Studies On Cresols Biotransformation By Recombinant E. Coli (PH_IND)

The purpose of this dissertation is to investigate the characteristics of cresols biotransformation by recombinant Escherichia coli expressing phenol hydroxylase. The enzymatic characteristics of phenol hydroxylase were investigated, and then the metabolites of cresols biotransformation were identified by HPLC, LC-MS and 1H-NMR. The metabolic pathways of cresols biotransformation were presumed as well. Finally, o-cresol was selected to study the optimal biotransformation condition by response surface methodology (RSM).The optimal conditions for cresols biotransformation by phenol hydroxylase were as follows:pH 8.0 and temperature 30℃. Phenol hydroxylase could tolerate to Ca2+, Fe2+, Mg2+ and Mn2+ in cresols biotransformation, while it was inhibited significantly by Cu2+, Ni2+, Hg2+ and Zn2+. According to the amino acid sequences analysis, the phylogentic tree of the N unit of phenol hydroxylase was constructed, and the homology modeling of phenol hydroxylase was obtained based on the crystal structure of Pseudomonas sp.OX1 (2INPA). Furthermore, the docking models of phenol hydroxylase and cresols were constructed, which could explain the difference of the binding energy on ligands and individual residues, and the results were in accordance with the Km of enzymatic reactions. Phenol hydroxylase was able to transform the cresols in the following order:p-cresol> m-cresol> o-cresol.Subsequently, the charge distribution of cresols was analyzed by Gaussian and the results showed that the ortho-position was the most active site, and 3-methylcatechol or 4-metnylcatechol was presumed as products. The products of cresols biotransformation were identified by HPLC, LC-MS and 1H-NMR, and the results suggested that 3-methylcatechol and 4-methylcatechol were the products of o-cresol and p-cresol respectively. While both 3-methylcatechol and 4-methylcatechol were found in the product of m-cresol biotransformation.The optimal conditions of cresols biotransformation were also determined using o-cresol as substrate. The optimal cell density of recombinant E. coli (PH_IND) was OD600 2.0. The time course of o-cresol biotransformation revealed that the speed of biotransformation was corresponded with first order kinetics. The RSM was used to optimize the main factors including glucose concentration, pH and o-cresol concentration. The optimal conditions were as following:30 mmol/L of glucose, pH 6.46, and 97.32 mg/L of o-cresol. In this condition, the biotransformation rate of o-cresol by was 87.5%, and the formation rate of 3-methylcatechol was 83.9 mg/L.