【作者】 玉崧成；

【导师】 冯雁；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 脂肪酶是具有甘油三酯水解活性的重要工业用酶,发现和表征新型脂肪酶对于促进相关工业发展具有重要意义。从嗜热菌Fervidobacterium nodosum基因组中发现基因Fond₁333编码302个氨基酸,其氨基酸序列含有脂肪酶A-X-S-X-G基序、负氧离子洞保守区域,因此推测基因Fond₁333可能为脂肪酶基因。本文应用PCR技术成功地克隆Fond₁333基因,构建了携带pET-28a+ Fond₁333质粒的E.coli BL21 CodonPlus工程菌,并对重组蛋白质（FnL）的酶学性质进行了较为系统的研究。FnL能水解各种不同碳链长度甘油三酯和对硝基苯酚酯,最适底物分别为三丁酸甘油酯和对硝基苯酚癸酸酯;其最适反应温度为70℃,最适pH值为9.0;具有良好的生物学稳定性。另外,重组FnL的酶活力易受金属离子、抑制剂和表面活性剂等影响。研究表明FnL是一种新型嗜热脂肪酶。更多还原

【Abstract】 Lipases, interfacially activated triacylglycerol hydrolases, have a wide existence in animal, plant and microorganism. They belong to serine hydrolase family with a triad made up of Ser, Asp and His, or Ser, Glu and His to achieve the catalytic activity. It shows a wide specificity and regioselectivity towards a variety of triglycerides. Owing to these characteristics, it is wildly applied in food industry, pharmaceuticals, chemical industry, polluted water curing, petroleum exploiting etc. However, the biological stability of lipases available could not meet the need of industry and limited its development. Therefore, it is a significant and urgent program to explore new promising lipases to cater to the fast developing industry.Up to now, a large quantity of methods has been set up to obtain new genes and proteins, and searching potential target genes in the genomes is a simple and effective one. A huge database has been made with the sequences of a variety of genomes. The interested genes can be predicted after analyzing the sequences in the database and cloned easily by molecular technology.In the present study, gene Fond₁333 in Fervidobacterium nodosum, a thermophilic microorganism, was selected as the objective to clone thermophilic lipase gene. Gene Fond₁333 is made up of 906 nucleotides and encodes 302 amino acids （FnL）. FnL contains the conserved motif A-X-S-X-G, which is common in most of lipases, and thus is predicted as a new lipase. The theoretical mass and isoelectric point of FnL are 34841.35 Da and 5.40, respectively. Conserved domains search at NCBI revealed that FnL had the conserved active domain and the conserved domain of oxyanion hole, which were common in most lipases. The prediction of the secondary structure indicated that FnL was a typicalα/βfolding hydrolase includingα-helix,β-sheet and coil. In addition, there was a longα-helix at the C terminal of FnL, which might make a great contribution to the high thermostability. Compared with various lipases belonging to different families, FnL had a low homology and considered to be likely belonging to lipase family VI. Therefore, FnL is a new member of lipase family VI.The thermophilic lipase gene Fond₁333 has been cloned, and overexpressed in the insoluble form in E. coli CodonPlus. Therefore, we established a simple and quick method to obtain target protein. The collected cells were first mixed with 50 mM PBS （pH 8.0）, ultrasonic cell disintegrated and centrifuged at 5000 rpm for 20 minutes. The deposit part was washed with the 0.2% DOC solution for 20 minutes at room temperature, and then centrifuged at 5000 rpm for 20 minutes again. The deposit part was resuspended with the 0.25% Tween-80 solution at 60℃for 20 minutes, and then centrifuged at 12000 rpm for 20 minutes. The supernatants were collected as the crude FnL solution, and the purity was very high. Further purification step could be performed by HiTrap Q Sepharose chromatography, depending on the research purpose.FnL could catalyze the hydrolysis of triglycerides and p-nitrophenyl esters, and its optimal substrates were tributyrin and pNP-C10, respectively. The optimal reaction temperature and pH value were 70℃and 9.0, respectively. The enzyme was stable at high temperature, and the t1/2 at 60℃was up to 100 hours.Furthermore, FnL possessed good resistance against organic solvents. All the organic reagents employed, including methanol, ethanol, propanol, acetone, DMSO and DMF, did not inhibit the catalytic activity. Surprisingly, the enzyme could be activated to some extent in some of them （Propanol, Acetone, DMSO and DMF）. Therefore, thermophilic lipase FnL was considered to have the potential to be applied in the synthesis of chemicals in organic medium.The effects of pH value, metal ions, surfactants and inhibitors were also investigated in the thesis. FnL kept high catalytic ability at a relative broad pH value, from 8.0 to 10.0, but it was stable in the pH range 8.0-9.0. Metal ions, inhibitors and surfactants had significant effects on the catalytic activity of FnL. Among them, Mg2+, Na+, Ca2+, Co2+ and Triton X-100 could activate FnL. On the other hand, Zn2+, Cu2+, MoO42-, DOC, SDS, PMSF and high concentration of Tween-80 would inhibit its catalytic ability. In a word, proper conditions should be controlled when thermophilic FnL was employed in the catalysis.In this work, FnL was successfully cloned, expressed and characterized. Our research showed that FnL was a new thermophilic lipase of high thermostability and resistance against organic reagents. Therefore, FnL was a potential target to be widely used in the biochemical industry.更多还原