Protein engineering of the relative specificity of glucoamylase from Aspergillus awamori based on sequence similarities between starch-degrading enzymes.

Aspergillus glucoamylase catalyzes hydrolysis of D-glucose from non-reducing ends of starch with an approximately 300-fold (kcat/Km) preference for the alpha-1,4- over the alpha-1,6-glucosidic linkage determined using the substrates maltose and isomaltose. It is postulated that as most amylolytic enzymes act on either the alpha-1,4- or alpha-1,6-linkages, sequence comparison between active-site regions should enable the correlation of the substrate bond specificity with particular residues at key positions. Therefore, the already high bond-type selectivity in Aspergillus glucoamylase could theoretically be augmented further by three single mutations, Ser119-->Tyr, Gly183-->Lys and Ser184-->His, in two separate active-site regions. These mutants all had slight increases in activity as compared with the wild-type enzyme towards the alpha-1,4-linked maltose; this was due to lower Km values as well as small decreases in activity towards isomaltose. This latter decrease in activity was a result of higher Km values and a decrease in kcat for the Ser184-->His mutant. As a consequence, the selectivity of the three glucoamylase mutants for alpha-1,4- over alpha-1,6-linked disaccharides is enhanced 2.3- to 3.5-fold. In addition, the introduction of a cationic side chain in Gly183-->Lys and Ser184-->His glucoamylase, broadens the optimal pH range for activity towards acidic as well as alkaline conditions.