Expression and Biochemical Characterization of a Thermostable Branching Enzyme from Geobacillus thermoglucosidans

Abstract

The branching enzyme (EC 2.4.1.18) catalyzes the formation of α-1,6 branch points in starch. In this study, the <i>Geobacillus thermoglucosidans</i> gene-encoding branching enzyme was expressed in <i>Escherichia coli </i>BL21 (DE3) and the protein was isolated and characterized. <i>G. thermoglucosidans </i>branching enzyme is a thermostable enzyme with an optimal reaction temperature of nearly 60°C and a half-life at 65°C of approximately 1.1 h. The activity of the recombinant enzyme is optimal at pH 7.5, with broad stability between pH 5.5 and 9.0. Its thermostability, relatively broad pH stability and optimal temperature near the temperature at which starch begins to gelatinize may make it easy to use in industrial production. Furthermore, the enzyme is activated by Mg²⁺, Ba²⁺, K⁺ and Na⁺ in a concentration-dependent manner and dramatically inhibited by Ni²⁺ and Co²⁺. Its substrate dependence, using amylopectin as the substrate, could be adequately fitted using the Michaelis-Menten equation, yielding a<i> K</i>_m of 0.99 mg/ml. High-performance anion exchange chromatography results showed that the chain length distribution of branching enzyme-treated waxy corn starch is indistinguishable from that of the branching enzyme-treated common corn starch. This enzyme may therefore be a promising tool for the enzymatic modification of starch.