The Study Of Directed Evolution Of Proteus Vulgaris T6 Lipase

Biodiesel is a form of bioenergy with the advantages of environmental friendly, regeneration, low energy consumption. It can resolve the crisis brought by the depletion of oil and environmental contamination, so 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. However, it is difficult for the development and industrialization of biodiesel produce 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 screening of strain which could produce lipase, cloning and expression of the lipase gene, directional evolution, abstracting of lipase in large scale and producing of biodiesel by lipase in order to acquire the lipase fitted for the producing of biodiesel. Main study results as following:1 109 strains which could produce lipase were obtained from the sample of soil, 57 strains from the endogenesis bacterium of rape, 58 from ocean microbiology with the screening method of Tween 80 plate and rhodamineB plate. A small bank of the lipase producing strains was established. 37 strains with high yield of lipase were obtained by shake flask fermentation. The hydrolysis of tributyrin was tracked by TLC and a strain named T6 which can produce lipase with non special catalysis was obtained. It is identified as Proteus vulgaris by the identification method of configuration, physiology, biochemistry, molecular biology. A strain named M36 which could produce lipase with the enduring of organic solvent was obtained using benzene and toluol as the only carbon source. It was identified as Staphylococcus saprophyticus by the identification method of configuration, physiology, biochemistry, molecular biology. The remained activity of lipase produced by M36 was 32% at l/4(v/v) methanol and 36% at 1/4(v/v) ethanol respectively after 200rpm, 30min at 37℃.2 The full-length cDNA was obtained by PCR amplification with primers desigend according to the Proteus Vulgaris lipase gene (U3348). Blast searches of the NCBI database for the cDNA sequence with U33485 revealed three base mutation: 55 A-G, 87 T-G, and 285 C-T. The reconmbinant plasmid pLLP-STII—PL1 containing ST- II and His₆ tag was transformed into E. coli stain TOP 10F'. After induced with 100μg/ml IPTG, it showed that His6-T6 lipase was produced in cytoplasm. His6-T6 lipase was further purified with Ni—NTA His Tag Kit, and the purification ratio was 83.79%, special activity was 7011.23U/mg. The optimal pH of His₆-T6 lipase was 9.0 and it was stable at pH 7.0—11.0 with 82% residual activity; The optimal temperature was 50℃and it could keep the 99% residual activity when t≤40℃. It could't endure the organic solvent with special catalyzation.3 The directional evolvement of lipase produce Proteus Vulgaris T6 were carried by Error-PCR and DNA shuffling. The mutation EF 3.3 with special enzyme of 47600U/mg was obtained after one circle Error-PCR and three circle DNA shuffling. The enzyme activity was increased about 5.8 fold. There were three mutation of amino acid in EF3.3, Val become He at 102, Gly become Ser at 197, Arg become His at 229. It was founded that the amino acid residue of 102 and 197 were located at the combine part of substrate by predicting the three-dimensional structure of protein. The mutation of amino acid increased the combining capability between enzyme and substrate and thereby increased the lipase activity. The hydrogen bond length between Ser 79 and He 102 which mutated from Val was increased from 2.92 A to 2.93 A. As the increase of bond length, the effect of bond decreased. It was more easily for Ser anion to attack the substrate and completed the acylation and increase the catalyze efficiency. The mutation from Arg to His at 229 may decrease the effect of "cover" formed by cc-helix and is good for the open of cover. The effect of interface activation was more easily occurred and thereby the enzyme activity was increased. The express plasmid of EF3.3 was still stable after 50 generations. The optimal conditions of induce express of mutant EF3.3 is 28℃, 150rpm, 100μg/ml IPTG for 1.5h. The molecular mass of EF3.3 is 31.5 KDa. The enzyme activity was increased at pH 5-8 compared with wild lipase and there were no deviation at temperature, organic solvent and non special character.4 Influence of PEG on the distribution of lipase EF3.3 and background protein on an aqueous two-phase system was studied. Results showed that PEG2000 had good purification effect of lipase. After influence of phase concentration, pH and KC1 on the distribution of lipase in PEG2000/phosphate aqueous two-phase system were studied, the optimal PEG systems was is the 17.5%PEG/12.5%phosphate, pH7.0, 1.5% KCl. IN this system, the distribution coefficient, the purification coefficient and the enzyme returns-ratio are 6.5, 5.8, 89% respectively.5 Transestenfication of rapseeds oil catalized by liapse EF3.3 for biodiesel production was studied. Result indicated the addition of methanol, addition way , lipase dosages , the reaction temperature , organic solvent, rotation rate and the addition of silica gel had obvious effect to productivity. The optimal reaction condition was: 40℃,125 rpm , 7.5% enzyme base on oil weight, methanol put in with three batches, methanol added every time is equal to rapeseed oil molar ratio 1:1,0.1 Mol hexane, 1.0 g silica gel, reaction time 36 h, methyl ester yield was 80%.