Study of protein adsorption on indigo particles confirms the existence of enzyme--indigo interaction sites in cellulase molecules

Adsorption of several crude and purified cellulases (from Trichoderma reesei, Penicillium verruculosum and Chrysosporium lucknowense) on indigo particles and Avicel cellulose was studied. Much higher amounts of protein were bound to indigo than to cellulose under similar conditions. For different purified enzymes, the quantity of bound protein per mg of adsorbent (indigo or cellulose) varied in the range of 57-111 and 0-62 microg x mg(-1), respectively. However, in general, the enzyme adsorption on indigo was less specific than the adsorption on cellulose. Three endoglucanases, having the highest indigo-binding ability, demonstrated the best washing performance in the process of enzymatic denim treatment. These data confirmed our previous findings that certain cellulases, which have indigo-binding sites (clusters of closely located aromatic and other non-polar residues) on the surface of their molecules, may remove indigo from the denim fabric better than cellulases with lower content of hydrophobic residues exposed to solvent.     

Application of microassays for investigation of cellulase abrasive activity and backstaining

Model microassays were used for testing the denim-washing performance and indigo backstaining for Trichoderma reesei and Chrysosporium lucknowense commercial cellulase preparations on a ‘test-tube scale’. C. lucknowense preparation demonstrated a higher potential in the denim biostoning process. The performance of four purified cellulases (two endoglucanases and two cellobiohydrolases) from C. lucknowense on cotton textiles was assayed, and the key enzyme (endoglucanase with a molecular weight of 25 kDa) responsible for high abrasion effects on denim fabrics was found.     

Specific quantification of trichoderma reesei cellulases in reconstituted mixtures and its application to cellulase-cellulose binding studies

Specific quantifications of the major cellulolytic components of the Trichoderma reesei enzyme complex, i.e., endoglucanases I and III and cellobiohydrolases I and II, are described and, employing a defined mixture of these four cellulases reconstituted according to the composition of the native Trichoderma cellulase complex, used to determine the binding of each individual component onto filter paper. During substrate degradation by this enzyme mixture, the specific adsorption of each individual cellulase gradually increases and no preferential binding of one enzyme component in any particular phase of cellulose hydrolysis is found. T. reesei cellobiohydrolases I and II admixed with endoglucanases I and III represent a "full-value" cellulase system that is capable of degrading semicrystalline cellulose efficiently. In comparison with the crude Trichoderma enzyme complex, almost identical adsorption properties and similar hydrolytic efficiency are found for the reconstituted mixture. (c) 1994 John Wiley & Sons, Inc.     

Cellulase finishing of woven, cotton fabrics in jet and winch machines

Some authors have reported that as the applied agitation rate increases, the apparent activity of the endoglucanases from Trichoderma reesei towards cotton cellulose increases more markedly than does the apparent activity of the cellobiohydrolases. This suggests that the quality of cellulase finishing effects on cellulosic textiles may be machine-type dependent. The present work using total crude, endoglucanase-rich and cellobiohydrolase-rich cellulases from T. reesei confirmed that the final properties of woven, cotton fabrics treated under realistic processing conditions in a jet machine, were measurably and perceivably different from those of the same fabrics, treated using the same processing conditions of temperature, time, pH, enzyme concentration and fabric to liquor ratio, but in a winch machine. The results are interpreted in terms of the effects of agitation rate on the adsorption–desorption behaviour of the T. reesei endoglucanases and cellobiohydrolases.     

The mechanism of fungal cellulase action. Synergism between enzyme components of Penicillium pinophilum cellulase in solubilizing hydrogen bond-ordered cellulose.

Studies on reconstituted mixtures of extensively purified cellobiohydrolases I and II and the five major endoglucanases of the fungus Penicillium pinophilum have provided some new information on the mechanism by which crystalline cellulose in the form of the cotton fibre is rendered soluble. It was observed that there was little or no synergistic activity either between purified cellobiohydrolases I and II, or, contrary to previous findings, between the individual cellobiohydrolases and the endoglucanases. Cotton fibre was degraded to a significant degree only when three enzymes were present in the reconstituted enzyme mixture: these were cellobiohydrolases I and II and some specific endoglucanases. The optimum ratio of the cellobiohydrolases was 1:1. Only a trace of endoglucanase activity was required to make the mixture of cellobiohydrolases I and II effective. The addition of cellobiohydrolases I and II individually to endoglucanases from other cellulolytic fungi resulted in little synergistic activity; however, a mixture of endoglucanases and both cellobiohydrolases was effective. It is suggested that current concepts of the mechanism of cellulase action may be the result of incompletely resolved complexes between cellobiohydrolase and endoglucanase activities. It was found that such complexes in filtrates of P. pinophilium or Trichoderma reesei were easily resolved using affinity chromatography on a column of p-aminobenzyl-1-thio-beta-D-cellobioside.     

