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Biochemical Characterization of Raw-starch-digesting Alpha Amylase Purified from Bacillus amyloliquefaciens

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Abstract

Alpha amylase (E.C. 3.2.1.1) of Bacillus amyloliquefaciens produced by submerged fermentation was purified to near homogeneity by ion exchange chromatography. Through the process 38.6-fold increase in purity with a specific activity of 72 U/mg proteins was obtained. The apparent molecular weight of the purified enzyme was found to be 58 kDa by SDS-PAGE. The enzyme was relatively stable between pH 5.0–8.0 and temperature between 50 and 60°C. The enzyme did not show any obligate requirement of metal ions but Ca2+ and Cu2+ enhanced the enzyme activity marginally and the thermostability was enhanced in the presence of Ca2+ ions. The purified enzyme exhibited maximal substrate specificity for amylose and efficiency in digesting various raw starches. The K m and V max of the enzyme was determined using both amylose and soluble starch as substrate. The analysis of the hydrolyzed products of soluble starch by thin layer chromatography showed the yield of maltosaccharides after 6 h of hydrolysis.

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Acknowledgment

The study was financially supported by Department of Biotechnology, New Delhi.

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Authors and Affiliations

  1. Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST), CSIR, Trivandrum, 695 019, India

    Dhanya Gangadharan, K. Madhavan Nampoothiri, Swetha Sivaramakrishnan & Ashok Pandey

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  1. Dhanya Gangadharan
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  2. K. Madhavan Nampoothiri
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  3. Swetha Sivaramakrishnan
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  4. Ashok Pandey
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Correspondence to K. Madhavan Nampoothiri.

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Gangadharan, D., Nampoothiri, K.M., Sivaramakrishnan, S. et al. Biochemical Characterization of Raw-starch-digesting Alpha Amylase Purified from Bacillus amyloliquefaciens . Appl Biochem Biotechnol 158, 653–662 (2009). https://doi.org/10.1007/s12010-008-8347-4

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