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Production, purifi cation, and characterization of α-amylase by Bacillus subtilis and its mutant derivates

The effects of various carbon and nitrogen sources on production of α-amylase by Bacillus subtilis and its mutant derivates were investigated. The maximum production of α-amylase by all strains was obtained in the presence of mesoinositol as the carbon source. | E. DEMİRKAN Turk J Biol 35 (2011) 705-712 © TÜBİTAK doi:10.3906/biy-1009-113 Production, purification, and characterization of α-amylase by Bacillus subtilis and its mutant derivates Elif DEMİRKAN Biology Department, Faculty of Sciences and Arts, Uludağ University, 16059 Bursa – TURKEY Received: 24.09.2010 Abstract: The effects of various carbon and nitrogen sources on production of α-amylase by Bacillus subtilis and its mutant derivates were investigated. The maximum production of α-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. α-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 Km and Vmax values of both amylases were also compared. Enzymes were strongly inhibited by Cu2+, Hg2+, and Ag2+, but activated by Ca2+, Ba2+, Mg2+, Li2+, and Mn2+. 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. Key words: Bacillus subtilis, mutant, α-amylase, production, purification, characterization Bacillus subtilis ve mutant türevleri tarafından α-amilazın üretimi, saflaştırılması ve karakterizasyonu Özet: Çalışmada, B. subtilis ve onun mutant türevleri tarafından α-amilazın üretimi üzerine çeşitli karbon ve azot kaynaklarının etkisi araştırıldı. Tüm suşlar .