Purification and Properties of Neutral-cyclodextrin Glycosyl-transferase of an Alkalophilic Bacillus sp.

Neutral-cyclodextrin glycosyltransferase (EC 3.2.1.19) of alkalophilic Bacillus sp. (ATCC 21783) was purified by starch adsorption, DEAE-cellulose chromatography and Sephadex G–150 gel filtration chromatography followed by preparative polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was 85,000-88,000 by SDS-disc gel electrophoresis. The enzyme was most active at pH 7 and 50°C, and stable up to 60°C at pH 7 and in the range of pH 6~8 at 60°C by 30 min incubation. The apparent V_max and Km values for α- and β-cyclodextrin at a constant concentration of sucrose were 417, 70 µmoles glucose/min · mg protein and 10, 0.83 nm, respectively. About 85~90% of amylose, 75~80% of potato starch, 65~70% of amylopectin, 55~60% of glycogen, 45~50% of amylopectin β-limit dextrin, 20~25% of maltotriose and 10~15% of maltose were converted to cyclodextrins with 0.5~1% (w/v) of each substrate.

Schardinger β-dextrin was preferentially produced from starch, and α- or γ-dextrin was gradually formed after prolonged incubation. After 20 min incubation, about 0.4, 14 and 2.5% of α-, β- and γ-dextrin were formed from starch, respectively.