重组兽疫链球菌透明质酸酶在大肠杆菌中的表达及其酶活力

Expression of Recombinant Hyaluronidase from Streptococcus zooepidemicus in Escherichia coli and Its Enzyme Activity

以兽疫链球菌(Streptococcus zooepidemicus MF002)基因组为模板,利用PCR方法扩增透明质酸酶编码基因(hyl),将其插入温敏型质粒p BLMVL2,并导入大肠杆菌(Escherichia coli BL21),获得高效表达重组透明质酸酶的基因工程菌MF005。升温诱导试验和SDS-PAGE结果表明,重组蛋白的相对分子量约9.7×104,最适诱导表达温度为41.5℃。经过包涵体复性等步骤获得的粗酶液具有透明质酸水解活性,比活力为2.3×106 u/mg,最适反应温度为37℃,最适反应p H值为5.5。

ABSTRACT： A hyaluronidase encoding gene （hyl） was amplified by PCR method using Streptococcus zooepidemicus MF002 genome as the template. The amplified fragment was inserted into a temperature-inducible expression vector named pBLMVL2. The recombinant plasmid was transformed into Escherichia coli BL21. Then, the recombinant hyaluronidase, which had a relative molecular mass of 9.7× 10^4, was successfully expressed in recombinant E. coli named MF005. The results of temperature induction experiment and SDS-PAGE showed that the optimum temperature for hyaluronidase expression was 41.5 ℃. The crude hyaluronidase solution showed hydrolytic activity to hyaluronic acid after renaturation of inclusion body. The specific activity of crude hyaluronidase solution reached 2.3×10^6 u/mg. The optimum enzymatic activity was achieved at 37 ℃ and pH 5.5.