Expression And Activity Study Of Alkaline Lipase Gene From Penicillium Expansum

Lipase (EC 3.1.1.3.Triacylglycerol hydrolase) is a kind of carboxyl group esterase that catalyzes hydrolysing long chain acylglycerol. The lipase has biological catalysis characteristic in both the aqueous phase medium and the non-aqueous phase medium,moreover the enzyme respond has the mild condition, the low energy consumption, the few poisonous coproduct and so on. The alkaline lipase produced from Penicillium expansum is the only enzyme strain industrialized at present in our country.It has already been widely applied in the flour additive, detergent additive, resolution of chiral medicine, leather and fur degreasing, scales of fish degreasing and so on.At present the method of lipase production mainly has the extractio and the fermentation method by microorganism,concerning the economical and the craft, the fermentation method by microorganism is convenient. Because the microorganism resources are rich,and it not influenced by the natural environment, and massive accumulation of target enzyme by manual control is available. The cycle is short, the production cost is low. Therefore the fermentation method is the economy practical method of industrialized production for lipase.Because the Penicillium expansum is bacterium which has the high production of lipase through multigeneration mutagenesis, but it also has imperfection, for example, the condition is unstable, easy to become original condition to change suddenly, the fermentation condition is difficultly to control and so on. The development of recombinate technology of DNA enables us to construct the highly effective expressing genetic engineering strain. As the improvement of vectors as well as the development of technology ,methyl nutrition Pichia pastoris expression system has become the the favorite one for laboratory research and the production of the commercial recombination protein. It is as easy as Saccharomyces ceresiviae to operate,it has a powerful promoter AOX1 which is induced by methanol,so it can express foreign protein effectively. The fermentation level is very high; the secretary ability is strong, the product is easy to be epurated;the expression strain is stable; translation and posttranslational processing par example the N-acetylate glycosylation is more similar to eukaryote than Saccharomyces ceresiviae, the chain of glucose has 8-14 mannose residue, which is lessn than Saccharomyces ceresiviae.In this article alkaline lipase gene cDNA fragment was cloned by RT-PCR method using Biospin RNA Simply P extrac kit total RNA extracted total RNA from Penicillium expansum. Cloned this gene into the prokaryotic expression vector pET-DsbA and pkk223-3, and transformed it into E.coli BL21(DE3) and JM105, the expressed product has no activity after IPTG induction.The gene was cloned into eukaryotic expression vector pPIC9, electrotransformed into His4 mutant Pichia pastoris host strains GS115, through MD-MM plate and PCR method identification. The expression products of PEL gene was analysis by SDS-PAGE, molecular weight of the expressed protein was estimated to be 28ku. With the method of using olive oil plate and the titration method by NaOH the activity of lipase was up to 225u/mL. The result indicated that PEL gene was functionally overexpressed in Pichia pastor.The possible reason is that expressing eucaryotic gene in E.coli was influenced by powerful promoter of the vector , transcription termination sequence, the distance from the SD sequence to the start signal ATG, the preference of codon, the glycosylation and so on. When it was expressed in Pichia pastoris the expressed protein was directly secreted to supernatant, occupied 30% of all supernatant protein, moreover expresse protein has lipase activity, the activity reached as high as 225u/mL.Our country have a ability to consume many abstergent, adding the enzyme and non-phosphorus detergent conforms to the current trend, we may reduce the cost greatly using the genetic engineering to produce alkalinity lipase, the market prospect is broad.To study and research the enzymology nature of lipase the consequence indicate that the recombinate lipase PEL-PIC9 is stable in pH7~11 scopes, optimization pH was 9, it belongs to the alkalinity lipase which may serve as the detergent enzyme, the lipase is stable below 40℃, most suitable reactive temperature was 25~30℃, K+, Mg2+ is the promoter ,Ag+,Gu2+ and Fe2+ are the enzyme inhibitor, Triton X-100 is the lipase promoter , SDS is the enzyme inhibitor.In this experiment using PCR method screening recon which is easy and feasible.The study is the preliminary research to the enzymology nature of lipase. In order to elevate the activity of lipase we could optimize the fermentation time, the fermentation temperature, the pH value, the culture medium ingredient, methanol induction density, induction time and so on. Thats would conduce to the large scale industrial production of the lipase, and have the condition to further studied its structure and function .