Isolation, Purification and Biochemical Characterization of CGTase from Bacillus halodurans

A novel Cyclomaltodextrin glucanotransferase (CGTase) producer, Bacillus halodurans was isolated from soil obtained from sugarcane fi elds. CGTase was produced in bulk through submerged batch fermentation in Horikoshi’s Media II. Soluble starch was used as carbon source and a combination of yeast extract and peptone were used as nitrogen source in the media, along with MgSO4.7H2O, K2HPO4 and Na2CO3, as they were found to be ideal for CGTase production. The enzyme was purified through acetone precipitation and starch adsorption methods, which proved to be simple and effi cient methods of purifi cation. Starch adsorption purifi ed sample was found to be homogenous on performing SDS-PAGE and the yield of the method was 49.44% with fold purifi cation of 17.34. The enzyme had appreciable affi nity for starch with a Km of 1.1mM and a turnover number of 10.9s-1 and was found to have an apparent molecular weight of ≈33 KDa. CGTase had two pH optima at pH 7.0 and pH 9.0 and a temperature optimum of 600C. There was no effect of metal-chelating agents on enzyme activity indicating that the enzyme is not a metalloenzyme, however it is a metal-activated enzyme as activity was enhanced by Mn²+. Inhibitory effects of group specific reagents indicate that serine and histidine residues may be involved in enzyme activity. The microorganism isolated grows in a wide range of temperatures, pH and salt concentrations, which are useful attributes in industrial applications requiring versatile organisms. The enzyme isolated also has appreciable activity at higher temperature and pH and is easily purified; making it valuable for use in industry