Maltogenic amylase CoMA from Corallococcus sp. strain EGB catalyzes the hydrolysis and transglycosylation of maltooligosaccharides and soluble starch into maltose, the sole hydrolysate. This process yields pure maltose with potentially wide applications. Here, we identified and evaluated the role of phenylalanine 314 (F314), a key amino acid located near the active center, in the catalytic activities of the CoMA. Site-directed mutagenesis analysis showed that the activity of a F314L mutant on potato starch substrate decreased to 26% of that of wild-type protein. Compared with the wild-type, F314L exhibited similar substrate specificity, hydrolysis pattern, pH, and temperature requirements. Circular dichroism spectrum data showed that the F314L mutation did not affect the structure of the folded protein. In addition, kinetic analysis demonstrated that F314L exhibited an increased Km value with lower substrate affinity. Homology modeling showed that the benzene ring structure of F314L was involved in π-π conjugation, which might potentially affect the affinity of CoMA toward starch. Taken together, these data demonstrated that F314 is essential for the hydrolytic activity of the CoMA from Corallococcus sp. strain EGB.
Keywords: Corallococcus sp; maltogenic amylase; substrate affinity; π-π conjugation.
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