【作者】 贾彬；

【导师】 刘曼西； 闫云君；

【作者基本信息】 华中科技大学 ， 微生物学， 2010， 博士

【摘要】 生物柴油是解决目前石化能源危机的重要途径之一,其中脂肪酶催化法制备生物柴油技术具有反应条件温和、转化效率高、绿色环保无污染等优点,符合国家的新能源发展战略,前景广阔。但昂贵的脂肪酶价格限制了酶法生物柴油制备技术的推广和工业化。本研究筛选到一株产耐热、耐短链醇脂肪酶的洋葱伯克霍尔德菌(Burholderia cepacia) G63,分子生物学鉴定其属于B. cenocepacia。B. cenocepacia G63脂肪酶水解橄榄油的最适pH为9.0,最适温度为60℃,在pH 6.0-10.0以及65℃以下都十分稳定,60℃温育4 h后仍保留80%以上的酶活力。该酶对乙醇、丙醇和异丙醇等多种短链醇的耐受性极佳。这些特性表明B. cenocepacia G63脂肪酶是催化制备生物柴油的理想用酶。摇瓶发酵的G63平均产酶量为12.4 U／mL。论文就提高G63脂肪酶的表达量进行了一系列分子生物学研究,B. cenocepacia G63脂肪酶在重组大肠杆菌中的表达量达到50 mg/g cell-wet weight,在B. cenocepacia同源重组工程菌BC-T7Aliplif中水解橄榄油的酶活最高达32.7 U／mL,在毕赤酵母基因工程菌中水解pNPP酯的酶活最高达184.3 U／mL。上述结果表明本研究所建立的脂肪酶基因改造和表达技术路线是正确的。本论文的主要工作及创新点如下：1.建立了TB-TA平板定向筛选方法。该法筛选洋葱伯克霍尔德菌(Burholderia cepacia)阳性率高达100%,非常适合该菌的规模化筛选。并使用Haelll-recA RFLP (restriction fragment length polymorphism)和亚种特异性PCR的方法将G63鉴定为B. cenocepacia,是首次鉴定到亚种水平的BCC脂肪酶生产菌。2.扩增了G63脂肪酶及其折叠酶的基因并在大肠杆菌中实现了大量表达。根据生物信息学方法对信号肽预测的结果,将脂肪酶基因去除信号肽部分后在pet系统中表达,在胞内表达成包涵体,随后采用超声波破碎、脱氧胆酸钠溶解细胞碎片的简单方法制备出高纯度脂肪酶包涵体。蛋白质含量测定表明脂肪酶的表达量达到50mg/g cell-wet weight。根据蛋白质跨膜区域预测的结果,删除折叠酶N端跨膜的70个氨基酸,成功以pet系统在E. coli中实现折叠酶的可溶性表达,随后利用Ni-NTA金属亲和层析法一步纯化出N端携带6×His标签的折叠酶。3.进行脂肪酶的体外复性研究。证实折叠酶辅助法的复性效率明显优于大量稀释法。在对折叠酶-脂肪酶摩尔比、复性pH和复性时间等因素进行研究后得出,B.cenocepacia脂肪酶体外最佳复性条件为pH7.2,折叠酶-脂肪酶摩尔比1：1,复性时间12 h,复性后脂肪酶比活力最高为473.8 U／mg,复性效果良好。4.将T7蛋白质表达系统导入B. cenocepacia菌并实现了脂肪酶的高效同源表达。该系统以B. cenocepacia G63为宿主菌,通过同源重组的方法把T7RNA聚合酶基因定向插入到B. cenocepacia基因组中脂肪酶启动子的后面,使T7RNA聚合酶的表达受到脂肪酶启动子的调控,然后将受T7启动子调控的脂肪酶基因以质粒形式进行表达,最终实现了脂肪酶的高效表达。整个表达系统可分成T7 RNA聚合酶整合型重组菌和脂肪酶表达载体两部分。T7重组菌的构建通过自杀质粒pJQ200SK介导。先把脂肪酶操纵子前后两段500bp序列分别融合到T7 RNA聚合酶基因两端,再将此杂合基因克隆到pJQ200SK中,然后通过三亲本杂交将构建的自杀质粒导入野生菌。自杀质粒与野生菌基因组在500bp同源区域发生同源重组,从而将T7 RNA聚合酶基因整合于B. cenocepacia基因组中,获得T7重组菌。T7启动子型表达载体共有4种,其中pBBR221ip和pBBR221iplif采用脂肪酶自身信号肽,pBBR22△lip和pBBR22△liplif采用pelB信号肽。实验结果表明pelB分泌信号比脂肪酶原始分泌信号更适合于脂肪酶的表达与分泌。通过电转化将表达质粒转入T7重组菌获得4种脂肪酶工程菌。B. cenocepacia脂肪酶工程菌的这种构建方法系本文首创。5.在毕赤酵母中初步实现了B. cenocepacia脂肪酶的活性分泌表达。在生物信息学分析的基础上,采用重叠PCR技术改造了B. cenocepacia脂肪酶基因结构,获得适于在毕赤酵母中表达的优化基因。使用pGAPZa和pPIC9K分别构建了组成型和诱导型毕赤酵母基因工程菌。发酵结果表明,GAP启动子更适合该脂肪酶的高效表达,脂肪酶水解pNPP酯的最高酶活力达到184.3 U／mL。初步酶学性质研究表明,毕赤酵母表达的脂肪酶与野生型脂肪酶性质基本相同,可以用于规模生产。更多还原

