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Journal of Chemistry
Volume 2013, Article ID 673173, 11 pages
http://dx.doi.org/10.1155/2013/673173
Research Article

Production, Purification, and Characterization of Thermostable α-Amylase Produced by Bacillus licheniformis Isolate AI20

1Bioprocess Development Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City for Scientific Research and Technology Applications (SRTACity), Universities and Research Institutes Zone, New Borg El-Arab, Alexandria 21934, Egypt
2Pharmaceutical Bioproduct Research Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City for Scientific Research and Technology Applications (SRTACity), New Borg El-Arab, Alexandria 21934, Egypt

Received 11 May 2012; Revised 20 June 2012; Accepted 16 July 2012

Academic Editor: Peter Chang

Copyright © 2013 Yasser R. Abdel-Fattah et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

An optimization strategy, based on statistical experimental design, is employed to enhance the production of thermostable α-amylase by a thermotolerant B. licheniformis AI20 isolate. Using one variant at time (OVAT) method, starch, yeast extract, and CaCl2 were observed to influence the enzyme production significantly. Thereafter, the response surface methodology (RSM) was adopted to acquire the best process conditions among the selected variables, where a three-level Box-Behnken design was employed to create a polynomial quadratic model correlating the relationship between the three variables and α-amylase activity. The optimal combination of the major constituents of media for α-amylase production was 1.0% starch, 0.75% yeast extract, and 0.02% CaCl2. The predicted optimum α-amylase activity was 384 U/mL/min, which is two folds more than the basal medium conditions. The produced α-amylase was purified through various chromatographic techniques. The estimated enzyme molecular mass was 55 kDa and the α-amylase had an optimal temperature and pH of 60–80°C and 6–7.5, respectively. Values of and for the purified enzyme were 454 mU/mg and 0.709 mg/mL. The α-amylase enzyme showed great stability against different solvents. Additionally, the enzyme activity was slightly inhibited by detergents, sodium dodecyl sulphate (SDS), or chelating agents such as EDTA and EGTA. On the other hand, great enzyme stability against different divalent metal ions was observed at 0.1 mM concentration, but 10 mM of Cu2+ or Zn2+ reduced the enzyme activity by 25 and 55%, respectively.