倍半萜合酶C末端在其功能中的作用
發(fā)布時(shí)間：2018-11-25 10:59
【摘要】：萜類化合物數(shù)量眾多,結(jié)構(gòu)多樣,普遍存在于植物和微生物的次級(jí)代謝產(chǎn)物中。隨著越來越多的萜類化合物的發(fā)現(xiàn),對(duì)萜類合酶的研究也更加深入,這不僅可以幫助我們從理論上認(rèn)識(shí)萜類化合物的合成機(jī)制,理解萜類合酶結(jié)構(gòu)和功能的關(guān)系,還有助于進(jìn)一步研究其在藥物、香精等方面的應(yīng)用。以本實(shí)驗(yàn)室前期初步實(shí)驗(yàn)結(jié)果(ADS的C末端6個(gè)氨基酸截短活性下降)為基礎(chǔ),利用軟件對(duì)催化機(jī)制不同的三種倍半萜合酶進(jìn)行比較,在相應(yīng)位置進(jìn)行截短,明確C末端保留幾個(gè)氨基酸能夠保留其原有活性,以探討此效應(yīng)是否有普遍性,具體工作如下:1.為探討C末端對(duì)倍半萜環(huán)化酶功能的影響,我們選擇對(duì)黃花蒿紫穗槐二烯合酶(amorpha-4,11-diene synthase,ADS)C末端進(jìn)行點(diǎn)突變,取純化后的蛋白在相同酶濃度下對(duì)相同濃度底物進(jìn)行體外催化,催化生成的產(chǎn)物經(jīng)GC-FID分析,同時(shí)用MG法測(cè)定酶動(dòng)力學(xué)參數(shù)Km和Kcat,發(fā)現(xiàn):C1-C10截短不改變酶催化生成的產(chǎn)物,C1-C6截短從酶動(dòng)力學(xué)參數(shù)上來看,其活性沒有明顯變化,C7-C10活性降低,沒有用此方法測(cè)出動(dòng)力學(xué)參數(shù)。2.為探討C末端對(duì)非環(huán)化倍半萜合酶功能的影響,我們選擇對(duì)非環(huán)化酶法呢烯合酶((E)-β-farnesene,BFS)C末端進(jìn)行點(diǎn)突變,取純化后的蛋白在相同酶濃度下對(duì)相同濃度底物進(jìn)行體外催化,催化生成的產(chǎn)物經(jīng)GC-FID分析,同時(shí)用MG法測(cè)定酶動(dòng)力學(xué)參數(shù)Km和Kcat,發(fā)現(xiàn):C1-C10截短不改變酶催化生成的產(chǎn)物,C1-C6截短從酶動(dòng)力學(xué)參數(shù)上來看,其活性沒有明顯變化,C7-C10活性降低,沒有用此方法測(cè)出動(dòng)力學(xué)參數(shù)。3.為探討C末端對(duì)反式倍半萜環(huán)化酶功能的影響,我們選擇反式環(huán)化酶煙草馬兜鈴烯合酶(5-epi-aristolochene synthase,EAS)C末端進(jìn)行點(diǎn)突變,取純化后的蛋白在相同酶濃度下對(duì)相同濃度底物進(jìn)行體外催化,催化生成的產(chǎn)物經(jīng)GC-FID分析,同時(shí)用MG法測(cè)定酶動(dòng)力學(xué)參數(shù)Km和Kcat,發(fā)現(xiàn):C1-C10截短不改變酶催化生成的產(chǎn)物,C1-C6截短從酶動(dòng)力學(xué)參數(shù)上來看,其活性沒有明顯變化,C7-C10活性降低,沒有用此方法測(cè)出動(dòng)力學(xué)參數(shù)。結(jié)合三種倍半萜合酶的結(jié)構(gòu)信息,我們發(fā)現(xiàn)三種酶C末端1-10個(gè)氨基酸的截短不會(huì)改變產(chǎn)物,α螺旋上的氨基酸會(huì)降低酶活性,而α螺旋以外的氨基酸的截短與野生型相比,在活性上沒有明顯變化。
[Abstract]:Terpenoids are abundant in quantity and diverse in structure. They are commonly found in secondary metabolites of plants and microorganisms. With the discovery of more and more terpenoids, the research on terpenoids synthase is more and more in-depth, which can not only help us to understand the synthesis mechanism of terpenes theoretically, but also understand the relationship between the structure and function of terpenoids synthase. It is also helpful to further study its application in medicine, essence and so on. Based on the preliminary experimental results of our laboratory (the reduced activity of 6 amino acids at the C-terminal of ADS), three sesquiterpene synthase with different catalytic mechanisms were compared by software, and the corresponding sites were truncated. It is clear that several amino acids in C terminal can retain their original activity in order to explore the universality of the effect. The specific work is as follows: 1. In order to investigate the effect of C-terminal on the function of sesquiterpene cyclase, we selected the point mutation of C-terminal of Amorpha fruticosa diene synthase (amorpha-4,11-diene synthase,ADS) from Artemisia annua. The purified protein was used to catalyze the substrate of the same concentration at the same enzyme concentration in vitro. The product was analyzed by GC-FID, and the kinetic parameters of Km and Kcat, were determined by MG method. It was found that C1-C10 truncation did not change the enzyme catalyzed product, but C1-C6 truncation did not change the activity of the enzyme from the kinetic parameters of the enzyme, but the activity of C7-C10 decreased, and the kinetic parameters were not measured by this method. 2. In order to investigate the effect of C-terminal on the function of acyclic sesquiterpene synthase, we selected a point mutation on the C-terminal of (E)-尾-farnesene,BFS. The purified protein was used to catalyze the substrate of the same concentration at the same enzyme concentration in vitro. The product was analyzed by GC-FID, and the kinetic parameters of Km and Kcat, were determined by MG method. It was found that C1-C10 truncation did not change the product catalyzed by the enzyme, but C1-C6 truncation did not change the activity of the enzyme from the kinetic parameters of the enzyme, but the activity of C7-C10 decreased, and the kinetic parameters were not measured by this method. 