Isolation Identification and Mutation of a Novel Native Alkaline and Thermophile Bacillus Sp V 41 Containing Amylase Activity


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Khodayarı F., Cebeci Z., Özcan B. D.

Advances in Bioresearch, vol.5, no.3, pp.57-64, 2014 (Peer-Reviewed Journal)

Abstract

Alkaline, halophilic and thermophilic Bacillus sp. strain V-41, which produces extracellular α-amylase was isolated from Van Lake area located in Turkey. Characterization of strain V-41 by 16S rRNA gene analysis found it closest member of Bacillus and related to uncultured bacterium clone HAV7D9G02CMKTO with a similarity of >97%. This bacterial isolate was mutagenized by treatment with Ethidium bromide (EtBr) and 4 mutant variants, V41-M1, V41-M2, V41-M3, and V41-M4 were obtained. The mutant α-amylase from V41-M4 displayed more than 91% activity according to the wild type enzyme. The α-amylase activities from Bacillus sp. V-41, and its mutant variants V41-M1, V41-M2, V41-M3 and V41M4 were 0.65, 0.84, 0.73, 0.6 and 1.27 U mL-1 min-1, respectively. Bacillus sp. wild type strain V-41 and its mutant variant V41-M4 were selected for partial characterization. Maximum α-amylase productions were achieved at the end of 60 and 48 h of growth for V-41 and V41-M4, respectively. The optimum temperatures of the V-41 and V41-M4 α-amylases were found to be 60 and 40 °C, respectively. The optimum pH of the both α-amylases was found to be 7.5. An analysis of the enzyme for molecular mass was carried out by SDS-Starch-PAGE electrophoresis revealed a single band with molecular weight of 70 kDa for wild type and mutant variants.