Cellulase crude, 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),... 

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 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. 

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.

Specific Adsorption on Different Cellulose Columns. Purification of cellulolytic enzymes, including the Ci enzyme, has been carried out by Li et al. (32) by specific adsorption. By passing the crude enzyme through a column of Avicel the Ci fraction was retained by the column while 95% of the cellulase activity could be eluted. The cellulase fraction was then further purified by passing a column of alkali-swollen cellulose where the cellulase was retained. The enzyme could, however, be eluted by buffer in a high yield from the column. The adsorption on an alkali-swollen cellulose column was then repeated. The yield of enzyme obtained with the specific adsorption method is surprisingly high if compared with the yield on Sephadex and on polyacrylamide gel columns. 

For enzymes for commercial use, the purification process may be rather rough and proteins and enzymes with similar properties to the objective enzyme may be included in the preparation. Therefore, there is the possibility that the some chitosanolytic enzymes may be present in the crude lipase. According to the previous literature mentioned above and results of this present work, among the commercial enzymes which exhibit both their labeled activity and chitosanolytic activity, we speculate that carbohydrases such as pectinases and cellulases are more likely to be bifunctional enzymes than lipases and proteases, because the structures of the substrates of these carbohydrases are similar to each othe, which is confirmed from the experiments. 

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