摘要
Phospholipase A1(PLA_(1))is a member of the hydrolase family with applications in various fields,especially in the food industry.A calcium-independent PLA_(1) from Streptomyces albidoflavus was expressed in E.coli BL21(DE3)in this study.The results indicated that the soluble expression of PLA_(1) was at low level,which was possibly due to the toxicity of PLA_(1) to the host.In contrast,the expression of the enzyme as inclusion bodies exhibited a high-level expression and 0.3 mg inclusion bodies protein could be derived from 1 mL culture medium.Furthermore,the renaturation of PLA_(1) was achieved through a direct dilution method,yielding 29.6 U/mL PLA_(1) activity after 16 h of renaturation at 4℃.For improving the efficiency of the dilution refolding process,a continuous refolding strategy was established,and 155 U/mL PLA_(1) activity was derived from the continuous refolding process.With soybean lecithin as the substrate,the specific activity of purified renatured PLA_(1) was 1380 U/mg and the optimal temperature and pH was found to be 60℃ and 6.5.In addition,the renatured PLA_(1) was observed with better activity towards phosphatidyl inositol,whilst lipase activity was detected when the catalyzing temperature was below 55℃.Overall,this study provides a possible solution to obtain calcium-independent PLA_(1) with high yield by heterologous expression in E.coli and hence to promote its further application in the field of food industry.
基金
supported by the National Key Research&Developmental Program of China(2021YFC2100303,2018YFA0900300).
