Characterization And Reaction Kinetic Of Biphenyl Hydrolase From Rhodococcus Sp. R04

Polychlorinated biphenyls (PCBs) are serious environmental pollutants that threaten both the natural ecosystem and human health. Nowadays, among many of methods for pollutant treatment, one of the most efficient and popular method is biological treatment which is economic and efficient, especially, not produces secondary pollution. However, it is founded that lots of yellow substances accumulated in the microbial degradation of PCBs. Analysis shows that they are chlorinated 2-hydroxy-6-oxo-6-phenylhexa -2,4-dienoic acids (C1-HOPDA), which are the substrate of hydrolase (BphD), an enzyme of the biphenyl biodegradation pathway encoded by the bphD of Rhodococcus sp. R04. The further degradation of PCBs was limited because of the feedback inhibition of these yellow materials. Due to its key role in biphenyl biodegradation, researchers are more devoted to studying the structure and function of hydrolase.In this paper, the hydrolase gene was amplificated from the genome of Rhodococcus sp. R04, and ligated to expression vector pBV220 to generate a recombinant plasmid, pBV220-bphD. The recombinant plasmid was introduced into E. coli BL21 (DE3), and was expressed heterologously. Then the products were purified to apparent homogeneity with a final purification fold of 5.02 by anion exchange and exclusion chromatography. Analysis of SDS-PAGE and gel filtration demonstrated that BphD is a hometetramer with a subunit molecular mass of 32 kDa. Some properties of BphD were investigated, and the results revealed that the optimum temperature and pH of BphD are 80℃and 9, respectively. The enzyme has remaining 90% of activity incubating for 1 hour at 60℃, and has a half-life of 1 hour at 70℃, making it the most thermostable biphenyl hydrolase reported in Rhodococcus. The information of BphD from circular dichroism indicated that the enzyme is a-helix dominated, and its secondary structure has undergone great changes with the temperature rising to 70℃, and has been severely damaged at 80℃. The susceptibility of BphD towards several surfactants, inhibitors and metal cations was also assessed. The enzyme activity was restrained strongly by SDS at the concentration of 1 mmol/L,1 mmol/L of metal ions also inhibited BphD activity besides Mn2+. Further more, BphD have weak metal-dependent and relatively strong antioxidant ability.The relationship between thermostability and the secondary structure of BphD was further analysed by circular dichroism, and the results reflected that the activity depends on the integrity of secondary structure. The secondary structure of BphD was destroyed step by step with the temperature increasing, accordingly the activity declined gradually. The results of sequence alignment between BphD and other homologous hydrolases indicated that the Ser, His, Asp and Trp residues are highly conserved, besides, some amino acids such as S46, G53, V65, A180, V207, S213 and M252 were considered characteristic of BphD in R04 because they were different from those of other hydrolases at the same sites. Chemical modification of these conserved amino acids suggested that the activity of modified BphD was reduced markedly. It is speculated that they were likely to play critical roles in the hydrolysis of HOPDA or near the active center of enzyme. To further determine the function of these conserved and characteristic amino acids, site-directed mutagenesis and expression were performed respectively. However, there were little soluble products harvested of these mutants.In addition, the steady-state and pre-steady-state kinetics of BphD had been studied respectively. Steady-state results showed that BphD has a Km of 0.44μmol/L and a kcat of 2.8 s-1 for HOPDA, the enzyme has higher specificity for HOPDAs than catechol materials, meanwhile, the specificity of BphD for Cl-HOPDA decreased markedly with the size of substituent. Single turnover stopped-flow analysis showed that the HOPDA was transformed rapidly at the beginning of reaction, and ultimately reached a constant level. The enzyme-substrate complex (E:S) was appeared in the initial stage, then decayed sharply and balanced finally. The amount of product HPD increased gradually with the time extended, and eventually reached equilibrium. The whole reaction was found to obey classic Michaelis-Menten kinetics, moreover, the reaction rate of every response element was accelerated with the rising of reaction temperature and enzyme concentration. These relevant results will build a basis for more learnings of the kinetics of BphD.