Cellulase purification

However, we have observed that values obtained with crude extracts were only qualitative. Often, they did not accurately estimate the quantities of the individual enzymes present. Inhibitors were typically present that caused the underestimation of certain enzymes ie.g., ligninases Table II) and that could potentially mask less dominant enzymes. Also, certain polysaccharidases e.g., hemicellulases) were often overestimated due to the action of non-specific or synergistic enzymes e.g., other hemicellulases or cellulases) (9,14), This artifact resulted in low apparent recovery of a given activity and only moderate increases in specific activity upon purification of the major corresponding enzyme present, in spite of the fact that SDS polyacrylamide gels indicated good recovery and substantial removal of contaminants (14),... 

Cellobiohydrolase I (CBH I, 1,4-jS-D-glucan-cellobiohydrolase, E.C. 3.2.1.91) is the main protein (ca. 60%) of the cellulase complex produced by T. reesei strains. CBH I hydrolyses crystalline cellulose, acid swollen cellulose and 4-methylumbelliferyl-cellodex-trins by cleaving off the terminal cellobiose unit from the non reducing end of the chain. It operates with retention of configuration in the reaction products 19,20. The abundance of this enzyme and its stability has facihtated its purification to homogeneity... 

The construction of hybrid proteins containing bacterial CBDs may provide a cheap generic method for enzyme immobilization and/or purification using cellulosic matrices. The CBD can be fused at the amino or carboxyl terminus, as in the parent cellulase, to suit individual applications. We have constructed model fusion proteins using the C. fimi CBDs to demonstrate this potential. 

These results demonstrate several useful properties of the C. fimi CBDs for enzyme immobilization and purification. Presumably, the CBDs of other bacterial cellulases (Figure 3) can be used in similar ways. It remains to be seen whether these differ significantly in their affinity or capacity for adsorption if so, certain CBDs may be preferable for specific purposes. 

Our chromophoric substrates proved to be valuable in the study of several aspects of the enzymology of these cellulases. A rapid and specific method for purification (affinity chromatography) has been developed. Following our collaboration with several groups, new insights into the domain arrangement and tertiary structures of two cellulases were obtained. Contributions to the elucidation of the synergistic action (adsorption-hydrolysis) of these enzymes were achieved. 

A given microorganism may produce one or more enzymes of each type. An understanding of the role of each enzyme in cellulose biodegradation requires their purification and characterization, and an analysis of the ways in which they interact with the substrate and with each other. However, it is often quite difficult to determine the number and type of truly different enzymes produced by an organism. Many cellulolytic microorganisms secrete proteases, which may degrade some or all of the cellulases to smaller,... 

Compound (44 g, NHAc form) (Scheme 14) was found to be a competitive inhibitor for CBHI cellulase (family 7) from Trichoderma reesei, when 4-methyl-umbelliferyl / -lactoside was used as substrate. Therefore (44 g, NHj form) was coupled to CH-Sepharose 4B, and the affinity gel was very effective for the purification of cellobiohydrolases from a crude commercial cellulolytic extract of T. reesei [40c]. Using the same approach aryl 1,4-dithioxylobioside and l,4,4 -trithioxylotrioside (44 h, NH2 form) were coupled to CH-Sepharose 4B to give affinity gels which were used for the purification of xylanases [40a,b]. 

The minor content of impurities found in the starch slurry are connected to the starch granules themselves. To facilitate the purification of the starch during filtration, cellulases, pentosanases, glucanases, proteases, and pectinases are sometimes used. Wheat starch is known to form precipitates or hazes that are difficult to filter. Arabinoxylan, pentosanes, andlysophospholipids are claimed to be responsible for this problem (73). 

Fractionation and Purification of Ex-1 Cellulase Component from Driselase. Driselase powder (50g) was extracted with several aliquots of water and the precipitate formed upon salting out with ammonium sulfate (on a saturation between 20% and 80%) was fractionated on a DEAE-Sephadex A-50 column. Each fraction was tested for -glucosi-dase, xylanase, CMCase, Avicelase activities, and protein content. The elution patterns are shown in Figures 1 and 2. 

The E-3 peak was high in Avicelase activity and in protein content as compared with CMCase activity. This peak was further fractionated on a Bio-gel P-100 column five protein peaks (E-3-1 to E-3-5) were obtained, of which E-3-2 peak was highest among them in Avicelase activity and protein content. The elution patterns are shown in Figure 3, and the time course of hydrolysis of CMC by these cellulase fractions measured by a decrease in the viscosity is shown in Figure 4. Randomness of them is in the order of E-3-5 < E-3-2 < E-3-1 E-3-4 E-3-3. The E-3-2 fraction was subjected to further purification on a CM-Sephadex C-50 column because E-3-5 was very low in the Avicelase activity. 

The sequential and cooperative roles of the enzymes are shown in Figure 1. Endoglucanases, often called cellulases or carboxymethylcellu-lases, provide nonreducing chain ends from which the exoCBH s cleave cellobiosyl residues. The cellobiose, which is inhibitory to the depoly-merizing enzymes, is cleaved to glucose by the / -glucosidase. Since the product of one enzyme may become the substrate for another, enzyme purification is an essential prerequisite to any clear description of the system. 

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... 

The cellulase components that are synthesized in the presence of sophorose were investigated by the basic procedures previously described (1,2,4) for the isolation of cellulolytic components from commercial cellulase preparations. The purification to homogeneity of the proteins that yield the three predominant bands when the crude preparation is subjected to disc gel electrophoresis was accomplished by ion exchange chromatography. 

Biosynthesis, Purification, and Mode of Action of Cellulases of Trichoderma reesei... 

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. 

Physical Properties. All of the cellulase (CMCase) activity which develops in auxin-treated pea apices dissolves in salt solutions (e.g., phosphate buffer, 20mM, pH 6.2, containing 1M NaCl). Gel chromatography of such extracts indicates the presence of two cellulase components with similar levels of activity and elution volumes corresponding to molecular weights of about 20,000 and 70,000 (Figure 1). If the tissue is extracted with buffer alone, only the smaller cellulase dissolves (referred to as buffer-soluble or BS cellulase). The larger buffer-insoluble (BI) cellulase can then be extracted from the residue by salt solutions. This simple extraction procedure effectively separates the two cellulases, and can be used as an initial step for their estimation or purification. 

Figure 1. Elution profiles of cellulase activity from Sephadex G-100 gel chromatographs of crude extracts of auxin-treated pea apices. BS cellulose activity has an elution volume corresponding to a molecular weight of 20,000. BI cellulase activity dissolves in 1M NaCl and elutes with a molecular weight of 70,000. These values correspond to those observed for purified cellulases (3), indicating that the enzymes were not altered in molecular weight during purification, and could be effectively separated by differential extraction.

Beldman, G., Searle-Van Leeuwen, M., Rombouts, F., and Voragen, F., The cellulase of Trichoderma viride Purification, characterization and comparison of all detectable endoglucanases, exoglucanases and B-glucosi-dases. EurJ. Biochem 1985, 146, 301-8. 

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