addition of fresh enzymes, glucose is generated at a high rate. When filtration was applied multiple times, the concentration of inhibitory reaction products such as glucose and cellobiose was reduced from 70 to 10 g/L. However, an enhanced saccharification performance was not observed, most likely because the concentration of the inhibitory products remained too high. Further reduction in the product concentration was not investigated, because it would make the reaction unnecessarily complex and result in a product stream that is much too dilute to be useful. Finally, an economic analysis shows that reuse of cellulase can reduce glucose production costs, especially when the enzyme price is high. The most economic performance is shown to occur when the cellulase enzyme is reused and a small amount of fresh enzyme is added after each separation step to replace lost or deactivated enzyme.

Biomass↗

PubMed2005

Enhancing cellulase foam fractionation with addition of surfactant.

Foam fractionation cannot be used to recover cellulase from an aerated water solution effectively because cellulase by itself can produce only a small amount of foam. The addition of a surfactant can, however, increase the foamate volume and enhance the concentration of cellulase. We studied three detergents individually added to a 200 mg/L cellulase solution to promote foaming. These detergents were anionic, cationic, and nonionic surfactants, respectively. Although contributing to foam production, it was observed that nonionic surfactant (Pluronic F-68) barely concentrated cellulase, leaving the enrichment ratio unchanged, near 1. With anionic surfactant, sodium dedecyl sulfate, and cationic surfactant, cetyltrimethylammonium bromide (CTAB), the enrichment ratio became much larger, but cellulase denaturation occurred, reducing the activity of the enzyme. When CTAB was used to help foam cellulase, beta-cyclodextrin was subsequently added to the foamate to help restore the enzyme activity.

Cellulase↗

PubMed2006

Affinity foam fractionation of Trichoderma cellulase.

Cellulase could not be selectively collected from fermentation broth by simple foam fractionation, because of the presence of other more surface-active compounds. A new approach of affinity foam fractionation was investigated for improvement. A hardwood hydrolysate (containing cellulose oligomers, substrates to cellulase) and two substrate analogs, i.e., carboxymethyl cellulose (CMC) and xylan hydrolysate, were added before the foaming process. The substrates and substrate analogs were indeed found to bind the cellulase selectively and form more hydrophobic complexes that partition more readily onto bubble surfaces. In this study, the effects of the type and concentration of substrate/analog as well as the presence of cells at different growth stages were examined. The foam fractionation properties evaluated included foaming speed, foam stability, foamate volume, and enrichment of filter paper unit (FPU) and individual cellulase components (i.e., endoglucanases, exoglucanases, and beta-glucosidases). Depending on the broth and substrate/analog employed, the foamate FPU could be more than fourfold higher than the starting broth FPU. Addition of substrate/analog also deterred the enrichment of other extracellular proteins, resulting in the desired cellulase purification in the foamate. The value of E/P (enzyme activity-FPU/g/L of proteins) in the foamate reached as high as 18, from a lactose-based fermentation broth with original E/P of 5.6. Among cellulase components, exoglucanases were enriched the most and beta-glucosidases the least. The study with CMC of different molecular weights (MW) and degrees of substitution (DS) indicated that the CMC with low DS and high MW performed better in cellulase foam fractionation.

Cell Culture Techniques↗

PubMed1980

[Hydrolysis of microcrystalline cellulose by multienzyme cellulase complexes of various origins].

