Modification of enzyme function by carbohydrate chain addition

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Two chimeric enzymes were engineered from highly homologous rice alpha-amylase isozymes Amy1A and Amy3D.Although these two isozymes show high homology in amino acid sequences, they showed different reaction properties in soluble starch and oligosaccharide hydrolysis. The reaction properties of chimeric enzymes Amy1A/3D and Amy3D/1A were studied. Since one isozyme Amy1A and one chimeric enzyme Amy3D/1D have N-linked carbohydrate chain, effects of carbohydrate chain were extensively studied.(1) Thermostability of Amy3D/1A,which has the carbohydrate chain, were lower than other enzymes (Amy1A,Amy3D and Amy1A/3D). Although our previous study suggests that the carbohydrate chain significantly affects thermostability, Amy3D/1A did not show high thermostability because of a loose conformational packing.(2) Amy3D/1A showed higher reactivity to soluble starch than Amy3D.This result suggested that the carbohydrate chain enhances the enzyme reaction.(3) Tertiary structure near N-terminal end of the enzyme has significant effect on the "stiffness" of the enzyme structure, and affects the hydrolysis efficiency. The barrel structure, on the other hand, affects the hydrolysis efficiency of oligosaccharides. Since the carbohydrate chain is positioned outer surface of the barrel structure near the active cleft, the carbohydrate chain might interact with long substrate soluble starch.Thus, it was suggested that reaction property of alpha-amylase, especially that to soluble starch, can be improved by creating N-glycosylation site at the outer surface of the barrel structure near the active cleft.