Functional Characterization Of A Phenol Hydroxylase Gene In Corynebacterium Glutamicum

Aromatic compounds constitute one of the largest groups of environmental pollutants.They will enrich in higher organisms and human body, which could make a great damage topeople when they reached a certain concentration. However, aromatic compounds are hardlydegraded by physical and chemical methods due to their structural stability. A number ofmicroorganisms have the ability of utilizing various aromatic compounds as their sole carbonand energy source, providing intensive pathways to degrade aromatic compounds—microorganism degradation. Corynebacterium glutamicum RES167is a phenol-degradingbacterium expressing high phenol-oxygenating activity. Ncgl2588gene is the putative genethat encodes phenol hydroxylase in Corynebacterium glutamicum RES167. In this study, wecloned the gene encoded phenol hydroxylase from Corynebacterium glutamicum RES167andoverexpressed in E. coli to confirm its function. Moreover, we constructed deletion mutantsand detected the difference of growth curve. In enzymatic property, we detected variousproperties in detail. Some site-directed mutations were constructed to identify the key aminoacids in enzyme activity. Details were shown as below:1. Recombinant plasmid pk18mobsacB-Δ2588through overlapping PCR wasconstructed successfully to knockout the ncgl2588gene in Corynebacterium glutamicumRES167, and the deletion mutant was constructed by homologous recombination. When thebacteria strains were cultured by mudium that use phenol as the sole carbon and energy source,the deletion mutant lacking ncgl2588gene could not grow; In contrast, the wild-type straincould utilize phenol. This result indacted that phenol hydroxylase in Corynebacteriumglutamicum RES167is encoded by ncgl2588gene.2. We constructed the recombinant plasmid of pET28a-ncgl2588successfully, BL21(DE3) cells containing a plasmid with ncgl2588gene were expressed successfully. Thenprotein was purified by His-tag resin. SDS-PAGE analysis of the purified phenol hydroxylaseprotein revealed the presence of a single polypeptide chain corresponding to a molecular massof73KDa and this value is in good agreement with the molecular mass predicted from genesequence analysis. The specific activity of highly purified phenol hydroxylase determined was 0.3mmol·min-1·mg-1when assayed at room temperature (about22°C) and pH7.4.3. The enzymatic properties of phenol hydroxylase from Corynebacterium glutamicumwas detected in detail:3.1The pH and temperature optima of the phenol hydroxylases activity were pH8.0and45°C; Km values for the two substrates of phenol hydroxylase were determined byLevenberg-Marquardt algorithm: Km(Phenol)=256μM, Km(NADPH)=284μM, respectively. Thephenol hydroxylase in Corynebacterium glutamicum RES167has very high substratespecificity to phenol, other substrates are only attacked at10-30percent except hydroquinoneand m-dihydroxybenzene remaining fifty percent activity to phenol.3.2The enzyme was very sensitive to low temperature, catalyze activity will decrease2-fold and5-fold at-80°C and-20°C for24h，respectively. But it can store few weeks in4°C with no activity loss.3.3Metal ions Fe2+,Fe3+,Zn2+and Ni2+could severely inhibit the activity of phenolhydroxylase while Mn2+and Mg2+slightly increase the activity. The activity will decreaseapproximately50percent with Co2+or Cu2+.3.4Some site-directed mutations including P241S, P261S, R262S, C349S and C476Swere constructed to identify the key amino acids of phenol hydroxylase activity. As expected,mutation of C349and C476residues led to a great loss of enzymatic activity towards phenol,the activity maintained are30percent and45percent respectively, while enzymatic activityincreased sharply of mutation P241S and R262S, about2-fold and4-fold. Mutation P261Smaintained15percent activity comparing with wild-type enzyme.