The kinetic regularities of glucose and cellobiose formation from microcrystalline cellulose (MCC) under the action of cellulase complexes from eight different sources were studied. By means of successive addition of selected components of the cellulase complexes (endoglucanase and cellobiase) to the reaction system the rate-limiting steps for multienzymatic hydrolysis of MCC were determined. It was shown that in most cases the rate-limiting step of glucose formation (via hydrolysis of the intermediate cellobiose) is the cellobiase action. In a single case only (with a cellulase complex from Aspergillus foetidus enriched with cellobiase) the rate of glucose formation from MCC was limited by the endoglucanase action. In accordance with the kinetic theory developed it was shown that the addition of cellobiase excess to the reaction system resulted in changes of the rate-limiting step over to endoglucanase attack on the non-soluble cellulose for all cellulase complexes under study. Under the given experimental conditions a linear correlation between the steady-state ready of glucose formation from MCC under the action of all cellulase complexes on the on hand, and the endoglucanase activity of these complexes, on the other, was established. It was shown that the action of all cellulase (arbitrarily selected ones) is described by principally the same kinetic regularities, which, in turn, is indicative of identical mechanisms for hydrolysis of the insoluble cellulose under effects of cellulase complexes of various origin.

Actinomycetales↗

PubMed1989

Isolation, characterization and manipulation of cellulase genes.

The complete hydrolysis of cellulose requires a number of different enzymes including endoglucanase, exoglucanase and beta-glucosidase. These enzymes function in concert as part of a 'cellulase'complex called a cellulosome. In order (i) to develop a better understanding of the biochemical nature of the cellulase complex as well as the genetic regulation of its integral components and (ii) to utilize cellulases either as purified enzymes or as part of an engineered organism for a variety of purposes, researchers have, as a first step, used recombinant DNA technology to isolate the genes for these enzymes from a variety of organisms. This review provides some perspective on the current status of the isolation, characterization and manipulation of cellulase genes and specifically discusses (i) strategies for the isolation of endoglucanase, exoglucanase and beta-glucosidase genes; (ii) DNA sequence characterization of the cellulase genes and their accompanying regulatory elements; (iii) the expression of cellulase genes in heterologous host organisms and (iv) some of the proposed uses for isolated cellulase genes.

Journal Article↗

PubMed1999

Wastepaper hydrolysate as soluble inducing substrate for cellulase production in continuous culture of trichoderma reesei

The enzymatic hydrolysate of wastepaper was evaluated for its cellulase-inducing capability and production characteristics in continuous culture of Trichoderma reesei RUT C30. Under the study conditions, i.e., pH 5.0, temperature 25 degreesC, and typical medium C:N ratio, the apparent cell yield constant was found to be 0. 76 (g of dry cell weight/g of reducing sugar), and the maximum specific cell growth rate was 0.26 h-1. The study on the effects of medium C:N ratio confirmed an important role of N sources in the cellulase synthesis. The cellulase production decreased significantly when the feed concentrations of N sources were reduced. An experiment at pH 7.5 with 4-fold N source concentrations also led to poorer cellulase production. When compared with cellulose, the wastepaper hydrolysate was found to have similar cellulase-inducing strength and to induce an apparently complete set of cellulase components. The hydrolysate was also concluded to be a better soluble inducer than sophorose. While comparable at a low dilution rate (0.012 h-1), the specific cellulase productivities of the hydrolysate-supported and the sophorose-induced systems exhibited opposite trends with increasing dilution rates. The specific productivity in sophorose-induced systems decreased with an increase in the dilution rate. On the other hand, with increasing dilution rate the specific productivity in the hydrolysate-supported systems increased from 2.2 FPU/g.h at D = 0.012 h-1 to 12.2 FPU/g.h at D = 0.122 h-1 before beginning to decline. The initial increasing trend was attributed to the higher concentrations of inducing oligomer intermediates at larger dilution rates.

Journal Article↗

PubMed1975

The Effects of pH, Ionic Strength, and Ethylene on the Extraction of Cellulase from Abscission Zones of Citrus Leaf Explants.

