Gene Cloning, Heterologous Expression, and Characterization of a High Maltose-Producing α-Amylase of Rhizopus oryzae

Abstract

A putative α-amylase gene, designated as RoAmy, was cloned from Rhizopus oryzae. The deduced amino acid sequence showed the highest (42.8%) similarity to the α-amylase from Trichoderma viride. The RoAmy gene was successfully expressed in Pichia pastoris GS115 under the induction of methanol. The molecular weight of the purified RoAmy determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was approximately 48 kDa. The optimal pH and temperature were 4–6 and 60 °C, respectively. The enzyme was stable at pH ranges of 4.5–6.5 and temperatures below 50 °C. Purified RoAmy had a K _m and V _max of 0.27 mg/ml and 0.068 mg/min, respectively, with a specific activity of 1,123 U/mg on soluble starch. Amylase activity was strongly inhibited by 5 mM Cu²⁺ and 5 mM Fe²⁺, whereas 5 mM Ca²⁺ showed no significant effect. The RoAmy hydrolytic activity was the highest on wheat starch but showed only 55% activity on amylopectin relative to soluble corn starch, while the pullulanase activity was negligible. The main end products of the polysaccharides tested were glucose and maltose. Maltose reached a concentration of 74% (w/w) with potato starch as the substrate. The enzyme had an extremely high affinity (K _m = 0.22 mM) to maltotriose. A high ratio of glucose/maltose of 1:4 was obtained when maltotriose was used at an initial concentration of 40 mM.