Cloning, expression and characterization of a mesophilic catechol 1,2-dioxygenase from rhodococcus ruber OA1

Abstract

Background and Objective: Salicylic Acid (SA) is widely used in medicine and food. Rhodococcusruber OA1 utilizes salicylate as the sole carbon and energy source for growth and catechol 1,2-dioxygenase (C120) is detected in R. ruber OA1 grown on salicylate. However, C120 has not previously been physiologically or biochemically characterized in R. ruber. The aim of this study was to characterize C120 from R. ruber OA1 (OA1-C120). Methodology: The catechol 1,2-dioxygenase gene (catk) from R. ruber OA1 [OA1-catA) was cloned into the pEASY-E1 vector to obtain the recombinant plasmid pEASY-N-catA and CI20 was expressed in Escherichia coli BL21 (DE3). The heterologously expressed OA1-C12O was purified and its physiological and biochemical characteristics were further studied. Results: Based on the phylogenetic analysis of catA gene, it was found that OM-catA clustered with other catA genes from Gram-positive bacteria including Nocardia sp. C-14-1, Streptomyces ghanaensis ATCC 14672 and Amycolatopsis orientalis HCCB10007. After the expression and enzymatic characterization of C120, it was revealed that the expressed OA1-C12O had the ability to degrade catechol to cis, cis-muconic acid with a specific enzyme of 231.4 Umg-1 protein. The optimal reaction conditions of OA1-C120 were 25°C and pH 7.0. Besides, Mn2+ could increase the activity of OA1-C12O, while Mg2+ and NH4+ inhibited its activity. Conclusion: In this study the C120 from R. ruber OA1 was successfully expressed in E coli BL21 (DE3) for the first time and its catalytic characteristics were explored in detail.