PROTEIN ENGINEERING OF ASPERGILLUS-AWAMORI GLUCOAMYLASE TO INCREASE ITS PH OPTIMUM

To increase the pH optimum of glucoamylase (GA), five mutations-S411G, S411A, S411C, S411H and S411D-were designed to destabilize the carbox ylate ion form of Glu400, the catalytic base, by removing or weakening the hydrogen bond between Ser411 and Glu400, and thereby raising its pK. The substitution of alanine, histidine and aspartate were also des igned to study the additional effects of polarity and both positive an d negative charges, respectively. S411G GA had catalytic efficiencies like those of wild-type GA for isomaltose, maltose and maltoheptaose h ydrolysis at pH 4.4, while S411A and S411C GAs had 54-74% and S411H an d S411D GAs had only about 6-12% of wild-type catalytic efficiencies. All five mutations increased the pH optimum in the enzyme-substrate co mplex, mainly by raising pK(1) values. S411A is the best performing an d most industrially promising of the pH mutants isolated to date. S411 A GA increased the pH optimum by 0.8 units for both maltose and maltoh eptaose hydrolysis while maintaining a high level of activity and cata lytic efficiency. In hydrolysis of 28% DE 10 maltodextrin, S411A GA ha d a pH optimum of 7 compared with pH 5.6 for wild-type GA, and had hig her initial rates of glucose production than wild-type GA at all pH va lues tested above pH 6.6.