Effects of cellulase on the modification of cellulose

Multicomponent cellulases, purified endoglucanases and cellobiohydrolases were assayed and shown to modify pure natural cellulose (softwood pulp). Changes in structure and properties of the cellulose caused by enzymatic treatment depend on the composition, the type of enzyme, and the treatment conditions. The reactivity of cellulose for some dissolving and derivatization processes may be improved by enzymatic hydrolysis. Endoglucanases decreased the average degrees of polymerization (DP) and improved the alkaline solubility of cellulose most efficiently. The variation in the supramolecular structure estimated from the infrared spectra of the cellulose samples was found to be correlated with the reactivity and might represent wide variations in conformation caused by the breakdown of the hydrogen bonds.     

Cross-reactive and specific monoclonal antibodies against cellobiohydrolases I and II and endoglucanases I and II of Trichoderma reesei.

Splenocytes derived from mice inoculated with a commercial cellulase preparation or purified cellulases were fused with a stable myeloma cell line (SP2/0). Specific monoclonal antibodies to cellobiohydrolases I and II and endoglucanases I and II were established. In addition to specific monoclonal antibodies, we were also able to establish stable hybridoma cell lines which produced monoclonal antibodies that recognized similar epitopes possessed by two or more of the above cellulases. By obtaining monospecific antibodies for all four individual cellulases, the role and function of the individual cellulases can thus be studied in greater detail.     

The physical action of cellulases revealed by a quartz crystal microbalance study using ultrathin cellulose films and pure cellulases

The effects of fungal cellulases on model cellulose films were studied using a high-resolution quartz crystal microbalance (QCM) sensitive to minute changes of the nanometer thick model cellulose films. It was found that endoglucanases not only produce new end groups but also cause a swelling of the cellulose film. The cellobiohydrolases degraded the films quickly, which was detected as a rapid decrease in the remaining amount of cellulose on the QCM crystal. However, changing viscoelastic properties of the films also indicated a softening of the film during the degradation. A defined mixture of selected cellulases caused a significantly higher rate of degradation than only cellobiohydrolases. Cellulase synergism is discussed with the endoglucanase swelling effects and film softening added.     

Cross-reactive and specific monoclonal antibodies against cellobiohydrolases I and II and endoglucanases I and II of Trichoderma reesei

Splenocytes derived from mice inoculated with a commercial cellulase preparation or purified cellulases were fused with a stable myeloma cell line (SP2/0). Specific monoclonal antibodies to cellobiohydrolases I and II and endoglucanases I and II were established. In addition to specific monoclonal antibodies, we were also able to establish stable hybridoma cell lines which produced monoclonal antibodies that recognized similar epitopes possessed by two or more of the above cellulases. By obtaining monospecific antibodies for all four individual cellulases, the role and function of the individual cellulases can thus be studied in greater detail.     

Analysis of Molecular Size Distributions of Cellulose Molecules during Hydrolysis of Cellulose by Recombinant Cellulomonas fimi β-1,4-Glucanases

Four β-1,4-glucanases (cellulases) of the cellulolytic bacterium Cellulomonas fimi were purified from Escherichia coli cells transformed with recombinant plasmids. Previous analyses using soluble substrates had suggested that CenA and CenC were endoglucanases while CbhA and CbhB resembled the exo-acting cellobiohydrolases produced by cellulolytic fungi. Analysis of molecular size distributions during cellulose hydrolysis by the individual enzymes confirmed these preliminary findings and provided further evidence that endoglucanase CenC has a more processive hydrolytic activity than CenA. The significant differences between the size distributions obtained during hydrolysis of bacterial microcrystalline cellulose and acid-swollen cellulose can be explained in terms of the accessibility of β-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.     

The effect of yellow affinity substance on cellulases of Ruminococcus flavefaciens

Cellulolytic cultures of Ruminococcus flavefaciens produced a yellow affinity substance (YAS) with a strong affinity to microcrystalline cellulose (MC). YAS was bound to MC in the range of pH from 5 to 8 and at temperatures from 10°C to 60°C. The positive effect of YAS on adsorption of ruminococcal cellulases was demonstrated by comparing the adsorption behaviour of endoglucanases and cellobiohydrolases onto MC and YAS-treated MC. HPLC chromatography proved the presence of two yellow compounds with affinity to cellulose as well as to ruminococcal cellulases. Both YAS compounds were sensitive to oxidation. The observed YAS properties showed a close relation to YS of Clostridium thermocellum.     

Identification and purification of the main components of cellulases from a mutant strain of Trichoderma viride T 100-14

A new mutant strain of fungus Trichoderma viride T 100-14 was cultivated on 1% microcrystalline cellulose (Avicel) for 120h and the resulting culture filtrate was prepared for protein identification and purification. To identify the predominant catalytic components, cellulases were separated by an adapted two-dimensional electrophoresis technique. The apparent major spots were identified by high performance liquid chromatography electrospray ionization mass (HPLC-ESI-MS). Seven of the components were previously known, i.e., the endoglucanases Cel7B (EG I), Cel12A (EG III), Cel61A (EG IV), the cellobiohydrolases Cel7A (CBH I), Cel6A (CBH II), Cel6B (CBH IIb) and the beta-glucosidase. The seven major components in the fermentation broth of T. viride T 100-14 probably constitute the essential enzymes for crystalline cellulose hydrolysis and they were further purified to electrophoretic homogeneity by a series of chromatography column. Hydrolysis studies of the purified elements revealed that three of the cellulases were classified as cellobiohydrolases due to their main activities on p-nitrophenyl-beta-d-cellobioside (pNPC). Three of the cellulases, with the abilities of hydrolyzing both carboxymethyl-cellulose (CMC) and Avicel indicate their endoglucanase activities. It deserved noting that the beta-glucosidase from the T 100-14 displayed an extremely high activity on p-nitrophenyl-beta-D-glycopyranoside (pNPG), which suggested it was a good candidate for the conversion of cellobiose to glucose.     

Cellulase Complex of the Fungus Chrysosporium lucknowense: Isolation and Characterization of Endoglucanases and Cellobiohydrolases

Using different chromatographic techniques, eight cellulolytic enzymes were isolated from the culture broth of a mutant strain of Chrysosporium lucknowense: six endoglucanases (EG: 25 kD, pI 4.0; 28 kD, pI 5.7; 44 kD, pI 6.0; 47 kD, pI 5.7; 51 kD, pI 4.8; 60 kD, pI 3.7) and two cellobiohydrolases (CBH I, 65 kD, pI 4.5; CBH II, 42 kD, pI 4.2). Some of the isolated cellulases were classified into known families of glycoside hydrolases: Cel6A (CBH II), Cel7A (CBH I), Cel12A (EG28), Cel45A (EG25). It was shown that EG44 and EG51 are two different forms of one enzyme. EG44 seems to be a catalytic module of an intact EG51 without a cellulose-binding module. All the enzymes had pH optimum of activity in the acidic range (at pH 4.5-6.0), whereas EG25 and EG47 retained 55-60% of the maximum activity at pH 8.5. Substrate specificity of the purified cellulases against carboxymethylcellulose (CMC), beta-glucan, Avicel, xylan, xyloglucan, laminarin, and p-nitrophenyl-beta-D-cellobioside was studied. EG44 and EG51 were characterized by the highest CMCase activity (59 and 52 U/mg protein). EG28 had the lowest CMCase activity (11 U/mg) amongst the endoglucanases; however, this enzyme displayed the highest activity against beta-glucan (125 U/mg). Only EG51 and CBH I were characterized by high adsorption ability on Avicel cellulose (98-99%). Kinetics of Avicel hydrolysis by the isolated cellulases in the presence of purified beta-glucosidase from Aspergillus japonicus was studied. The hydrolytic efficiency of cellulases (estimated as glucose yield after a 7-day reaction) decreased in the following order: CBH I, EG60, CBH II, EG51, EG47, EG25, EG28, EG44.     

