Proteolysis during enzyme purification

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. 

E. coli RNase HI does not bind to DEAE-cellulose or DEAE-Sephacel, in contrast to the calf thymus enzyme. This is apparently due to the difference in isoelectric point. PMSF (0.1-1 mM) is often included in buffers to avoid possible proteolysis during purifications. 

A common problem associated with rupture of yeast cells and protein extraction is proteolysis. Yeast cells contain a full complement of intracellular proteolytic enzymes which may be liberated after the cells are broken either by autolysis or by mechanical disruption. These liberated proteolytic enzymes, unless inactivated during the isolation and purification of yeast proteins, hydrolyze the proteins causing poor yields of intact protein (55, 69,70). 

Protein contaminants can occur as natural isoforms or can arise during purification. Adventitious proteolysis and cysteine oxidation are probably the most common sources of microheterogeneity that occur during isolation (Lorber et al, 1987). This has frequently motivated the inclusion of protease inhibitors and/or reducing agents in crystallization solutions, as well as during purification. In many cases modifications that produce molecular heterogeneity are reflected in enzyme activity. For... 

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