Heterologous expression of cyclodextrin glycosyltransferase from Bacillus stearothermophilus in Bacillus subtilis and its application in glycosyl rutin production

In this paper, the cgt gene encoding cyclodextrin glycosyltransferase (CGTase) from Bacillus stearothermophilus was cloned into pWB980 plasmid for extracellular expression in Bacillus subtilis SCK6. Through adding a six-histidine affinity tag fused to the C-terminus, the recombinant CGTase could be purified by nickel ion affinity chromatography, and its molecular weight was approximately 76 kDa on SDS-PAGE. Then, the enzymatic properties were determined, and results were as follows: the optimum temperature and pH were identified as 40 degrees C and pH 5.0, respectively. CGTase had good tolerance to metal ions of Mn2+, Ca2+, and Mg2+. The enzyme activity was activated by Na+, Al3+, Fe3+, and Ni+, and it was remarkably inhibited by Cu2+ and Zn2+. To improve the aqueous solubility of rutin, CGTase was used to catalyze the transglycosylation reaction, and the conversion rate could reach as high as 80.13% under optimal conditions. Furthermore, the reaction mixture was treated with glucoamylase and microporous adsorbent resin. The yield of glycosyl-rutin was 56.1%, and its purity was 74.3%, which further improved the value of the product.