基于組合設(shè)計(jì)策略提高鹵醇脫鹵酶HheC降解2，3-DCP的活性
發(fā)布時(shí)間：2018-10-20 08:42
【摘要】：1,2,3-三氯丙烷(TCP)作為表氯醇工業(yè)生產(chǎn)中的主要副產(chǎn)物,已經(jīng)成為一種持久性的地下水污染物,對(duì)人體具有潛在致癌性,急需建立一種廉價(jià)高效的技術(shù)將TCP從污染源中清除。相比傳統(tǒng)的手段,生物降解環(huán)境污染物的方法更加環(huán)保高效,同時(shí)也能節(jié)約成本帶來(lái)更多的效益。鹵醇脫鹵酶(HHDHs)是一種能夠催化鹵代醇的碳-鹵鍵斷裂的裂解酶,因此可以降解環(huán)境中的TCP。同時(shí),它們也可以在親核試劑存在的情況下催化逆向的環(huán)氧化物開(kāi)環(huán)反應(yīng),可以用來(lái)制備多種具有重要應(yīng)用價(jià)值的手性鹵代醇、環(huán)氧化物和β-取代醇。改造鹵醇脫鹵酶的脫鹵活性和立體選擇性是本論文研究的兩個(gè)重要方面。最新研究顯示,在生物降解1,2,3-三氯丙烷(TCP)的多酶體系中,HheC酶催化中間產(chǎn)物2,3-二氯-1-丙醇(2,3-DCP)發(fā)生脫鹵反應(yīng)的活性極低,嚴(yán)重制約了TCP的生物轉(zhuǎn)化。為此,本論文借助組合式改造策略來(lái)提高HheC酶對(duì)2,3-DCP的催化活性。首先運(yùn)用分子動(dòng)力學(xué)模擬獲得HheC在對(duì)2,3-DCP進(jìn)行脫鹵時(shí)相關(guān)的氨基酸殘基信息,預(yù)測(cè)篩選出得到可能發(fā)揮作用的10個(gè)關(guān)鍵位點(diǎn);然后借助半理性設(shè)計(jì)的策略,將這10個(gè)位點(diǎn)進(jìn)行合理組合構(gòu)建了6個(gè)飽和突變文庫(kù);篩選約8200個(gè)克隆子后,最終獲得了30個(gè)針對(duì)底物2,3-DCP脫鹵活性提高的突變體,其中突變體P84A活性提高了21.8倍,F86I提高16.7倍,F12Q/F186L和W249P分別提高17.4倍和16.9倍。篩選得到對(duì)映體選擇性提高的突變體F86S/L142Y(由ER=7.6提高到ER=200),W139A(由野生態(tài)的ER=7.6提高到ER=184),P84A也從ER=7.6提高到ER=72。通過(guò)分子對(duì)接和MD分析進(jìn)一步探究上述突變體催化特性大幅改進(jìn)的結(jié)構(gòu)基礎(chǔ)。結(jié)果表明:P84、F86、F12、F186和W249這些位點(diǎn)對(duì)提高HheC降解2,3-DCP的活性至關(guān)重要,且這些位點(diǎn)突變?yōu)樾?cè)鏈氨基酸時(shí)活性更高;氨基酸P84,F86,L142,T134,N176和W139在調(diào)控HheC酶對(duì)底物的立體選擇性方面也起著關(guān)鍵作用,P84,F86和W139突變成小側(cè)鏈氨基酸時(shí),鹵醇脫鹵酶HheC對(duì)(R)-2,3-DCP有很強(qiáng)的偏好性,而當(dāng)T134和N176發(fā)生突變時(shí),HheC酶對(duì)(R)-2,3-DCP的偏好性全部喪失,有逆轉(zhuǎn)HheC偏好性的趨勢(shì)。綜上所述,鹵醇脫鹵酶HheC的脫鹵活性和立體選擇性的調(diào)控是由多個(gè)氨基酸共同完成的。因此,組合式改造策略對(duì)HheC相關(guān)催化特性的改造具有很大的優(yōu)勢(shì)。
[Abstract]:As the main by-product in the industrial production of epichlorohydrin, (TCP) has become a persistent groundwater pollutant, which has potential carcinogenicity to human body. It is urgent to establish a cheap and efficient technology to remove TCP from pollution sources. Compared with traditional methods, biodegradation of environmental pollutants is more environmentally friendly and efficient, but also cost savings bring more benefits. Halogenol dehalogenase (HHDHs) is a kind of lyase that can catalyze the breaking of carbon-halogen bond of halogenated alcohols, so it can degrade TCP. in environment. At the same time, they can also catalyze the reverse epoxide ring opening reaction in the presence of nucleophilic reagents, and can be used to prepare many kinds of chiral halogenated alcohols, epoxides and 尾 -substituted alcohols with important application value. The dehalogenation activity and stereoselectivity of halogenated alcohol dehalogenase are two important aspects in this paper. The latest studies have shown that in the multi-enzyme system of biodegradation of (TCP), HheC enzyme catalyzes the dehalogenation of the intermediate product 2O3-dichloro-1-propanol (2C3DCP), which seriously restricts the biotransformation of TCP. Therefore, the combined modification strategy was used to improve the catalytic activity of HheC. Firstly, the information of amino acid residues related to the dehalogenation of 2n3-DCP by HheC was obtained by molecular dynamics simulation, and the 10 key sites that could play a role were predicted and screened, and then the semi-rational design strategy was used. Six saturated mutants were constructed by reasonable combination of these 10 loci. After screening about 8200 clones, 30 mutants with enhanced dehalogenation activity were obtained. The activity of P84A, F86I, F12Q/F186L and W249P increased 21.8 fold, 16.7 fold, 17.4 fold and 16.9 fold, respectively. F86S/L142Y (from ER=7.6 to ER=200), W139A (from wild ecological ER=7.6 to ER=184) and P84A from ER=7.6 to ER=72. were screened for enantioselectivity. Molecular