A newly identified glutaminase-free &#671 -asparaginase (&#671 -ASPG86) from the marine bacterium Mesoflavibacter zeaxanthinifaciens

Abstract

&#671 -Asparaginase (EC 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 &#671 -asparaginase gene (&#671 -ASPG86) in the genus Mesoflavibacter, which consists of a 1035-bp open reading frame (ORF) encoding 344 amino acids (aa). Following phylogenetic analysis, the deduced amino acid sequence of &#671 -ASPG86 (&#671 -ASPG86) grouped as a type I asparaginase with respective homologs in Escherichia coli and Yersinia pseudotuberculosis. The &#671 -ASPG86 gene was cloned into the pET-16b vector to express the respective protein in E. coli BL21 (DE3) cells. Recombinant &#671 -asparaginase (r-&#671 -ASPG86) showed optimum conditions at 37-40oC, pH 9. Moreover, r-&#671 -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-&#671 -ASPG86 was 687.1 units/mg under optimum conditions (37oC, pH 9 and 5 mM MnSO4).bacter, which consists of a 1035-bp open reading frame (ORF) encoding 344 amino acids (aa). Following phylogenetic analysis, the deduced amino acid sequence of &#671 -ASPG86 (&#671 -ASPG86) grouped as a type I asparaginase with respective homologs in Escherichia coli and Yersinia pseudotuberculosis. The &#671 -ASPG86 gene was cloned into the pET-16b vector to express the respective protein in E. coli BL21 (DE3) cells. Recombinant &#671 -asparaginase (r-&#671 -ASPG86) showed optimum conditions at 37-40oC, pH 9. Moreover, r-&#671 -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-&#671 -ASPG86 was 687.1 units/mg under optimum conditions (37oC, pH 9 and 5 mM MnSO4).