Overexpression of Rhizomucor miehei Lipase and Activity Assay

Abstract:

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:1) 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.