假单胞菌HS-D36偏苯三酚1,2-双加氧酶克隆表达与催化特性研究

Expression,purification,and characterization of the recombinant hydroxyquinol-1,2-dioxygenase fromPseudomonas sp.strain.

由pnpC编码的偏苯三酚1,2-双加氧酶(Hydroxyquinol 1,2-dioxygenase,PnpC)是微生物分解硝基芳香族类环境污染物的关键酶.本研究从对硝基苯酚(p-nitrophenol,PNP)降解菌HSD38(Pseudomonas sp.)中克隆pnpC基因,利用E.coli BL21(DE3)高效表达重组PnpC,通过亲和色谱纯化并分析其催化特性.实验结果表明:HS-D36的pnpC开放阅读框长度为873bp,编码290个氨基酸,酶蛋白相对分子量33KD;20℃、异丙基硫代-β-半乳糖苷(Isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导可高效表达重组PnpC;重组酶经Ni-NTA亲和色谱一步分离可达到电泳纯;纯酶比活力9.3U/mg,纯化倍数37.2,活力收率23.8%;重组PnpC催化邻苯二酚开环反应的最适温度45℃、最适pH 5.0;Lineweaver-Burk双倒数作图表明PnpC对邻苯二酚降解的米氏常数(Km)为21.95mol/L、最大反应速度(Vmax)为2.68mol/(min·mg);Fe3+、Cu2+、Fe2+和Zn2+对该酶具激活作用,Ni 2+则显示抑制效应.

Hydroxyquinol 1, 2-dioxygenase （PnpC）, encoded by pnpC gene, is a key enzyme for bacteria to degrade a series of recalcitrant nitrophenol pollutants, such as p-nitrophenol （PNP）. In the present work, a pnpC gene from Pseudomonas sp. strain HS- D36 was cloned which was able to degrade PNP efficiently. The open reading frame of the pnpC contains 873 nucleotides and codes for 290 amino acids （PnpC） with the molecular weight of 33 KD. The recombinant PnpC was highly expressed in E. coli BL21 （DE3） as a histidine-tag fusion protein and purified to homogeneity by one step of NiNTA affinity chromatography. The specific activity of the purified protein reached at 9.3 U/mg. The optimum temperature and pH of the PnpC for catechol were 45℃ and pH 5.0, respectively. Lineweaver-Burk plot of the PnpC activity against catechol indicated that Km was 21.95 mol/L and Vmax was 2.68 mol/（min · mg）. In addition, the enzyme activity was activated by Fe^3＋ , Fe^2＋ , Cu^2＋ , Zn^2＋ , Mn^2＋ , and Co^2＋ , while inhibited by Ni^2＋.