Functional analysis of the threonine- and serine-rich Gp-I domain of glucoamylase I from Aspergillus awamori var. kawachi.
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, YEUp3H alpha, and expressed in Saccharomyces cerevisiae. Wild-type GAI expressed in yeast cells (GAY), GAGpI, having an extra Gp-I, and GA delta 470-493, lacking the A-470-to-T-493 sequences of Gp-I, were successfully secreted into the culture medium. On the other hand, GA delta 470-507, 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.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics; D004795:Enzyme Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012694:Serine / Q000737:chemistry; D013213:Starch / Q000378:metabolism; D013696:Temperature; D013912:Threonine / Q000737:chemistry
D004271:DNA, Fungal; D013912:Threonine; D012694:Serine; D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase