Functional analysis of the threonine- and serine-rich Gp-I domain of glycoamylase I from Aspergillus awamori var. kawachi
1995
Semimaru, T. | Goto, M. | Furukawa, K. | Hayashida, S.
Glucoamylase I (GAI) from Aspergillus awamori var. kawachi hydrolyzes raw starch efficiently and is composed of three functional domains: the amino-terminal catalytic GAI' domain (A-1 to V-469), the threonine- and serine-rich O-glycosylated Gp-I domain (A-470 to V-514), and the carboxy-terminal raw starch-binding Cp domain (A-515 to R-615). In order to investigate the role of the Gp-I domain, an additional repeat of Gp-I and internal deletions of the entire Gp-I sequence or parts of the Gp-I sequence were introduced within Gp-I. All mutant genes as well as the wild-type gene were inserted into a yeast-secretion vector, YEUp3Halpha, and expressed in Saccharomyces cerevisiae. Wild-type GAI expressed in yeast cells (GAY), GAGpI, having an extra Gp-I, and GA-delta470-493, lacking the A-470-to-T-493 sequences of Gp-I, were successfully secreted into the culture medium. On the other hand, GA-delta470-507, lacking A-470 to S-507, and GA-delta-GpI, lacking the entire Gp-I (A-470-to-V-514) sequence, failed to be secreted and remained in the yeast cells. The carbohydrate content of GAGpI was 1.2 times higher than that of GAY and 2.4 times higher than that of the original GAI. The raw starch digestibility of GAGpI was almost the same as that of GAY but was 1.5 times faster than that of GAI. Furthermore, GAGpI gained enhanced thermal stability as well as pH stability compared with GAY and GAI. On the other hand, GA-delta470-493 had decreased carbohydrate content (29% of that of GAY), and the raw starch digestibility of this enzyme was one-third that of GAY. These results indicate that the C-terminal portion of the Gp-I domain is critically required for secretion of GAI and, together with the Cp domain, plays an important role in raw starch digestion.
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