The solubility of cellulase extracted from the abscission zones of citrus leaf explants (Citrus sinensis L. Osbeck) in sodium phosphate buffer depends on the pH of the extracting solution and, to a lesser extent, on the ionic strength. By increasing molarity from 0.01 to 0.16, the solubility of cellulase increased from 51% to 89% at pH 6.1 and from 70% to 98% at pH 7. In all cases, residual cellulase was further extracted from the pellet by buffer containing 1 m NaCl. Most of the enzymic activity was found in tissues proximal to the separation line, and activity of the cellulase which was soluble in phosphate buffer was closely correlated with abscission at both pH values. When extraction of cellulase at pH 6.1 with phosphate buffer was followed by a reextraction of the pellet with buffer containing 1 m NaCl, the activity of the cellulase soluble in the fortified buffer was also correlated with abscission. Pretreatment of explants with ethylene increased the solubility of cellulase in the phosphate buffer regardless of the pH used at the first extraction.

Journal Article↗

PubMed1978

Factors affecting the activity of cellulases isolated from the rumen digesta of sheep.

Sodium phosphate buffer was used to extract cellulases from the plant solids fraction of rumen contents. The mixed cellulase preparation had maximal activity at pH 6.9 and 49 degrees C. The V(max) and the apparent K(m) for wheaten hay cellulose were 19.8 glucose units/min and 6.35 mg/ml, respectively, and for microcrystalline cellulose (Sigmacell) at the same enzyme concentration, they were 33 glucose units/min and 27.5 mg/ml, respectively. For these assays a glucose unit was defined as nanomoles of glucose plus twice the nanomoles of cellobiose. Consideration of thermodynamic and kinetic data suggested that the hydrolysis of a relatively labile arabino-xylan comprising 3% of the wheaten hay cellulose was dependent on prior removal of the protecting beta-1,4-glucose chains at the outer surface of the cellulose preparation. Sequential removal of structural polysaccharides from the plant cell wall rendered the latter more susceptible to cellulase activity. Cellulase activity was stimulated by increasing the concentration of phosphate from 5 to 50 mM. The stimulation was magnified in the presence of cell-free rumen fluid. Cellulase activity was not stimulated by calcium, magnesium, iron, zinc, manganese, copper, or cobalt ions and was unaffected by the chelators ethylenediaminetetraacetic acid and ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid. O-phenanthroline inhibited activity by 30 to 50%, but this may have been due to nonchelate properties. Anaerobic conditions or thiol protective agents were not essential for either the activity or stability of the cellulases during assay. An ultrafiltrable inhibitor of cellulase activity was detected in cell-free rumen fluid.

Journal Article↗

PubMed1985

Solid-State Fermentation with Trichoderma reesei for Cellulase Production.

Cellulase yields of 250 to 430 IU/g of cellulose were recorded in a new approach to solid-state fermentation of wheat straw with Trichoderma reesei QMY-1. This is an increase of ca. 72% compared with the yields (160 to 250 IU/g of cellulose) in liquid-state fermentation reported in the literature. High cellulase activity (16 to 17 IU/ml) per unit volume of enzyme broth and high yields of cellulases were attributed to the growth of T. reesei on a hemicellulose fraction during its first phase and then on a cellulose fraction of wheat straw during its later phase for cellulase production, as well as to the close contact of hyphae with the substrate in solid-state fermentation. The cellulase system obtained by the solid-state fermentation of wheat straw contained cellulases (17.2 IU/ml), beta-glucosidase (21.2 IU/ml), and xylanases (540 IU/ml). This cellulase system was capable of hydrolyzing 78 to 90% of delignified wheat straw (10% concentration) in 96 h, without the addition of complementary enzymes, beta-glucosidase, and xylanases.

Journal Article↗

PubMed1992

Induction of Cellulase by Gentiobiose and Its Sulfur-Containing Analog in Penicillium purpurogenum.

