谷氨酸棒杆菌中苯酚羟化酶的鉴定及功能研究
Characterization and functional identification of phenol hydroxylase gene in Corynebacterium glutamicum

【目的】鉴定谷氨酸棒状杆菌中ncgl 2588基因编码的苯酚羟化酶，并验证其在苯酚降解中的作用。【方法】构建谷氨酸棒状杆菌ncgl 2588基因缺失突变株，比较突变株和野生型菌株利用苯酚能力的差异；同时将ncgl 2588基因克隆到表达质粒pET28a中并转化入大肠杆菌BL21(DE3)，利用IPTG诱导重组菌株原核表达，纯化并测定重组蛋白对苯酚的羟化作用。【结果】成功构建了谷氨酸棒状杆菌ncgl 2588基因缺失突变株，其在以苯酚为惟一碳源和能源的培养基中不能生长，而野生型谷氨酸棒状杆菌能正常生长；pET28a-ncgl 2588重组质粒在大肠杆菌BL21(DE3)中成功表达分子质量为73 ku的目的蛋白，此蛋白酶活为0.37 mmol/(min·mg)；在以苯酚为惟一碳源和能源培养谷氨酸棒状杆菌时，苯酚羟化酶得到最高表达，用其他芳香族化合物培养则苯酚羟化酶表达较少；进化树分析发现，谷氨酸棒状杆菌中的苯酚羟化酶与酵母Trichosporon cutaneum中的苯酚羟化酶序列同源性高达43%，远远高于与细菌中苯酚羟化酶的同源性。【结论】谷氨酸棒状杆菌中的ncgl 2588基因编码苯酚羟化酶，且该酶为可诱导酶，在苯酚降解中发挥着重要作用。
【Objective】This study aimed to identify the functional and biochemical characterizations of putative phenol hydroxylase encoded by ncgl 2588 in Corynebacterium glutamicum RES167.【Method】The ncgl 2588deleted mutant of C.glutamicum RES167 was constructed to determine the difference in phenol degradation compared to wild-type.The recombinant phenol hydroxylase was expressed in E.coli BL21(DE3) by IPTG and purified to determine the activity of catalyzing phenol hydroxylation.【Result】The obtained ncgl 2588 gene deletion mutant of C.glutamicum RES167 could not grow in the medium that used phenol as sole carbon and energy source,while the wild type strain grew very well.The pET28a-ncgl 2588 plasmid expressed was in E.coli BL21(DE3) as a single band with molecular mass of 73 ku and specific activity to phenol of 0.37 mmol/(min·mg).Phenol hydroxylase reached highest expression when cultured in phenol while it was in low level when cultured in other aromatic compounds.The phenol hydroxylase in C.glutamicum RES167 shared 43% phylogenetic identity with yeast Trichosporon cutaneum,much higher than the identity with bacteria.【Conclusion】This study confirmed that the ncgl 2588 gene encoded phenol hydroxylase in C.glutamicum RES167 as an induced enzyme.It could play an important role in phenol degradation