Study on soluble expression and characteristics of recombinant lipase from Serratia marcescens ECU1010.

Objective To enhance the soluble expression of recombinant lipase from Serratia marcescens ECU1010(GST-lipase) and do some research on its properties.Methods A lipase gene from Serratia marcescens ECU1010 was amplified by PCR and cloned into expression vector PGEX-4T-1,then the recombinant plasmid was transformed to E.coli BL21(DE3).The soluble expression of recombinant lipase was improved by optimizing culture conditions.The expressed protein was purified by Glutathione Sepharose Fast Flow Chromatography.The substrate specificity and effects of temperature and pH on the enzyme activity were studied.The kinetic resolution of(±)-MPGM was studied by means of HPLC.Results Through the optimization of culture conditions,the lipase activity was up to 8612.3 U/L.The specific activity was 3.3 U/mg protein after the purification and the purification factor was 9.8-fold.This recombinant lipase displayed stable below 30 ℃ and in the pH range 6.0~6.8,with the optimal temperature and pH value being 30 ℃ and 9.0,respectively.Its activity was found to increase in the presence of metal ions such as Ca2+,Fe2+and some nonionic surfactants.In addition,the recombinant lipase showed the maximum activity on p-nitrophenyl laurate(C12).With(±)-MPGM as the substrate,a high conversion,corresponding to conversion yield of 47.3 %,was achieved after 4 h,along with an ee value of 89.7 %.Conclusion The activity of soluble lipase can be greatly improved after optimization.The study of the related properties of the recombinant lipase lays a foundation for the industrial production of diltiazem.