Cellulase induction by beta-glucodisaccharides was investigated by using non-cellulase-induced mycelia of Penicillium purpurogenum P-26, a highly-cellulase-producing fungus. Gentiobiose induced significant amounts of cellulase compared with cellobiose when nojirimycin was added to the induction medium to inhibit extracellular beta-glucosidase activity. Thiogentiobiose (6-S-beta-d-glucopyranosyl-6-thio-d-glucose), a sulfur-containing analog of gentiobiose, was more effective for cellulase induction than gentiobiose even in the absence of nojirimycin. Thiogentiobiose appeared to be a gratuitous inducer since it was not metabolized during cellulase induction. Gentiobiose was formed from cellobiose by the intracellular beta-glucosidase of P. purpurogenum. These findings indicate that gentiobiose is an active inducer of cellulase for this fungus and may possibly be formed by intracellular beta-glucosidase from cellobiose.

Journal Article↗

PubMed2006

A functionally based model for hydrolysis of cellulose by fungal cellulase.

A new functionally based kinetic model for enzymatic hydrolysis of pure cellulose by the Trichoderma cellulase system is presented. The model represents the actions of cellobiohydrolases I, cellobiohydrolase II, and endoglucanase I; and incorporates two measurable and physically interpretable substrate parameters: the degree of polymerization (DP) and the fraction of beta-glucosidic bonds accessible to cellulase, F(a) (Zhang and Lynd, 2004). Initial enzyme-limited reaction rates simulated by the model are consistent with several important behaviors reported in the literature, including the effects of substrate characteristics on exoglucanase and endoglucanase activities; the degree of endo/exoglucanase synergy; the endoglucanase partition coefficient on hydrolysis rates; and enzyme loading on relative reaction rates for different substrates. This is the first cellulase kinetic model involving a single set of kinetic parameters that is successfully applied to a variety of cellulosic substrates, and the first that describes more than one behavior associated with enzymatic hydrolysis. The model has potential utility for data accommodation and design of industrial processes, structuring, testing, and extending understanding of cellulase enzyme systems when experimental date are available, and providing guidance for functional design of cellulase systems at a molecular scale. Opportunities to further refine cellulase kinetic models are discussed, including parameters that would benefit from further study.

Cellulase↗

PubMed1996

Cellulase formation by species of Trichoderma sect. Longibrachiatum and of Hypocrea spp. with anamorphs referable to Trichoderma sect. Longibrachiatum.

The cellulolytic potential of the wild-type strain of Trichoderma reesei was compared to other members of Trichoderma sect. Longibrachiatum and Hypocrea spp. that have anamorphs referable to that section. There was high diversity even within the same species (as defined by morphological and macromolecular characters). Differences, where notable, were more pronounced for carboxymethyl-cellulase activity than for filter paper activity. High cellulase activities were observed for several strains of T. longibrachiatum and T. citrinoviride, whereas T. parceramosum formed only low levels of activity. Among the corresponding teleomorphs, most strains of H. schweinitzii were comparatively poor producers, whereas the highest percentage of high producers was found among H. jecorina isolates, and many strains were even more active than the parent T. reesei QM 6a. Immunoblot analysis of corresponding culture filtrates of various H. jecorina strains showed that the three major cellulase proteins (cellobiohydrolase I, cellobiohydrolase II, and endoglucanase I) were present in culture filtrates and their M(r) was identical to that of the respective T. reesei proteins. ELISA analysis demonstrated that these enzymes were also present in the same relative proportions in culture filtrates from H. jecorina and T. reesei. With the aid of primers, corresponding to conserved sequences in the cellobiohydrolase I-encoding gene cbh1, a fragment of this gene was amplified from selected strains of H. jecorina, T. reesei, T. longibrachiatum, T. citrinoviride, and H. schweinitzii. The fragments had the same size in all fungi. Cleavage of this fragment with Hhal produced a RFLP pattern which was identical in H. jecorina and T. reesei, but different in the other species. In the latter, the RFLP pattern was also species specific. These results provide support for a close genetic similarity of T. reesei and H. jecorina cellulases. In the latter, an ascomycetous model system for cellulase biosynthesis is now available. The results further indicate that other anamorphs of Trichoderma section Longibrachiatum are promising sources of high cellulase production.

