Mutations to alter Aspergillus awamori glucoamylase selectivity. I. Tyr48Phe49-->Trp, Tyr116-->Trp, Tyr175-->Phe, Arg241-->Lys, Ser411-->Ala and Ser411-->Gly

• Published in: Protein engineering Vol. 11; no. 2; pp. 119 - 126
• Main Authors: Fang, T Y, Coutinho, P M, Reilly, P J, Ford, C
• Format: Journal Article
• Language: English
• Published: England 01.02.1998
• Subjects: Arginine; Aspergillus - enzymology; Binding Sites; Carbohydrate Conformation; Catalysis; Glucan 1,4-alpha-Glucosidase - chemistry; Glucan 1,4-alpha-Glucosidase - genetics; Glucan 1,4-alpha-Glucosidase - metabolism; Glucose - metabolism; Hydrolysis; Isomaltose - metabolism; Kinetics; Molecular Structure; Mutagenesis, Site-Directed; Polysaccharides - metabolism; Serine; Structure-Activity Relationship; Thermodynamics; Tyrosine
• ISSN: 0269-2139; 1741-0126; 1741-0134
• DOI: 10.1093/protein/11.2.119

Glucoamylase mutations to reduce isomaltose formation from glucose condensation and thus increase glucose yield from starch hydrolysis were designed to produce minor changes in the active site at positions not totally conserved. Tyr175-->Phe and Ser411-->Gly glucoamylases had catalytic efficiencies on DP 2-7 maltooligosaccharides like those of wild-type glucoamylase, while the catalytic efficiencies of Tyr116-->Trp, Arg241-->Lys and Ser411-->Ala glucoamylases were reduced by about half and Tyr48Phe49-->Trp glucoamylase had little remaining activity. Tyr175-->Phe, Ser411-->Ala and Ser411-->Gly glucoamylases had decreased ratios of the initial rate of isomaltose formation from glucose condensation to that of glucose formation from maltodextrin hydrolysis at both 35 and 55 degrees C compared with wild-type glucoamylase. Arg241-->Lys glucoamylase had a very similar ratio, while Tyr116-->Trp glucoamylase had a higher ratio. The highest glucose yields from maltodextrin hydrolysis were by the mutant glucoamylases having the lowest ratios of initial rates of isomaltose formation to glucose formation and this predicted high glucose yields better than the ratio of catalytic efficiency for maltose hydrolysis to that for isomaltose hydrolysis.