3. In order to investigate the effect of C-terminal on the function of trans-sesquiterpene cyclase, we selected the trans-cyclase (5-epi-aristolochene synthase,EAS) for point mutation at the C-terminal of tobacco aristolochene synthase (5-epi-aristolochene synthase,EAS). The purified protein was used to catalyze the substrate of the same concentration at the same enzyme concentration in vitro. The product was analyzed by GC-FID, and the kinetic parameters of Km and Kcat, were determined by MG method. It was found that C1-C10 truncation did not change the enzyme catalyzed product, but C1-C6 truncation did not change the activity of the enzyme from the kinetic parameters of the enzyme, and the activity of C7-C10 decreased, and the kinetic parameters were not measured by this method. Combined with the structural information of the three sesquiterpene synthase, we found that the truncation of 1-10 amino acids at the C-terminal of the three enzymes did not change the products, the amino acids on the 偽 helix decreased the enzyme activity, but the truncation of the amino acids other than the 偽 helix was compared with the wild type. There was no obvious change in activity.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q55
本文編號(hào)：2355858
[Abstract]:Terpenoids are abundant in quantity and diverse in structure. They are commonly found in secondary metabolites of plants and microorganisms. With the discovery of more and more terpenoids, the research on terpenoids synthase is more and more in-depth, which can not only help us to understand the synthesis mechanism of terpenes theoretically, but also understand the relationship between the structure and function of terpenoids synthase. It is also helpful to further study its application in medicine, essence and so on. Based on the preliminary experimental results of our laboratory (the reduced activity of 6 amino acids at the C-terminal of ADS), three sesquiterpene synthase with different catalytic mechanisms were compared by software, and the corresponding sites were truncated. It is clear that several amino acids in C terminal can retain their original activity in order to explore the universality of the effect. The specific work is as follows: 1. In order to investigate the effect of C-terminal on the function of sesquiterpene cyclase, we selected the point mutation of C-terminal of Amorpha fruticosa diene synthase (amorpha-4,11-diene synthase,ADS) from Artemisia annua. The purified protein was used to catalyze the substrate of the same concentration at the same enzyme concentration in vitro. The product was analyzed by GC-FID, and the kinetic parameters of Km and Kcat, were determined by MG method. It was found that C1-C10 truncation did not change the enzyme catalyzed product, but C1-C6 truncation did not change the activity of the enzyme from the kinetic parameters of the enzyme, but the activity of C7-C10 decreased, and the kinetic parameters were not measured by this method. 2. In order to investigate the effect of C-terminal on the function of acyclic sesquiterpene synthase, we selected a point mutation on the C-terminal of (E)-尾-farnesene,BFS. The purified protein was used to catalyze the substrate of the same concentration at the same enzyme concentration in vitro. The product was analyzed by GC-FID, and the kinetic parameters of Km and Kcat, were determined by MG method. It was found that C1-C10 truncation did not change the product catalyzed by the enzyme, but C1-C6 truncation did not change the activity of the enzyme from the kinetic parameters of the enzyme, but the activity of C7-C10 decreased, and the kinetic parameters were not measured by this method. 3. In order to investigate the effect of C-terminal on the function of trans-sesquiterpene cyclase, we selected the trans-cyclase (5-epi-aristolochene synthase,EAS) for point mutation at the C-terminal of tobacco aristolochene synthase (5-epi-aristolochene synthase,EAS). The purified protein was used to catalyze the substrate of the same concentration at the same enzyme concentration in vitro. The product was analyzed by GC-FID, and the kinetic parameters of Km and Kcat, were determined by MG method. It was found that C1-C10 truncation did not change the enzyme catalyzed product, but C1-C6 truncation did not change the activity of the enzyme from the kinetic parameters of the enzyme, and the activity of C7-C10 decreased, and the kinetic parameters were not measured by this method. Combined with the structural information of the three sesquiterpene synthase, we found that the truncation of 1-10 amino acids at the C-terminal of the three enzymes did not change the products, the amino acids on the 偽 helix decreased the enzyme activity, but the truncation of the amino acids other than the 偽 helix was compared with the wild type. There was no obvious change in activity.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q55
【參考文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 高瑞平;青蒿紫穗槐二烯合酶C-末端截短和兩個(gè)關(guān)鍵區(qū)域突變對(duì)其功能的影響[D];河北大學(xué);2014年
，本文編號(hào)：2355858
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