Research Journal of Biological Sciences

Year: 2010
Volume: 5
Issue: 10
Page No. 699 - 707

Isolation and Characterization of Cyclodextrin Glucanotransferase from Soil Bacterium

Authors : R. Geetha and Sunil S. More

Abstract: An alkalophilic bacterium was isolated from the soil sample of sugar cane fields. A sterilized culture medium was prepared and the bacteria were grown for 6 days at 28°C in static condition at pH 10.5. phenolphthalein assay was performed to confirm the presence of CGTase using soluble starch as substrate. The positive strain was identified based on morphological, physiological and biochemical tests and according to Bergey’s manual as Bacillus alcalophilus AF078812. Time course of CGTase production was studied in shake flasks for 20 days with the maximum activity observed on the 6th day. The enzyme was purified upto 44.06 fold by starch adsorption method with an yield of 6.4% and specific activity of 327 U mg-1. Among the starches examined, soluble starch was the best substrate. A low Km value (0.02 mg mL -1) suggested soluble starch as the preferred substrate to produce CDs with Vmax of 64.1 U mg -1 protein. The enzyme was a monomer with a molecular weight 28 kDa estimated by SDS-PAGE. Enzyme activity was observed over a broad range of pH 4.5-8.5, optimum being 8.5. Alkaline CGTase exhibited thermostability for a longer duration at its optimum temperature 45°C. SDS was found to be strong inhibitor of the enzyme wherein TLCK, NEM, IAA, DTT, TLCK and DAN has no effect on enzyme activity. Moderate inhibitory effect was seen with EDTA on CGTase, indicating it to be a metalloenzyme. Enhanced activity was observed in presence of activators like Cu+2, Mn+2, Ca+2 and Fe+2 whereas, Mg+2 and Zn+2 lowered it.

How to cite this article:

R. Geetha and Sunil S. More, 2010. Isolation and Characterization of Cyclodextrin Glucanotransferase from Soil Bacterium. Research Journal of Biological Sciences, 5: 699-707.

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