Production, purification, and characterization of \alpha-amylase by Bacillus subtilis and its mutant derivates

Authors: ELİF DEMİRKAN

Abstract: The effects of various carbon and nitrogen sources on production of \alpha-amylase by Bacillus subtilis and its mutant derivates were investigated. The maximum production of \alpha-amylase by all strains was obtained in the presence of mesoinositol as the carbon source. There was no more significant increase in enzyme yield in the case of the supplementation of nitrogen sources, whereas malt extract and tryptone were preferred nitrogen sources for amylase production by Bacillus subtilis and mutant U 2-6 strain, respectively. \alpha-Amylases of B. subtilis and its mutant strain (EBUE 5-3) were purified through a series of steps, and characterized. The optimum temperature and pH values of the purified amylases were found to be 45 °C and 6.0, respectively. The enzyme of mutant strain had more stability than the enzyme of the parental strain in alkaline conditions (85% at pH 8.0 for 1 h). The K_m and V_{max} values of both amylases were also compared. Enzymes were strongly inhibited by Cu^{2+}, Hg^{2+}, and Ag^{2+}, but activated by Ca^{2+}, Ba^{2+}, Mg^{2+}, Li^{2+}, and Mn^{2+}. Metal ion concentration of 1 mM had a greater effect on enzyme activities than 5 mM did. The estimated molecular weight of the purified enzymes was 56 kDa. The N-terminal amino acid sequence of amylases produced by the parental and the mutant strain showed homology.

Keywords: Bacillus subtilis, mutant, \alpha-amylase, production, purification, characterization

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