Optimal Expression Of Recombinant Lipase

Enzyme often show a high degree of selectivity, substrate specificity and regional selectivity, in the synthesis catalyzed chemical reactions. Lipase is a class of enzymes with various catalytic activities that can be catalytic triacylglycerols and other water-insoluble ester hydrolysis, alcoholysis, esterification reverse synthesis reaction. Their different activities to play depend on what the reaction system at. Lipase is mainly used for food, fine chemical synthesis, and pharmaceutical industries. People found that the expression of lipase is a big problem, especially in the E. coli system; the expression protein often formed inactive inclusion body or the active form of the precipitation of aggregates. It is necessary to refold or renaturedthe inclusion body or aggregates to obtain the active enzyme. Because lipases are so usefull, to get a high level in the supernatant of E. coli expression of lipase has been a staff scientist and biochemical goal.In this paper, we expressed three kinds of lipase gene (LipA, PFL and RDL) with ubiquitin fusion gene and the pHUE. The results showed that the expression level of RDL and the supernatant LipA has been improved. The expressed conditions, optimal temperature, induction time, IPTG induction concentration of OD values has been studied. For RDL and LipA, the purpose of supernatant protein was about 1.5 times and 1.2 times higher. Specific study as followed:1. RDL-pHUE and PFL-pHUE vector and expression optimizationThe vector PFL-pET28b and RDL-pET28b was constructed by our lab previously. In this experiment, they were used as templates. With PCR reaction, we successfully amplified two lipase genes, inset into pHUE vector, and transfected to E.coli BL21 to express the protein. The electrophoresis result showed that PFL was expressed in the deposition. The expression level of RDLwith pHUE in the supernatant increased respectively, and then the expression conditions were optimized. The final induced conditions: The media is 2YT medium; the induced temperature is 30℃; The OD600 value is over 1; the final concentration of IPTG is 0.5mM; The induced time is for 8 hours.2. RDL-pHUE and PFL-pHUE and LipA co-transfected molecular chaperone After RDL-pHUE co-transfect five chaperones pGro7, pkJE7, pG-Tf2, pG-KJE8 and pTf16, we found that pG-KJE8 can help RDL to express in active form, the expression level of RDL in the supernatant was increased, which was 1.5 times as the corresponding without molecular chaperone.When PFL-pHUE co-transfect with chaperones, we found the expressed form of PFL showed no change, which means these molecular chaperones are no use for PFL.LipA co-transfected chaperones helps the folding of LipA, in the presence of pG-KJE8 molecular chaperone, LipA was expressed in the supernatant, which was about 1.2 times as the expression without molecular chaperone.