A novel cyclodextrin glycosyltransferase from Bacillus sphaericus strain 41: Production, characterization and catalytic properties

Abstract

The alkalophilic Bacillus sphaericus strain 41 was isolated from soybean-soil culture, using a highly alkaline pH medium containing 1% Na 2CO 3. The cyclodextrin glycosyltransferase (CGTase) from this microorganism was purified up to 315-fold with a yield of 31%, by biospecific affinity-column chromatography using Sepharose 6B gel and β-cyclodextrin (β-CD) as the ligand. The molecular weight of the purified enzyme was estimated to be 59 kDa by SDS-PAGE. In addition to the cyclization, the CGTase showed disproportionation and coupling activities. For cyclization activity, the optimal pH was 6.0 and the temperature was 65 °C. The enzyme showed pH stability in the range of 6.0–7.0. Thermal deactivation was noticeable above 70 °C, and the enzyme was highly stable below 65 °C. The activation and deactivation energy for the production of β-CD were 9.4 kcal/mol and 28.0 kcal/mol, respectively. The influence of substrate concentration on the initial rate of CD production was studied, and the kinetic parameters were determined. The K m was 0.0008 mol/L and V max was 0.0631 mol of β-CD/(L h), using maltodextrin as substrate. The CGTase was strongly inhibited by the products, and produced a level of CDs reaching 22 g/L with a β-CD ratio of 54%. This enzyme produced α-, β- and γ-CD in the ratio of 0.40:1:0.45.