Functional characterization of multiple catechol dioxygenases in triclocarban degrader Sphingomonas sp. YL-JM2C

Abstract:

[Objective] We are aiming to study the growth characteristics of Sphingomonas sp. YL-JM2C when triclocarban acts as carbon source; to mine putative genes encoding catechol 1,2-dioxygenase and catechol 2,3-dioxygenase from strain YL-JM2C; and to express, purify and functionally identify the putative catechol dioxygenase, then revealing the characters of catechol dioxygenases in this strain. [Methods] The growth of S. sp. YL-JM2C was measured at different pH values with different triclocarban concentrations on the optimized R2A medium containing extremely low amount of single carbon source. The putative catechol dioxygenase genes were cloned and heterologous expressed in E. coli BL21(DE3), and purified through AKTA purifier system. The purified proteins were functionally identified based on their abilities to catalyze the ring cleavage of catechol as well as its derivatives 3-chlorocatechol and 4-chlorocatechol. Their enzymatic characters were also determined through enzyme kinetics parameters. [Results] The optimal pH value was 7.0?7.5 for the growth of strain YL-JM2C, and this strain was able to utilize triclocarban as the carbon source and the optimum concentration for its growth was in the range of 4?8 mg/L. With adding 4 mg/L triclocarban, the biomass of strain YL-JM2C increased over time when this strain grew in the optimized R2A medium containing 0.01% of yeast extract and mineral salts. Six putative catechol dioxygenase genes (stcA1, stcA2, stcA3, stcE1, stcE2 and stcE3) were found through bioinformatics analysis. All exhibited catechol dioxygenase activities through assay using crude enzymes expressed in E. coli, except StcE3. Further, after purification, the substrate range analyses revealed that StcA1, StcA2 and StcA3 belonged to type II catechol 1,2-dioxygenase while StcE1 and StcE2 were identified as the novel type catechol 2,3-dioxygenase, which were all able to catalyze the ring cleavage of catechol and chlorocatechol. The kinetics parameters obtained from purified enzymes revealed that the enzymes exhibited highest affinity and catalytic efficiency to catechol, followed by 4-chlorocatechol. [Conclusion] In this research, five active catechol dioxygenase genes are functional identified from a single strain, which all have the ability to catalyze the ring cleavage of catechol and chlorocatechol. Among them, StcA1, StcA2, and StcA3 belong to type II catechol 1,2-dioxygenase, and StcE1 and StcE2 are new type catechol 2,3-diocygenase. This study will be greatly helpful to explore the diverse capability for the microbial aromatic degradation of microbes using catechol and chlorocatechol as ring-cleavage substrates.