Cellulase↗

PubMed1997

Possible roles for a non-modular, thermostable and proteinase-resistant cellulase from the mesophilic aerobic soil bacterium Cellvibrio mixtus.

The widespread presence of cellulose-binding domains in cellulases from aerobic bacteria and fungi suggests the existence of a strong selective pressure for the retention of these non-catalytic modules. The complete nucleotide sequence of the cellulase gene, celA, from the aerobic soil bacterium Cellvibrio mixtus, was determined. It revealed an open reading frame of 1089 bp that encoded a polypeptide, defined as cellulase A (CelA), of M(r) 41,548. CelA displayed features characteristic of an endo-beta-1,4-glucanase, rapidly decreasing the viscosity of the substrate while releasing only moderate amounts of reducing sugar. Deletion studies in celA revealed that removal of 78 nucleotides from the 5' end or 75 from the 3' end of the gene led to the complete loss of cellulase activity of the encoded polypeptides. The deduced primary structure of CelA revealed an N-terminal signal peptide followed by a region that exhibited significant identity with the catalytic domains of cellulases belonging to glycosyl hydrolase family 5. These data suggest that CelA is a single-domain endoglucanase with no distinct non-catalytic cellulose-binding domain. Analysis of the biochemical properties of CelA revealed that the enzyme hydrolyses a range of soluble cellulosic substrates, but was inactive against Avicel, xylan or any other hemicellulose. CelA was resistant to proteolytic inactivation by pancreatic proteinases and surprisingly, in view of its mesophylic origin, was shown to be thermostable. The significance of these findings in relation to the role of single-domain cellulases in plant cell wall hydrolysis by aerobic microorganisms is discussed.

Amino Acid Sequence↗

PubMed2003

Detecting cellulase and esterase enzyme activities encoded by novel genes present in environmental DNA libraries.

A genomic DNA library was made from the alkaliphilic cellulase-producing Bacillus agaradhaerans in order to prove our technologies for gene isolation prior to using them with samples of DNA isolated directly from environmental samples. Clones expressing a cellulase activity were identified and sequenced. A new cellulase gene was identified. Genomic DNA libraries were then made from DNA isolated directly from the Kenyan soda lakes, Lake Elmenteita and Crater Lake. Crater Lake clones expressing a cellulase activity and Lake Elmenteita clones expressing a lipase/esterase activity were identified and sequenced. These were encoded by novel genes as judged by DNA sequence comparisons. Genomic DNA libraries were also made from laboratory enrichment cultures of Lake Nakuru and Lake Elmenteita samples. Selective enrichment cultures were grown in the presence of carboxymethylcellulose (CMC) and olive oil. A number of new cellulase and lipase/esterase genes were discovered in these libraries. Cellulase-positive clones from Lake Nakuru were isolated at a frequency of 1 in 15,000 from a library made from a CMC enrichment as compared to 1 in 60,000 from a minimal medium enrichment. Esterase/lipase-positive clones from Lake Elmenteita were isolated with a frequency of 1 in 30,000 from a library made from an olive-oil enrichment as compared to 1 in 100,000 from an environmental library.

Amino Acid Sequence↗

PubMed2000

Diverse genes of cellulase homologues of glycosyl hydrolase family 45 from the symbiotic protists in the hindgut of the termite Reticulitermes speratus.

Diverse genes encoding cellulase homologues belonging to glycosyl hydrolase family 45 were identified from the symbiotic protists in the hindgut of the termite Reticulitermes speratus through the use of consensus PCR and the screening of a cDNA library. Fifteen full-length cDNA clones were isolated and sequenced, which encoded polypeptides consisting of 218-221 amino acid residues showing up to 63% identity to known family 45 cellulases. The cellulase sequences of the termite symbiotic protists were phylogenetically monophyletic, showing more than 75% amino acid identity with each other. These enzymes consist of a single catalytic domain, lacking the ancillary domains found in most microbial cellulases. By whole-cell in situ hybridization using oligonucleotide probes specific for regions conserved in some of the sequences, the origin of the genes was identified as symbiotic hypermastigote protists. The presence of diverse cellulase homologues suggests that symbiotic protists of termites may be rich reservoirs of novel cellulase sequences.

