腐生葡萄球菌脂肪酶3制备及酶活性研究

Study on Purification and Enzymic Activity of Lipase 3 from Staph. saprophyticus M36

克隆得到腐生葡萄球菌M36的脂肪酶3编码基因,构建了Pet28-Ss-Lip3表达质粒,在大肠菌株中获得腐生葡萄球菌脂肪酶3的高效上清表达,应用Ni^(2+)-NTA亲和层析柱获得了纯度约90%的重组脂肪酶,分子筛检测发现其在溶液中主要以单体形式存在。酶活性分析纯化后的脂肪酶最适宜的反应pH值为7.0,温度为40℃。结果表明,腐生葡萄球菌脂肪酶3的碱性和温度耐受性较好,有望通过定点突变获得更好适应性的脂肪酶。

The lipase gene from Staph. saprophyticus M36 was cloned into pet28 a vector. The recombinant lipase 3 was expressed in the supernatant of the E coli lysate. The lipase was purified by Ni（2＋）-NTA affinity column with about 90% purity. The size exclusion chromatography experiment confirmed that Lipase 3 existed as monomer in solution. The optimum p H and temperature of the recombinant lipase were 7. 0 and 40 ℃,respectively. Collectively,the recombinant lipase 3 had excellent tolerance for alkaline buffer and temperature,implying it was a good candidate for directed evolution to solution to lipase for commercial purposes.