FUNCTIONAL-ROLE OF ASPERGILLUS-ORYZAE GLUCOAMYLASE C-TERMINAL DOMAIN INVESTIGATED USING ITS CDNA

The C-terminal domain of Aspergillus oryzae glucoamylase was analyzed by site-directed mutagenesis using glucoamylase cDNA. A mutant glucoam ylase cDNA lacking the region corresponding to the C-termlnal domain o f the wild-type glucoamylase was constructed by inserting two stop cod ons in the gene for the wild-type glucoamylase. The wild-type and muta nt glucoamylase cDNAs were expressed in Saccharomyces cerevisiae YPH 2 50, and then the produced wild-type and mutant glucoamylases were puri fied by acarbose affinity column chromatography. As compared to those of the wild-type glucoamylase, the K-m values of the mutant enzyme det ermined using maltose, maltotriose or maltopentaose as a substrate wer e similar, but that determined using soluble starch as a substrate was twofold higher. The mutant glucoamylase showed a low rate of hydrolys is of raw cornstarch, although the wild-type glucoamylase showed a hig h rate of raw cornstarch hydrolysis. These results indicated that the C-terminal domain is important in the affinity of the enzyme to raw st arch as reported for the glucoamylase of Aspergillus awamori.