A binding-site-deficient, catalytically active, core protein of endoglucanase III from the culture filtrate of Trichoderma reesei

From the culture filtrate of Trichoderma reesei we have isolated a novel endoglucanase (38 kDa) which was shown to be identical to endoglucanase III (E III, 50 kDa), but lacking the first 61 N-terminal amino acids. This core protein, designated E III core, is fully active against soluble substrates, such as carboxymethylcellulose, whereas both activity against and adsorption to microcrystalline cellulose (Avicel) is markedly decreased. Sedimentation velocity experiments revealed that the intact E III enzyme has much higher asymmetry than the E III core protein, suggesting that the N-terminal region split off constitutes a protruding part of the native enzyme. These results lead to the proposal that native E III consists of two functionally separated domains: a catalytically active core and a protruding N-terminal domain which acts in the binding to insoluble cellulose. The N-terminal peptide missing in E III core corresponds to the heavily glycosylated common structural element found also in the N-terminus of cellobiohydrolase II and in the C-termini of cellobiohydrolase I and endoglucanase I. A similar bifunctional organization could thus be the rule for Trichoderma cellulases, endoglucanases as well as cellobiohydrolases.     

Functional analysis of a gene encoding endoglucanase that belongs to glycosyl hydrolase family 12 from the brown-rot basidiomycete Fomitopsis palustris

The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and beta- glucosidases. A gene encoding endoglucanase, designated as cel12, was cloned from total RNA prepared from F. palustris grown at the expense of Avicel. The gene encoding Cel12 has an open reading frame of 732 bp, encoding a putative protein of 244 amino acid residues with a putative signal peptide residing at the first 18 amino acid residues of the N-terminus of the protein. Sequence analysis of Cel12 identified three consensus regions, which are highly conserved among fungal cellulases belonging to GH family 12. However, a cellulose-binding domain was not found in Cel12, like other GH family 12 fungal cellulases. Northern blot analysis showed a dramatic increase of cel12 mRNA levels in F. palustris cells cultivated on Avicel from the early to late stages of growth and the maintenance of a high level of expression in the late stage, suggesting that Cel12 takes a significant part in endoglucanase activity throughout the growth of F. palustris. Adventitious expression of cel12 in the yeast Pichia pastoris successfully produced the recombinant protein that exhibited endoglucanase activity with carboxymethyl cellulose, but not with crystalline cellulose, suggesting that the enzyme is not a processive endoglucanase unlike two other endoglucanases previously identified in F. palustris.     

Catalytically important amino acid residues in endoglucanases from a mutant strain Trichoderma sp. M7

Two endoglucanases, EG-III (49.7 kD) and EG-IV (47.5 kD), from a mutant strain Trichoderma sp. M7 were modified with several specific reagents. Water-soluble carbodiimide completely inactivated only one of the purified endoglucanases and kinetic analysis indicated that at least two molecules of carbodiimide bind to EG-IV for inactivation. The reaction followed pseudo-first-order kinetics with a second-order rate constant of 3.57 x 10(-5) mM(-1) x in(-1). Both endoglucanases were inhibited by iodoacetamide, but the absence of substrate protection excluded direct involvement of cysteine residues in the catalysis. N-Bromosuccinimide (NBS) showed a strong inhibitory effect on both endoglucanases, suggesting that tryptophan residues are essential for the activity and binding to the substrate, since the presence of substrates or analogs prior to NBS modification protected the enzymes against inactivation.     

