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KMID : 0545120160260061115
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Journal of Microbiology and Biotechnology
2016 Volume.26 No. 6 p.1115 ~ p.1123
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A Newly Identified Glutaminase-Free L-Asparaginase (L-ASPG86) from the Marine Bacterium Mesoflavibacter zeaxanthinifaciens
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Lee Su-Jin
Lee Young-Deuk
Park Gun-Hoo
Umasuthan Navaneethaiyer
Heo Soo-Jin
De Zoysa Mahanama
Jung Won-Kyo
Lee Dae-Won
Kim Han-Jun
Kang Do-Hyung
Oh Chul-Hong
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Abstract
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L-Asparaginase (E.C. 3.5.1.1) is an enzyme involved in asparagine hydrolysis and has the potential to effect leukemic cells and various other cancer cells. We identified the Lasparaginase gene (L-ASPG86) in the genus Mesoflavibacter, which consists of a 1,035 bp open reading frame encoding 344 amino acids. Following phylogenetic analysis, the deduced amino acid sequence of L-ASPG86 (L-ASPG86) was grouped as a type I asparaginase with respective homologs in Escherichia coli and Yersinia pseudotuberculosis. The L-ASPG86 gene was cloned into the pET-16b vector to express the respective protein in E. coli BL21 (DE3) cells. Recombinant L-asparaginase (r-L-ASPG86) showed optimum conditions at 37-40oC, pH 9. Moreover, r-L-ASPG86 did not exhibit glutaminase activity. In the metal ions test, its enzymatic activity was highly improved upon addition of 5 mM manganese (3.97-fold) and magnesium (3.35-fold) compared with the untreated control. The specific activity of r-LASPG86 was 687.1 units/mg under optimum conditions (37°C, pH 9, and 5 mM MnSO4).
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KEYWORD
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L-Asparaginase, Mesoflavibacter, cloning, expression, manganese, glutaminase-free
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