Purification and properties of cyclodextrin glycosyltransferase from Bacillus sp. AL-6

Abstract

Cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus sp. AL-6 was purified to a homogeneous protein, using ammonium sulfate fractionation, starch adsorption and ion-exchange chromatography on DEAE-Sephadex A-50. The enzyme, a monomeric protein with a molecular weight of 74,000, was determined by gel chromatography on Sephadex G-150 and SDS polyacrylamide gel electrophoresis. With soluble starch or potato starch as a substrate, the enzyme produced a considerable amount of γ-cyclodextrin with β-cyclodextrin. At the initial stage of the reaction, γ-cyclodextrin was predominant, and the amount of β-cyclodextrin increased gradually with time from the start of the reaction. The optimum pH for the formation of γ-cyclodextrin was 7 to 10 and the optimum temperature was 60°C. The addition of ethanol to the reaction mixture enhanced the formation of γ-cyclodextrin. When 4.4% soluble starch was used as the substrate with 20% ethanol, the maximum yield of γ-cyclodextrin was about 22% in comparison to 8% in the absence of ethanol. With 1.8% potato starch, the maximum yield was 25% at an ethanol concentration of 20%.