A Newly Identified Glutaminase-Free L-Asparaginase (L-ASPG86) from the Marine Bacterium Mesoflavibacter zeaxanthinifaciens

Abstract

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 L-asparaginase 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 37-40 degrees C, 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-L-ASPG86 was 687.1 units/mg under optimum conditions (37 degrees C, pH 9, and 5 mM MnSO4).