Purification,characterization and thermodynamic analysis of cellulases produced from Thermomyces dupontii and its industrial applications

Cellulases involved in the hydrolysis of cellulose and plays a vital role in different industries like textile, detergent paper and Feed industry. Cellulases have been a prospective target for research by both the academic and industrial sectors because of the intricacy of the enzyme system and the enormous industrial potential. In the present work Thermomyces dupontii, which had previously been isolated and recorded as a promising cellulase producer were used. Both endoglucanases and betaglucosidases were purified to its homogeneity by ammonium sulfate followed by anion exchange and gel filtration chromatography. The recovery and purification fold for endoglucanases and betaglucosidases were 13.7, 10.7 % and 5.9, 2.7, respectively. The molecular weight of endoglucanases and betaglucosidases were estimated as 37 and 66 kDa on SDS-PAGE. Upon kinetic analysis the purified endoglucanases and betaglucosidases showed Km 0.63; 28.56 mg/ml and Vmax 82; 80 U/ml/min, respectively. Characterization revealed that enzyme was found to be acidophilic cellulase having optimal pH of 5.5 and 70 ?C. Furthermore, cellulases were accelerated in the presence of Ca²⁺ and EDTA. The cellulases had activation energy (Ea) of ?44.55; ?50.02 kJ/mol for carboxy-methyl-cellulose hydrolysis and Enthalpy (ΔH) 42.20; 47.70 kJ/mol and entropy ΔS ?5.1 and ?5.7 kJ/mol for EG and BGL, respectively. In addition to this the enzyme had a secondary structure of protein as represented by FTIR spectrum The current study suggested that purified cellulases can be used as a detergent additive to improve washing. Furthermore, it shows the biostoning ability when applied on jean fabric.     

Measurement and characterization of cellulase activity in sclerophyllous forest litter

Cellulases are enzymatic proteins which hydrolyze cellulose polymers to smaller oligosaccharides, cellobiose and glucose. They consist in three major types of enzymes: endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91) and beta-glucosidases (EC 3.2.1.21) which play an essential role in carbon turnover of forest ecosystem. The aim of this study was firstly to determine the parameters (i.e. buffer type, pH, temperature, quantity of litter, incubation time and reagent type) which affect the measurement of cellulase activity in a sclerophyllous forest litter, and secondly to compare two methods for measuring cellulase activity: a direct method and an extraction method. In the direct method, the litter was directly incubated with a buffered solution containing the enzyme substrate, whereas in the extraction method, the cellulases were firstly extracted before measuring their activity. The results were compared with other studies about soil cellulase activity, and it appeared that several parameters (buffer type, pH, temperature and sample quantity) which influence the measurement of cellulase activity differ according to whether a soil or a litter is considered. Concerning the procedure used for the measurement of cellulase activity, results showed that the activity values were higher when using an extraction procedure than when using a direct procedure. The extraction procedure, combined with a concentration stage of the extract, also allowed electrophoretic analysis (PAGE) of the cellulases extracted from the litter. The electrophoretic pattern revealed two cellulase isoenzymes which may be related to the occurrence of two pH-activity peaks of these enzymes when citrate buffer was used for the measurement of cellulase activity in the litter.     

Characterization of a Neocallimastix patriciarum cellulase cDNA (celA) homologous to Trichoderma reesei cellobiohydrolase II.

The nucleotide sequence of a cellulase cDNA (celA) from the rumen fungus Neocallimastix patriciarum and the primary structure of the protein which it encodes were characterized. The celA cDNA was 1.95 kb long and had an open reading frame of 1,284 bp, which encoded a polypeptide having 428 amino acid residues. A sequence alignment showed that cellulase A (CELA) exhibited substantial homology with family B cellulases (family 6 glycosyl hydrolases), particularly cellobiohydrolase II from the aerobic fungus Trichoderma reesei. In contrast to previously characterized N. patriciarum glycosyl hydrolases, CELA did not exhibit homology with any other rumen microbial cellulases described previously. Primary structure and function studies in which deletion analysis and a sequence comparison with other well-characterized cellulases were used revealed that CELA consisted of a cellulose-binding domain at the N terminus and a catalytic domain at the C terminus. These two domains were separated by an extremely Asn-rich linker. Deletion of the cellulose-binding domain resulted in a marked decrease in the cellulose-binding ability and activity toward crystalline cellulose. When CELA was expressed in Escherichia coli, it was located predominantly in the periplasmic space, indicating that the signal sequence of CELA was functional in E.coli. Enzymatic studies showed that CELA had an optimal pH of 5.0 and an optimal temperature of 40 degrees C. The specific activity of immunoaffinity-purified CELA against Avicel was 9.7 U/mg of protein, and CELA appeared to be a relatively active cellobiohydrolase compared with the specific activities reported for other cellobiohydrolases, such as T. reesei cellobiohydrolases I and II.