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 carboxylate 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 designed to study the additional effects of polarity and both positive and negative charges, respectively. S411G GA had catalytic efficiencies like those of wild-type GA for isomaltose, maltose and maltoheptaose hydrolysis at pH 4.4, while S411A and S411C GAs had 54-74% and S411H and S411D GAs had only about 6-12% of wild-type catalytic efficiencies. All five mutations increased the pH optimum in the enzyme-substrate complex, mainly by raising pK1 values. S411A is the best performing and most industrially promising of the pH mutants isolated to date. S411A GA increased the pH optimum by 0.8 units for both maltose and maltoheptaose hydrolysis while maintaining a high level of activity and catalytic efficiency. In hydrolysis of 28% DE 10 maltodextrin, S411A GA had a pH optimum of 7 compared with pH 5.6 for wild-type GA, and had higher initial rates of glucose production than wild-type GA at all pH values tested above pH 6.6.
Department of Food Science and Human Nutrition, Iowa State University, Ames 50011, USA.
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D001230:Aspergillus / Q000201:enzymology*; D002384:Catalysis; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005936:Glucans / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D007534:Isomaltose / Q000378:metabolism; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D005936:Glucans; D011994:Recombinant Proteins; C031146:maltoheptaose; D007534:Isomaltose; D008320:Maltose; D005087:Glucan 1,4-alpha-Glucosidase; D005947:Glucose