The Study Of Multistep Conversion Of Para-Substituted Phenols

Phenol compounds are common organic pollutants in the water, which cause great threats to human health and the environment. Cresols are the majority compounds in phenolic wastewater. The presence of substituted groups in phenols (i.e. in nitro and bromo-phenols) increases the toxic effects exerted on the environmental life and on the human health and their characteristics of persistence in the environment. Generally, biological processes is used to treat large amount of phenolic wastewater with low concentration. However, the intermediate products in the biodegradation process of phenols, like catechols and semialdehydes, are valuable compounds in organic synthesis. The current studies investigated the multi-step conversion of p-cresol,4-nitrophenol and4-bromophenol by recombinant Escherichia coli expressing phenol hydroxylase and2,3-dihydroxybiphenyl1,2-dioxygenase.The metabolic pathway of para-substituted phenols biotransformation by PH_IND-BphCLA-4was investigated using molecular docking, charge distribution analysis and LC-MS. The binding modes of phenol hydroxylase with para-substituted phenols and2,3-dihydroxybiphenyl1,2-dioxygenase with para-substituted catechols were constructed, which indicated that the enzymes and their substrates bound well, and it was possible for the hydroxylation and ring cleavage. After analyzing the charge distribution, it could be concluded that the ortho-position of hydroxyl of para-substituted phenols and the meto-position of dihydroxyl of para-substituted catechols were the most easily to be attacked. The hypothetical products of multi-step conversion were further confirmed by the LC-MS analysis. Finally, the metabolic pathway was identified as follows:phenol hydroxylase transformed para-substituted phenols to para-substituted catechols, which could be further converted to semialdehydes by2,3-dihydroxybiphenyl1,2-dioxygenase.The biotransformation conditions, i.e. stain concentration and substrate concentration, were also optimized. The optimal stain concentration of multi-step conversion was OD6oo=2.5, while the optimal substrate concentrations were1mmol/L p-cresol,0.5mmol/L4-nitrophenol and0.25mmol/L4-bromophenol, respectively. Under the optimized conditions, the biotransformation by PH_IND could be completely done whin60min, while BphCLA-4within10min. It also indicated that both strains exhibited high conversion capability.The kinetic assays for strains PH_IND and BphCLA-4with para-substituted phenols and para-substituted catechols were determined by the whole cell assays. For the first step conversion, strain PH_IND exhibited the highest formation rate (Vmax=0.31μM/min/mg of dry weight) for p-cresol, which was1.4and2.4-fold higher than the formation of4-nitrophenol and4-bromophenol, respectively. The Ks values of para-substituted phenols transformation by IH_IND demonstrated that the affinity between strain PH_IND and substrate was in the following order:4-bromophenol>4-nitrophenol>4-methylphenol. For the second step conversion by strain BphCLA-4, the highest formation rate for5-nitro-HODA (Vmax=1.01μM/min/mg of dry weight) was1.6and2.1-fold higher than the formation rate of5-bromo-HODA and5-methyl-HODA, respectively. The Ks values of para-substituted catechols transformation by BphCCLA-4demonstrated that the affinity between strain BphCLA-4and substrate was in the following order:4-methylcatechol>4-bromocatechol>4-nitrocatechol.