对硝基酚磷酸酶的原核表达、纯化及性质研究

Expression,Purification and Characterization of p-Nitrophenylphosphatase

克隆到脂肪芽孢杆菌Bacillusstearothermophilus中的对硝基酚磷酸酶(p nitrophenylphosphatase,pNPPase)基因,将其编码区cDNA连接到pQE30表达载体中,在E.coliM15中经IPTG诱导得到高效表达.用Ni NTASuperflow层析柱对表达产物进行了分离纯化,初步测定了pNPPase的酶学性质.发现它的反应最适pH是10.0,最适反应温度是55℃,热稳定性Tm值为52℃,Mg2+、Mn2+和Co2+对pNPPase的活性有一定的促进作用,而Zn2+和Ni2+对pNPPase没有影响.纯化后其比活为120U/mg.

The pnitrophenylphosphatase(pNPPase) gene was cloned from Bacillus stearothermophilus. The code region of pNPPase cDNA was cloned into pQE30 vector and expressed in E.coli M15 with the induction of IPTG. After purified with NiNTA Superflow chromatography,the property of pNPPase was studied. The optimum pH value of pNPPase was 10.0. The optimum reaction temperature and Tm value were 55 ℃ and 52 ℃, respectively. The pNPPase could be stimulated by Mg2+、Mn2+and Co2+, but influenced less by Zn2+and Ni2+. The relative activity of the purified pNPPase was 120 U/mg protein.