Cellulase purified

Functional screening of a soil metagenomic library for cellulases revealed a total of eight cellulolytic clones, one of which was purified and characterized [58]. Despite the fact that this library had been generated from a soil sample collected from a... 

Irwin, D. C., Spezio, M., Walker, L P. and Wilson, D. B. (1993). Activity studies of eight purified cellulases specificity, synergism, and binding domain effects. Blotechnol Bioeng 42,1002-1013. 

Cellulase-gold was made and applied according to (Berg et al., 1988) with sections from material embedded in Quetol 651. Chromatographically purified cellulase complex from Trichoderma reesei was obtained from Worthington Enzymes (Cat. it CEL). 

Because of the availability of this remarkably extensive physical picture of the CBH I molecule (as well as the potential availability in adequate quantities of the purified enzyme itself), DSC studies can provide vduable insights into structure-function relationships for this important cellulase. We present here a comparison of DSC analyses of the intact CBH I molecule with those of the isolated catalytic-domain and tail-region proteolytic fragments. 

RunUnococcus albus and Ruminococcus flavefadens. These bacteria are important cellulose-degraders found in the rumen of cattle and sheep (2). Most isolated strains ferment cellulose and xylan and all ferment cellobiose. Fermentation of glucose and some other carbohydrates depends on the particular strain. R flavefadens and B. succinogenes can ferment the highly ordered crystalline cellulosic su trates but R albus cannot. No evidence has been found for extracellular cellulase production by R albus, but Ohmiya et al. purified cellobiosidase from this culture 17). Laboratory growth of R albus has been conducted at pH 7.0 and 37 C. 

Several methods to prepare high-purity xylanases for potential industrial applications have focused on eliminating the cellulase contamination instead of purifying the xylanase components. This appears to be a very effective approach as it precludes the need for very expensive biochemical procedures and focuses, rather, on a limited number of simple steps to eliminate cellulase activity. Since the remaining materials in the enzyme preparation are essentially inert with respect to the cellulose, their presence may often be ignored. 

However, in the polysaccharides obtained from some mutant strains, there are deviations from this idealized structure.44 Xanthan is relatively resistant to enzymic hydrolysis, but it has been cleaved by an enzyme preparation from a Bacillus sp. at moderate temperatures and in the presence of buffer salts, yielding mono- and oligo-saccharides 45 A partially purified, enzyme preparation46 hydrolyzed deacetylated or depyruvated xanthan, and also xanthan from several wild-type and mutant strains of Xanthomonas. The release of reducing material varied little with xanthan preparations having differences in O-acetyl and pyruvic acetal contents. Under similar conditions of incubation, cellulase acted only on xanthan from mutant strains that had defective side-chain formation. 

The most intensive fractionation studies have been performed on the cellulase elaborated by T. koningii (5,6,9,16,26,27) and T. viride (8,10, 28-32). In a typical separation, normally by chromatography on DEAE-Sephadex, three protein peaks are obtained (Figure 1). The first and second contain Cx Ci is in the third. Cx and / -glucosidase are separated by chromatography on SE-Sephadex and Ci is purified by repeated chromatography on DEAE-Sephadex and by isoelectric focusing. 

Component enzymes of the cellulase system have been purified from several microbial species (1-13), among which mutants of the imperfect fungus Trichoderma provide the highest levels of extracellular enzyme activity (14). From this organism have been purified / -glucosi-dases (EC 3.2.1.21), endo-l,4-/ -D-glucanases (EC 3.2.1.4) and 1,4-/ -d-... 

Since we had been able to purify the enzymes of the cellulase system of T. viride, the purification and identification of the T. reesei system was attempted. It was of interest to compare the enzymes produced during growth on cellulose with those produced in response to sophorose. The... 

Tt is a widely recognized fact that true cellulolytic microorganisms A produce three basic cellulase components IS), and that these enzyme components act in concert to hydrolyze crystalline cellulose to glucose (6). Many research laboratories have undertaken the task to purify cellulose components from various cellulolytic microorganisms and to study the mechanisms of cellulose hydrolysis. Much information has accumulated concerning the mode of action of cellulose hydrolysis since Reese et al. first proposed the Ci-C concept (7). In spite of this, however, conflicting reports still flourish concerning the composition of the "cellulase complex, the multiplicity of cellulase components, the biosynthesis of cellulose, and the mechanisms of cellulose hydrolysis. 

Previously, both in our laboratory and elsewhere, cellulases subjected to purification procedures were obtained from commercial sources (5,6, 8,9,10,13,39,46). Three cellobiases and several endoglucanases and cellobiohydrolases from commercial preparations were purified in our laboratory. While use of protease inhibitors in the fractionation procedures minimized proteolysis during enzyme purification, the existence of enzymes proteolytically modified, presumedly during prolonged fermentation (required for obtaining high titres for commercial production), was a source of confusion, as previously explained. Therefore, we prepared T. reesei cellulase harvested from young culture broth. This was used to carry out the enzyme purification procedures described below. 

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