脂肪酶-P450脂肪酸脱羧酶融合蛋白催化的α烯烃合成的研究

发布时间：2018-03-16 08:41

  本文选题：脂肪酶P450脂肪酸脱羧酶　切入点：融合蛋白　出处：《青岛科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：化石燃料资源的大量消耗和不可再生产生了很多问题,诸如油价高企、能源紧缺、环境污染等。而在可持续发展和节能减排的政策背景下,借助合成生物学中的方法,利用酶工程和代谢工程技术,生产可替代化石燃料的绿色燃料-生物燃料,是势在必行的趋势。作为传统液态化石燃料的重要组成成分,实现脂肪烃的生物合成具有重要意义。由P450脂肪酸脱羧酶催化的中长链(C12-C20)脂肪酸脱羧反应生成的α烯烃是一种独特的微生物天然产物,因其在生物燃料和生物材料领域的潜在应用前景倍受关注。围绕以上设想,本研究通过对脂肪酸水解酶和P450脂肪酸脱羧酶进行融合表达、纯化和酶学表征,在本研究中成功通过将脂肪酶T1L的甘油三酯水解活性与P450脱羧酶OleTJE的脂肪酸脱羧活性偶联,实现了将油脂原料两步高效转化为α烯烃。在本研究中,通过对融合蛋白中组成部件及组氨酸标签的位置进行排列组合,成功构建四种结构的质粒:pET28b-his-TlL-O1eTJE、pET28b-TlL-O1eTJE-his、pET28b-his-O1eTJE-TlL 和 pET28b-O1eTJE-TlL-his;转化至大肠杆菌 E.co/i BL21(DE3),经由IPTG诱导表达,实现四种不同结构的融合蛋白的表达。其中两种结构为his-OleTJE-TlL和his-TlL-OleTJE的融合蛋白,经镍柱层析纯化后成功实现双功能活性。其中,his-OleTJE-TlL和his-TlL-OleTJE水解十四酸甘油三酯产酸率分别为21.3%和19.5%,脂肪酸脱羧产烃率为62.3%和15.1%。融合蛋白his-OleTJE-TlL明显具有更高催化活性。以十四脂肪酸甘油三酯为底物,经气相色谱分析,融合蛋白his-OleTJE-TlL催化甘油三酯顺序水解—脱羧偶联反应的产烃率最高,达到31.7%,并且催化效率相对于双酶混合体系明显提升。两种酶混合体系催化甘油三肉豆蔻酸酯产酸率为3.3±0.3%,同等条件下FusC产酸率为14.8±1.9%;在脱羧活性试验中,双酶混合体系催化肉豆蔻酸产1-十三烯,产率为29.9±3.3%,同等条件下FusC催化肉豆蔻酸产烃率为61.2±3.9%。进行一步产烃的体外实验,实验结果表明FusC能够催化水解和脱羧反应同时进行,并且由于底物通道效应催化产烃效率明显提升,两种酶混合体系催化产烃率为14.7±1.1%,FusC为31.7±2.9%。若两种酶分步进行反应,需大于9小时的反应时长才可达到32.7±1.4%的产烃率,而FusC仅需3小时便可达到同等结果。证明融合蛋白his-OleTJE-TlL具有良好的底物通道效应。增加十二酸甘油三酯和十六酸甘油三酯为底物,考察双酶混合体系和融合蛋白his-OleTJE-TlL两种系统的底物范围。两种酶混合体系催化三月桂酰甘油酯,三肉豆蔻酸甘油三脂和软脂酸甘油酯产烃率为18.3±2.1%、14.7±1.1%和8.46±0.3%,而FusC为28.1±4.1%、31.7±2.9%和26.6±0.4%。实验表明融合蛋白较双酶混合系统,催化效率提升2倍,证明融合蛋白具有宽泛的底物选择范围,并且对不同底物均具有高催化效率,具有潜在的实际应用前景为了近一步降低酶纯化制备的成本,且基于前期关于脂肪酶以分泌蛋白形式在毕赤酵母中成功表达的研究,选择毕赤酵母为宿主菌株,通过将密码子优化后的融合蛋白TlL-OleTJE基因整合进毕赤酵母基因组,经由甲醇诱导表达,但并未得到融合蛋白,正在对融合蛋白在毕赤酵母中的转录表达情况进行研究。
[Abstract]:Consumption of fossil fuels and non renewable resources to produce a lot of problems, such as high oil prices, energy shortage, environmental pollution and so on. And in the sustainable development and energy-saving emission reduction policies in the background, with the method of synthetic biology, using enzyme engineering and metabolic engineering, green fuel production alternatives to fossil fuels and biofuels that is imperative trend. As an important component of traditional fossil fuels, has important significance to realize the biosynthesis of aliphatic hydrocarbons. P450 fatty acid decarboxylase catalyzed by long chain fatty acids (C12-C20) and alpha olefin carboxylic reaction is a unique microbial natural products, because of its in the field of bio fuels and bio the potential applications of materials have attracted much attention. Based on the above ideas, this study by the fusion expression of fatty acid hydrolase and P450 fatty acid decarboxylase, purification and enzymatic table In this study, the success of fatty acid triglyceride hydrolysis activity of lipase T1L and P450 decarboxylase OleTJE decarboxylation activity coupling, will realize the two step oil raw material efficiently converted into alpha olefin. In this study, based on the fusion protein components and histidine tag position permutation and combination, construct the structure of the four kinds of plasmids: pET28b-his-TlL-O1eTJE, pET28b-TlL-O1eTJE-his, pET28b-his-O1eTJE-TlL and pET28b-O1eTJE-TlL-his; BL21 was transformed into E. coli E.co/i (DE3), via IPTG induced expression of four