Amino Acid Sequence↗

PubMed2006

Factors affecting foaming behavior in cellulase fermentation by Trichoderma reesei Rut C-30.

Coupling fermentation with in situ foam fractionation may be beneficial to cellulase production in optimizing oligomer inducer generation, minimizing catabolite repression and reducing cellulase degradation by proteases. In this study, the potential factors that may affect the foaming behavior of broth from Trichoderma reesei Rut C-30 fermentation were examined. These factors included solid (both cell and cellulose) concentrations, cellulase activity and extracellular protein concentration. The loss of cellulase activity caused by the foaming process was minimal. The foamate generation was lower in the presence of higher solids (cell and/or cellulose) concentrations. Cellulase appeared to promote the broth foaming ability but its enrichment ratio was not high (lower than 1.2). The enrichment ratios for the individual component enzymes (beta-glucosidase, endo- and exo-glucanases) were found to be similarly low. None of the cellulase components were likely the primary foaming factors. The foam also carried out cells and cellulose solids. The hydrophobicity of cell surface, studied at various fermentation stages and in both media with and without cellulose, increased as the fermentation approached the stationary phase and then decreased gradually after entering the stationary phase.

Cellulase↗

PubMed2001

Failed expression of an endo-beta-1,4-glucanhydrolase (cellulase) in a non-abscinding mutant of Lupinus angustifolius cv Danja.

Cellulase expressions in a normal shedding wild-type and a non-abscinding single gene mutant of Lupinus angustifolius have been studied during ethylene treatments of leaf abscission zone explants. Of the range of different glycohydrolases investigated only the abscission cell-specific beta-1,4-glucanhydrolase (cellulase) was not produced in the non-abscinding mutant. An endo-polygalacturonase was induced equally in both wild-type and mutant and other glycohydrolases were equally up-regulated. The abscission cell-specific cellulase induced at shedding of wild-type is antigenically similar to the Phaseolus vulgaris induced leaf abscission pI 9.5 cellulase but with a higher molecular mass (50 kD compared with 48 kD) and like the bean abscission-specific cellulase that of lupin is not glycosylated. Causes of the loss of function of cellulase expression in the non-shedding mutant are discussed.

Cellulase↗

PubMed1999

Isolation of a cDNA encoding a putative cellulase in the red claw crayfish Cherax quadricarinatus.

Amino acid sequences of cellulases have been determined in insects, nematodes, plants, slime moulds and bacteria but not in crustaceans. However, cellulase activity has been demonstrated in the hepatopancreas of the red claw crayfish, Cherax quadricarinatus. In order to obtain information on the nature of this cellulase, a C. quadricarinatus hepatopancreas cDNA library was screened with a PCR product generated using degenerate oligonucleotide primers derived from conserved regions of known cellulases. Two identical 1.56kb cDNAs with sequence similarities to known cellulases, particularly the termite endoglucanases, were identified and sequenced. The clones contain the complete cDNA open reading frame for an endo-1, 4-beta-glucanase of 469 amino acids termed Cherax quadricarinatus endoglucanase (CqEG). The endogenous origin of the gene was confirmed by PCR amplification and sequencing of a 1012bp PCR product from genomic DNA. This fragment contains four exon sequences identical to the cDNA and is interrupted by three introns of 371, 102, 194bp respectively, with one intron exhibiting typical eukaryotic splice sites. The isolation of an endo-1,4-beta-glucanase encoding cDNA from the crayfish C. quadricarinatus provides the first endogenous cellulase sequence in a crustacean species.

Amino Acid Sequence↗