【Abstract】 Biodiesel is one of the most potential ways to solve the current fossil energy crisis. Using lipase as catalyst for biodiesel production has a broad application prospect because it is in line with the country’s new strategy for new energy development due to its mild reaction condition, high efficiency and environmental protection. However, expensive price of lipase reduces competition of biodiesel.In this research, Burkholdria cepacia strain G63, whose lipase is highly thermostable and short-chain alcohol tolerant, was directly screened, and was indentified as B. cenocepacia through molecular biological analysis, the produced lipase was stable in pH ranging from 6.0-10.0 and the optimal pH for lipolytic activity was 9.0. The optimal temperature for the lipase was 60℃, and kept fairly stable below 65℃and retained 80% activity after incubation at 60℃for 4 h. The lipase exhibited highly tolerance to a variety of short-chain alcohols for example alcohol, propanol and isopropanol. The above characteristics made it an ideal enzyme for biodiesel production. The highest lipase activity of the wild strain G63 was 12.4 U/mL in shaking flask. To improve lipase expression level, a series of lipase engineering strains were constructed. The expression level of lipase in E. coli BL21 (DE3) was 50 mg/g cell-wet weight, the highest hydrlysis activity toward olive oil in homologous combinant B. cenocepacia was 32.7 U/mL, and the highest hydrolysis activity of pNPP in heterogeneous combinant Pichia pastoris GS115 was 184.3 U/mL. These results demonstrated that strategies of gene modification and overexpression for lipase of G63 were appropriate.The main work and innovations for the research are listed below.1. Directly screening B. cepacia strain by TB-TA plate. Positive screening rate of this method reached nearly 100%, which was appropriate for large scale screening. G63 was indentified as B. cenocepacia by Haelll-recA RFLP (restriction fragment length polymorphism) and genomovar specific PCR. This was the first lipase-producing B. cepacia strain indentified to genomovar level.2. Genes of lipase and foldase were coloned and overexpressed in E. coli, respectively. Based on the result of signal sequence prediction by informatics, we removed the signal sequence from the lipase gene. Then, the obtained gene without signal sequence was expressed in E. coli pet system. However, lipase was expressed in inclusion body. Lipase inclusion bodies of high purity were prepared by sonication and sodium deoxycholate treatment. The measured lipase expression level in E. coli was 50 mg/g cell-wet weight. Interestingly, foldase, whose N-terminal 70 amino acid residues were cut off according to the prediction result of the protein transmembrane region, was expressed in soluble form in the E. coli pet system. The foldase was purified by Ni-NTA chromatography and used for later lipase refolding.3. Lipase refolding in vitro was detailedly examined. The refolding results confirmed that the efficiency of foldase-assisted refolding was much better than that of the dilution refolding. After detailed studies on folase-lipase molar ratio, refolding pH and refolding time, the optimal refolding conditions were pH7.2, foldase-lipase molar ratio 1:1 and refolding time 12 h, under which conditions, the highest specific activity of the refolded lipase was up to 473.8 U/mg.4. T7 protein expression system was for the first time introduced into B. cenocepacia, by which the lipase was homologously overexpressed. Using B. cenocepacia G63 as a host, T7 RNA polymerase was fistly integrated into its genome and was controlled by lipase promoter. Then, lipase gene controlled by T7 promoter was electroporated into T7 RNA polymerase containing strain. Consequently, the lipase was over-expressed. The whole lipase expression system was composed of T7 recombination strain and expression vetors. T7 recombination strain was constructed by suicide vetor pJQ200SK. Regions of 500 bp before and after lipase operon were coloned and then flanked T7 RNA polymerase gene. T7 RNA polymerase with 500 bp flanking region was inserted into pJQ200SK. Through three parental mating, the modified suicide vector was introduced into G63. After homologous recombination between genome and suicide vector, T7 RNA polymerase gene was integrated into B. cenocepacia genome and resulted in T7 recombination strain. On the other hand, lipase expression vectors with T7 promoter were constructed into four types:Vectors pBBR221ip, pBBR221iplif with lipase native signal sequence, and pBBR22△lip, pBBR22△liplif with pelB signal sequence. The above vectors were respectively electroporated into T7 recombination strains and obtained four lipase expression strains. This is the first report by employing T7 protein expression system to construct a homologous combinant B. cenocepacia lipase strain.5. Active expression of B. cenocepacia lipase in Pichia was preliminarily achieved. Based on bioinformatics analysis, we modified and optimized B. cenocepacia lipase gene by overlap PCR to be appropriately expressed in P. pastoris GS115. Using vectors pGAPZa and pPIC9K, constitutive expression and inducible expression GS115 strains were constructed. After fermentation, the highest hydrolysis activity of pNPP was 184.3 U/mL,129.5 U/mL, respectively, which indicatd that GAP promoter was more appropriate for B. cenocepacia lipase expression in Pichia than AOX1 promoter. Enzymatic properties studies showed recombinant lipase expressed in GS115 was identical to the wild type and can also satisfy the needs of industrial application.更多还原