different structure of the fusion protein. The two kinds of structure for the integration of his-OleTJE-TlL and his-TlL-OleTJE protein after purification after the successful implementation of dual functional activity which, his-OleTJE-TlL and his-TlL-OleTJE fourteen hydrolysis acid triglyceride acid production rate were 21.3% and 19.5%, fatty acid decarboxylation The hydrocarbon generation rate was 62.3% and the 15.1%. his-OleTJE-TlL fusion protein has higher catalytic activity. Fourteen fatty acid triglyceride as substrate, column chromatography, the fusion protein of his-OleTJE-TlL catalytic hydrolysis of triglyceride order hydrocarbon decarboxylation coupling. The highest rate reached 31.7%, and the catalytic efficiency compared with double enzyme mixture significantly improved. Two the catalytic enzyme mixture of glycerol three myristate acid production rate was 3.3 + 0.3%, under the same conditions of FusC acid production rate was 14.8 + 1.9%; in the decarboxylation activity test, double enzyme catalyzed by acid producing mixed nutmeg 1- thirteen ethylene, yield was 29.9 + 3.3%, under the same conditions of FusC catalytic hydrocarbon generation rate for myristic acid 61.2 + 3.9%. step of the hydrocarbon production in vitro experiments, the experimental results show that FusC can catalyze the hydrolysis and decarboxylation reaction at the same time, and because the substrate channel effect catalytic hydrocarbon production efficiency improved significantly, two The enzyme mixture catalytic hydrocarbon generation rate was 14.7 + 1.1%, 31.7 + two FusC 2.9%. if by the enzyme reaction, the hydrocarbon generation can be reached only 32.7 + 1.4% for more than 9 hours long when the reaction rate of FusC, and only 3 hours can achieve the same results. His-OleTJE-TlL has demonstrated that the fusion protein substrate channel good effect. An increase of twelve and sixteen acid triglyceride acid triglyceride as substrate of the double enzyme mixture system and fusion protein his-OleTJE-TlL two systems. Two kinds of enzyme substrate range hybrid system in March Guangxi glycerol ester, three myristate, glycerin three greases and soft fatty acid glycerol ester of the hydrocarbon generation rate was 18.3 + 2.1%. 14.7 + 1.1% and 8.46 + 0.3%, and FusC was 28.1 + 4.1%, 31.7 + 2.9% and 26.6 + 0.4%. experiments show that the fusion protein was mixed double enzyme system, the catalytic efficiency 2 times, proving that the fusion protein has the broad substrate scope, and on different substrates were With high catalytic efficiency, has potential practical application prospect in order to further reduce the cost of preparation and purification of enzyme, and based on the preliminary research about lipase secreted proteins to form successfully expressed in Pichia pastoris, Pichia pastoris as host strains, the fusion protein TlL-OleTJE gene codon optimized integrated into Pichia pastoris genome, through the induction of methanol, but did not get the fusion protein on the transcription of the fusion protein was expressed in Pichia pastoris were studied.

【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O621.251
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本文编号：1619154