米黑霉脂肪酶基因的高效表达及活性测定

Overexpression of Rhizomucor miehei Lipase and Activity Assay

为了实现米黑霉脂肪酶（Rhizomucor miehei Lipase,RML）基因在大肠杆菌中的高效表达,并得到大量的具有生物活性的脂肪酶,首先通过3种方式提高RML在大肠杆菌中可溶性蛋白的表达量：1）低温诱导（16℃）RML表达;2）新型分子伴侣（Skp）与RML N端融合表达;3）载体pET32a上的Trx.tag与RML N端融合表达。其次对各蛋白质进行纯化及活性测定,各种条件得到的RML比活在（226±10~247±10）U/mg。蛋白质表达结果显示：经低温诱导RML的效果最好,纯蛋白质量浓度为0.86 mg/mL,表明诱导温度是影响可溶性蛋白质表达量的关键因素;活性测定结果表明,Skp和Trx.tag与目的基因N端的融合没有影响RML活性中心（C末端）对底物的结合和催化能力。因此,RML不仅在大肠杆菌中得到高效表达,并且保持原有生物学活性,在工业生产中有应用价值。

In order to overexpress the Rhizomucor miehei Lipase （RML） gene and obtain a large number of bioactive proteins in Escherichia coli. Firstly,the research improved the amount of the soluble protein through three methods：l ） RML was introduced at 16℃. 2） RML was fused with a new chaperone （Skp） which was located at the N terminal of RML;3） RML was fused with the Trx. tag in pET32a and the tag was located at the N terminal of RML. Secondly,the specific activity of purified RML derived from different methods was detected as （226＋10-247＋10） U/mg. The expression result showed that the low-temperature inducing was best method and the concentration of RML was 0.86 mg/mL. It was proposed that the temperature was the key factor of controling the amount of soluble protein. The activity test showed that fusion of Skp/Trx.tag and the N terminal of RML did not change the activity center （C terminal） and the catalytic ability of RML Therefore,the strategy for overexpression of RML in E.coli kept the biologycal activity and was valuble in the practice.