Biodegradation Of Pyrene By Bacillus Cereus And Cloning And Expression Of Degrading Gene

The biodegradation of pyrene by a baeterial strain(Bacillus cereus) were studied in this research. The pyrene degradation mechanism was expected to be clarified from a biological point of view via revealing the biodegradability of pyrene, the biological characteristics of bacteria in the process of degradation, and through analyzing the metabolite, functional genes and enzymes involved in the biodegradation.The optimal concentration of inorganic salt medium was listed as follows:K2HPO4 100 mg·L-1,KH2PO4 100 mg·L-1,Mg SO4·7H2O 20 mg·L-1 and sodium citrate100 mg·L-1. Obvious promotion was found in degradation of pyrene with the addition of Mn2+, Fe3+ and glucose at the concentrations of 1 mg·L-1, 0.1 mg·L-1 and 10 mg·L-1,respectively. Within 120 h, the degradation rate of 2.5 μmol·L-1pyrene by 1 g·L-1biomass was 61.4%.Four hydroxy metabolites of pyrene, 1-naphthol, 2-naphthol, 9-phenanthrol and1-pyrenol were detected by LC-MS/MS, which suggested that the ring-cleavage of pyrene was initiated by monooxygenase, and these four hydroxy metabolites were utilized by B. cereus effectively. The utilization rates of theses metabolites by B.cereus were 100%, 90.3%, 98.3% and 52.7%, respectively. The results of enzyme activities analysis showed that salicylate hydroxylase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase of B. cereus played a key role in degradation of pyrene, and the enzyme activities were obviously enhanced after pyrene treatment. Based on the product analysis and enzymatic determination, it was speculated that the degradation pathway of pyrene by B. cereus was due to the combined effects of monooxygenase and dioxygenase system.Catechol 2,3-dixygenase gene was cloned by PCR amplification using plasmid extracted from B. cereus as template. Gene sequencing and bioinformatics analysis on the results of sequencing were carried out. C23 O gene sequence is 924 bp in length with a complete open reading frame, encoding 307 amino acids. The theoretical protein molecular weight was 35.16 k Da, and the isoelectric point was 5.46. The percentage of G+C content was 61.8%, and A+T content was 38.2% in coden area.Compared the homology among C23 O gene of B. cereus and that of other bacteria in NCBI database, the C23 O gene of B. cereus and Pseudomonas bacteria have the highest similarity.The C23 O gene of B. cereus was expressed by p ET 32a(+) vector and expressed in Escherichia coli BL21(DE3) successfully. The enzyme activity characteristic research also demonstrated that the recombinant protein existed mainly by a intracellular enzyme form in host cell.