Cloning And Expression Of Organic Solvent Tolerant Lipase Gene From Staphylococcus Saprophyticus M36

Biodiesel is a form of biomass energy with the advantages of environmental friendly, regeneration, and low energy consumption. It can resolve the crisis yielded by the depletion of oil and environmental contamination, so biodiesel become more attractive recently. The synthesize technology of biodiesel by bioconversion method have the advantages of moderation condition, simple procedure, low energy consumption and environmental friendly compared with the traditional catalyze method. However, it is difficult for the development and industrialization of biodiesel produced by bioconversion method due to the high cost of lipase, toxicity effect of organic solvent to lipase and the special catalysis of lipase. This thesis mainly studied the cloning of the organic solvent-tolerant lipase gene lip3 from Staphylococcus saprophyticus M36, and the expression of the lipase in Escherichia coli and Bacillus subtilis. It also studies the site-specific mutagenesis of lip5 gene to get organic solvent-tolerant lipase. Main study results are as following:1. Genomic DNA was extracted form Staphylococcus saprophyticus M36. Then specific primers were designed according to the tentative lipase III sequence and the lip3 with the full length of 741bp was amplified. According to the conserved region sequence of lipase V, the specific primers were designed. With the specific primer and degenerate oligonucleotides, the lip5 of 804bp was amplified and then sequenced by Site finding-PCR. GenBank Accession Numbers of lip3 and lip5 are FJ979867 and FJ979868 respectively.2. Lip3 was fused with the expression vector pET-DsbA, and transformed into Escherichia coli BL21 (DE3), and finally obtained the recombinant pET-DsbA-lip3. It was certified that lip3 was expressed correctly through SDS-PAGE electrophoresis; active stain and chromogenic agar plate. With the induction by 0.4 mmol/L of Isopropyl beta-D-thiogalactopyranoside at pH 8.0, OD600 1.0,25℃for 12 h, the recombinase activity reached up to 25.8 U/mL. The fusion protein ligand of BL21/pET-DsbA-lip3 was removed by Nickel column, and the electrophoretically pure lip3 protein was obstained. The optimal pH of the recombinase is 8.0 and the optimal temperature is 25℃, and it was stable in the presence of methanol, n-hexane, and isooctane, n-heptane. The activity of BL21/pET-lip3 recombinase activity was raised by Mg2+ andβ-mercaptoethanol.3. Lip3 was highly expressed in B. subtilis DB104 using pHT43 as the vector. The optimal pH of pHT43-lip3 is 8.0 and the optimal temperature is 50℃. Nearly 50% of the activity was maintained in the presence of ethanol, isopropanol and acetone after 1 h. The recombinase activity was raised by isoamylol, Na+, K+ and P-mercaptoethanol.4. We constructed the mutant gene lip5D93S, via over-lap extension PCR. Organic solvent-tolerant lipase was produced by expression of the mutant in B. subtilis. It was proved that Asp in the active centre of lip5 was replaced by Ser, which resulted in inactive lip5 recombinase. Lip5D93s was ligated with the expression vector pHT43, and transformed into B. subtilis DB104. The properties of recombinase pHT43-lip5D93S were studied. The optimal pH is 8.0 and the optimal temperature is 40℃. And 50% of activity was retained in the presence of isopropanol, n-hexane, n-heptane and acetone after 1 h.