Glucoamylase (EC 3.2.1.3) (GA), an enzyme that catalyzes the release of [beta]-D-glucose from the nonreducing ends of starch and related oligo- and polysaccharides, is widely used in industry to produce high-glucose syrups. Site-directed mutagenesis was used to construct mutations to obtain mutant enzymes with industrially desired alterations in pH dependence and substrate specificity;An increased pH optimum for GA would be industrially desirable so that it could be used in process conditions more similar to that of other enzymatic steps in starch processing. Five mutations were designed to remove or weaken the hydrogen bond between Ser411 and Glu400 (the general catalytic base), in order to destabilize the carboxylate ion form of Glu400, and thereby raise its pK. All five mutations, Ser411→Cys, Ser411→Ala, Ser411→His, Ser411→Asp and Ser411→Gly, increased the optimal pH of GA. Ser411→Ala is the best performing pH mutant of GA isolated to date. Ser411→Ala GA increased the optimal pH by 0.84 units while maintaining a high level of catalytic activity (k cat) and catalytic efficiency ( kcat/Km);For substrate specificity, reducing isomaltose formation from glucose condensation would be industrially desirable so that the glucose yield in starch processing could be increased. The substrate specificity of ten single mutations and a double mutation were investigated. Compared to wild-type GA, Ser119→Glu, Ser119→Gly, Ser119→Trp, Tyr175→Phe, Arg241→Lys and Ser411→Gly GAs were highly active. Tyr116→Trp, Gly121→Ala, Ser411→Ala and Gly121→Ala/Ser411→Gly GAs had moderately decreased activity. Tyr48Phe49→Trp GA, however, had severely decreased activity. The ratio of the initial rate of isomaltose production (from glucose condensation reactions) to that of glucose production (from the hydrolysis of DE 10 maltodextrin) was used to represent the ability to synthesize isomaltose at a normalized level DE 10 maltodextrin hydrolytic activity. Compared to wild-type GA, mutant Ser119→Gly, Ser119→Trp, Gly121→Ala, Tyr175→Phe, Ser411→Ala, Ser411→Gly and Gly121→Ala/Ser411→Gly GAs showed a decrease in this ratio at 35°C. Only Tyr175→Phe, Ser411→Ala, Ser411→Gly and Gly121→Ala/Ser411→Gly GAs, however, showed a decrease in this ratio at 55°C. Mutant Tyr175→Phe, Ser411→Ala, and Ser411→Gly GAs have great potential for increasing the glucose yield in industrial starch processing applications.