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African Journal of Biotechnology

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Cloning, expression and purification of cold adapted acetate kinase from Shewanella species AS-11

Md. Abul Kashem Tang, Hiroyuki Motoshima, Keiichi Watanabe

Abstract


A psychrotrophic bacterium, Shewanella sp. AS-11 was isolated from a buccinid (shell) Neobuccinum living in the Antarctic ice-covered sea. An open reading frame of 1203 bp, coding for acetate kinase gene, called ack, was amplified, cloned into the expression vector, pETY-16b, and the enzyme was overproduced by using T7 system in Escherichia coli BL21 (DE3). After extraction of crude recombinant acetate kinase, the desired enzyme was able to be purified on a Blue Sepharose CL-6B and Super-Q affinity column chromatography. The molecular mass of the enzyme is about 86 kDa, which is associated with two monomers. In respect of pH, the enzyme was stable between 6 to 8 and maximum activity was obtained at 7.5. The purified enzyme was stable at 30°C but ligand bound enzyme was stable at 40°C. The structural comparison to mesophilic and thermophilic acetate kinases demonstrates that the psychrophilic one contains lower number of salt bridges and cation-pi interaction. So, it can be suggested that the enzyme is cold adapted with thermolabile and flexible structure.

Keywords: Acetate kinase, thermolabile, cold adapted, flexible, activity

African Journal of Biotechnology Vol. 11(29), pp. 7454-7463, 10 April, 2012



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