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

• Main Authors: Tang, Md. Abul Kashem, Motoshima, Hiroyuki, Watanabe, Keiichi
• Format: Article in Journal/Newspaper
• Language: English
• Published: Academic Journals (Kenya) 2014
• Subjects: Antarctic; The Antarctic; Antarc*
• Online Access: http://www.ajol.info/index.php/ajb/article/view/102392

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