Abstract
Most of the glucoamylases (GA), which catalyze the hydrolysis of α-1,4 and α-1,6 glycosidic linkages, have a distinct region called a starch-binding domain (SBD). We have developed a powerful method for screening a library of GA mutants by a combination of GA display and SBD mutagenesis on the yeast-cell surface. In the case of Rhizopus oryzae glucoamylase (RoGA), three amino acids (63S, 71T, 73S) of the SBD were combinatorially mutated to enhance the degradation activity toward cooked corn starch and the mutated RoGAs were displayed on yeast-cell surface by cell-surface engineering. After the first screening by halo assay using an iodine-starch reaction, about 200 of the 8000 colonies formed clear halos. Incubation of the yeast with the mutated and displayed RoGAs caused direct degradation of cooked corn starch. Repeated screening revealed that some of the mutants produced a degradation rate around ∼1.4-fold higher than did wild type. The results obtained from the DNA sequences of the mutated SBDs indicated that amino-acid residues with a carbonyl group (D, E, Q, N) in the SBD enhance the degradation ability of the GA by enhancing the binding activity of the SBD.
Published Version
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