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 ˜300-fold {k^J Km) preference for the a-1,4- over the a-l,6-glucosidic linkage determined using the substrates maltose and iso-maltose. It is postulated that as most amylolytic enzymes act on either the a-1,4- or a-l,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, Serll9 Tyr, Glyl83 Lys and Serl84 His, in two separate active-site regions. These mutants all had slight increases in activity as compared with the wild-type enzyme towards the a-l,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 fc^, for the Serl84→ His mutant As a consequence, the selectivity of the three glucoamylase mutants for a-1,4- over a-l,6-linked disaccharides is enhanced 2.3- to 3.5-fold. In addition, the introduction of a cationic side chain in Glyl83 → Lys and Serl84 → His glucoamylase, broadens the optimal pH range for activity towards acidic as well as alkaline conditions.