Modulating the pH-activity profile of cellulase by substitution: replacing the general base catalyst aspartate with cysteinesulfinate in cellulase A from Cellulomonas fimi.

摘要: Cellulase A (CenA) from Cellulomonas fimi is an inverting glycoside hydrolase and a member of family 6 the CAZy database classification system. We replaced its putative catalytic base aspartyl residues, Aps392 Asp216, with cysteinesulfinate using combination site-directed mutagenesis chemical modification to investigate applicability this approach for modulation enzymatic properties. The substituted cysteinyl residues were oxidized cysteinesulfinic acid hydrogen peroxide, resulting protein products demonstrated retain their native structure. Oxidation Asp392Cys mutant enzyme restored 52% wild-type activity when assessed at pH 7.5, whereas Asp216Cys CenA remained inactive. This suggests that Asp216 not provides further support Asp392 performing role. Similar substitution residue Asp252 or nucleophile retaining Cel5A Thermobifida fusca failed produce active enzymes. indicates potential utility uniquely identifying residues. replacement induced acidic shift in profile such derivative was more than below 5.5. These data demonstrate combining as viable cellulases, potentially other hydrolases, low pH.