| In this paper, the 828bp ORF 154 gene and 825bp ORF 454 gene, which was presumed as bacterial lipase genes, were first amplified by PCR from Saccharopolyspora spinosa S08-4. The termination codon TGA of the two genes was mutated to CTC, then cloned into pET30a(+) through NdeⅠand XhoⅠdigest for construct recombinant expression vectors and were transformed into Escherichia coli strain BL21(DE3). Yielded a high expression of soluble protein induced with the IPTG. Each of the protein had a His6-tag on its carboxyl terminal and apparent molecular weigh were 32 kDa and 33 kDa, respectively. The expressed protein was purified by Co2+ affinity chromatography and Vivaspin. Two of the proteins were storaged in TNG Buffer. According to calculation, per mg of the expression engineered bacteria cell (wet weight) can received Iip154 protein 18.8μg and lip454 protein 1μg. Taking p-Nitrophenyl palmitate (pNPP) as substrate. Under the condition of 60癈, pH8.0, the specific activities of Iip154 and Iip454 are 0.65 IU/mg and 0.05IU/mg, respectively.According to the DNA sequences and the amino acid sequences of lipase gene ORF 154 and ORF454, BLAST was carried out in NCBI, finding regions of similarity among published biological sequences. It had found a high similarity between the published lipase gene sequences and protein sequences; especially ORF 154 had a 88% of coverage rate and a 75% identity with the lipase gene from Saccharopolyspora erythraea NRRL 2338. The amino acid sequence of the two lipase analysis revealed the presence of the conserved pentapeptide G-X1-S-X2-G essential for lipase activity, It demonstrated that the two lipase belonged to FamilyⅠ. Moreover, it had predicted a typical region of lid sequence, I-V-A-F-R-G, for lip 154.The enzymatic properties of lipase lip154 and lip454 had been investigated. It was proven that the optimum reaction temperature of lip 154 was 100℃, and its optimum pH was 9.0, being a ultra-thermophilic lipase, with 24% residual activity after 40min under 120℃, which could tolerate acetonitrile and concentration below 25% (v/v) of isopropanol, with Hg2+ (1mM) and DTT increasing activity, and Co2+ strongly inhibiting enzymatic activity; its optimal substrate was the p-Nitrophenyl octanoate (C8). While the optimum reaction temperature of lip454 was 70℃, and its optimum pH was 10.0, being thermophilic alkaline lipase, with a much stronger tolerance of organic solvent than lip 154, with 1mM Cu2+ and 10mM of Li+ increasing activity, and Co2+ strongly inhibiting enzymatic activity; its optimal substrate was p-Nitrophenyl myristate (C14).Lip154 and lip454 had special features in enzymology, which indicated enzyme had particular industrial application. We had obtained biodiesel by putting lip454 in organic solvent system, under the condition of 2:1 molar ratio, and adding methanol in batches. with five Fatty acid methyl esters types analyzed by Gas Chromatography Analysis. The conversion rate was about 30%.Lipase is a key molecule of glyceride lipase derived by hydrolysis, with the advantages of ultra-thermophilic lipase, alkaline heat, alkali and other substances from the inhibition and so on, which makes it a more practical application widely, reflecting the huge potential of development and application, being an important study at home and in abroad. The lipase, which is not only thermotolerant but also anti-alkali, is unusual. This thesis has established a theoretical foundation for future study. |
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