CDNA cloning and functional expression of ?-glucosidase from Mortierella alliacea

Abstract

We recently purified an α-glucosidase comprising 61-kDa and 31-kDa subunits from the fungus Mortierella alliacea and characterized its soluble starch-hydrolyzing activity. Here, the cDNA coding for this enzyme was cloned, revealing that it encodes a single polypeptide of 1,053 amino acids, with a calculated molecular mass of 117 kDa. Comparison between the deduced amino acid sequence and the partial sequences of the purified enzyme suggested that an immature protein can be converted into the two subunits of mature enzyme by post-translational processing at least three cleavage sites. Heterologous expression of recombinant α-glucosidase in yeast gave rise to a significant increase in hydrolytic activity toward maltose and soluble starch, in both intracellular and extracellular fractions. Immunoblot analysis using antiserum against the α-glucosidase revealed that the active enzyme expressed in yeast is also composed of two subunits. The yeast expression system provides a model suitable for investigating the polypeptide-processing event and structure–function relationship of the α-glucosidase with unique substrate specificity.