本文選題：脂肪酶P450脂肪酸脫羧酶　切入點：融合蛋白　出處：《青島科技大學》2017年碩士論文　論文類型：學位論文

【摘要】：化石燃料資源的大量消耗和不可再生產(chǎn)生了很多問題,諸如油價高企、能源緊缺、環(huán)境污染等。而在可持續(xù)發(fā)展和節(jié)能減排的政策背景下,借助合成生物學中的方法,利用酶工程和代謝工程技術,生產(chǎn)可替代化石燃料的綠色燃料-生物燃料,是勢在必行的趨勢。作為傳統(tǒng)液態(tài)化石燃料的重要組成成分,實現(xiàn)脂肪烴的生物合成具有重要意義。由P450脂肪酸脫羧酶催化的中長鏈(C12-C20)脂肪酸脫羧反應生成的α烯烴是一種獨特的微生物天然產(chǎn)物,因其在生物燃料和生物材料領域的潛在應用前景倍受關注。圍繞以上設想,本研究通過對脂肪酸水解酶和P450脂肪酸脫羧酶進行融合表達、純化和酶學表征,在本研究中成功通過將脂肪酶T1L的甘油三酯水解活性與P450脫羧酶OleTJE的脂肪酸脫羧活性偶聯(lián),實現(xiàn)了將油脂原料兩步高效轉化為α烯烴。在本研究中,通過對融合蛋白中組成部件及組氨酸標簽的位置進行排列組合,成功構建四種結構的質粒:pET28b-his-TlL-O1eTJE、pET28b-TlL-O1eTJE-his、pET28b-his-O1eTJE-TlL 和 pET28b-O1eTJE-TlL-his;轉化至大腸桿菌 E.co/i BL21(DE3),經(jīng)由IPTG誘導表達,實現(xiàn)四種不同結構的融合蛋白的表達。其中兩種結構為his-OleTJE-TlL和his-TlL-OleTJE的融合蛋白,經(jīng)鎳柱層析純化后成功實現(xiàn)雙功能活性。其中,his-OleTJE-TlL和his-TlL-OleTJE水解十四酸甘油三酯產(chǎn)酸率分別為21.3%和19.5%,脂肪酸脫羧產(chǎn)烴率為62.3%和15.1%。融合蛋白his-OleTJE-TlL明顯具有更高催化活性。以十四脂肪酸甘油三酯為底物,經(jīng)氣相色譜分析,融合蛋白his-OleTJE-TlL催化甘油三酯順序水解—脫羧偶聯(lián)反應的產(chǎn)烴率最高,達到31.7%,并且催化效率相對于雙酶混合體系明顯提升。兩種酶混合體系催化甘油三肉豆蔻酸酯產(chǎn)酸率為3.3±0.3%,同等條件下FusC產(chǎn)酸率為14.8±1.9%;在脫羧活性試驗中,雙酶混合體系催化肉豆蔻酸產(chǎn)1-十三烯,產(chǎn)率為29.9±3.3%,同等條件下FusC催化肉豆蔻酸產(chǎn)烴率為61.2±3.9%。進行一步產(chǎn)烴的體外實驗,實驗結果表明FusC能夠催化水解和脫羧反應同時進行,并且由于底物通道效應催化產(chǎn)烴效率明顯提升,兩種酶混合體系催化產(chǎn)烴率為14.7±1.1%,FusC為31.7±2.9%。若兩種酶分步進行反應,需大于9小時的反應時長才可達到32.7±1.4%的產(chǎn)烴率,而FusC僅需3小時便可達到同等結果。證明融合蛋白his-OleTJE-TlL具有良好的底物通道效應。增加十二酸甘油三酯和十六酸甘油三酯為底物,考察雙酶混合體系和融合蛋白his-OleTJE-TlL兩種系統(tǒng)的底物范圍。兩種酶混合體系催化三月桂酰甘油酯,三肉豆蔻酸甘油三脂和軟脂酸甘油酯產(chǎn)烴率為18.3±2.1%、14.7±1.1%和8.46±0.3%,而FusC為28.1±4.1%、31.7±2.9%和26.6±0.4%。實驗表明融合蛋白較雙酶混合系統(tǒng),催化效率提升2倍,證明融合蛋白具有寬泛的底物選擇范圍,并且對不同底物均具有高催化效率,具有潛在的實際應用前景為了近一步降低酶純化制備的成本,且基于前期關于脂肪酶以分泌蛋白形式在畢赤酵母中成功表達的研究,選擇畢赤酵母為宿主菌株,通過將密碼子優(yōu)化后的融合蛋白TlL-OleTJE基因整合進畢赤酵母基因組,經(jīng)由甲醇誘導表達,但并未得到融合蛋白,正在對融合蛋白在畢赤酵母中的轉錄表達情況進行研究。
[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.

【學位授予單位】：青島科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O621.251
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本文編號：1619154