docking and MD analysis were used to further explore the structural basis of the improved catalytic properties of these mutants. The results showed that the sites P84 F86, F12, F186 and W249 were important to enhance the activity of HheC in degradation of 23-DCP, and the activity of these sites was higher when they were mutated to small side chain amino acids. Amino acids P84, F86, L146, T134N176 and W139 also played a key role in regulating the stereoselectivity of HheC to substrates. When P84, F86 and W139 mutated to small side chain amino acids, HheC had a strong preference for (R) 2N 3-DCP. However, when T134 and N176 mutated, the preference of HheC to (R) 23-DCP was all lost, which reversed the tendency of HheC preference. In conclusion, the dehalogenation activity and stereoselectivity of halogenated alcohol dehalogenase HheC were regulated by multiple amino acids. Therefore, the combined modification strategy has great advantages in the modification of HheC related catalytic properties.
【學(xué)位授予單位】：電子科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：X592;Q814
本文編號(hào)：2282636
[Abstract]:As the main by-product in the industrial production of epichlorohydrin, (TCP) has become a persistent groundwater pollutant, which has potential carcinogenicity to human body. It is urgent to establish a cheap and efficient technology to remove TCP from pollution sources. Compared with traditional methods, biodegradation of environmental pollutants is more environmentally friendly and efficient, but also cost savings bring more benefits. Halogenol dehalogenase (HHDHs) is a kind of lyase that can catalyze the breaking of carbon-halogen bond of halogenated alcohols, so it can degrade TCP. in environment. At the same time, they can also catalyze the reverse epoxide ring opening reaction in the presence of nucleophilic reagents, and can be used to prepare many kinds of chiral halogenated alcohols, epoxides and 尾 -substituted alcohols with important application value. The dehalogenation activity and stereoselectivity of halogenated alcohol dehalogenase are two important aspects in this paper. The latest studies have shown that in the multi-enzyme system of biodegradation of (TCP), HheC enzyme catalyzes the dehalogenation of the intermediate product 2O3-dichloro-1-propanol (2C3DCP), which seriously restricts the biotransformation of TCP. Therefore, the combined modification strategy was used to improve the catalytic activity of HheC. Firstly, the information of amino acid residues related to the dehalogenation of 2n3-DCP by HheC was obtained by molecular dynamics simulation, and the 10 key sites that could play a role were predicted and screened, and then the semi-rational design strategy was used. Six saturated mutants were constructed by reasonable combination of these 10 loci. After screening about 8200 clones, 30 mutants with enhanced dehalogenation activity were obtained. The activity of P84A, F86I, F12Q/F186L and W249P increased 21.8 fold, 16.7 fold, 17.4 fold and 16.9 fold, respectively. F86S/L142Y (from ER=7.6 to ER=200), W139A (from wild ecological ER=7.6 to ER=184) and P84A from ER=7.6 to ER=72. were screened for enantioselectivity. Molecular docking and MD analysis were used to further explore the structural basis of the improved catalytic properties of these mutants. The results showed that the sites P84 F86, F12, F186 and W249 were important to enhance the activity of HheC in degradation of 23-DCP, and the activity of these sites was higher when they were mutated to small side chain amino acids. Amino acids P84, F86, L146, T134N176 and W139 also played a key role in regulating the stereoselectivity of HheC to substrates. When P84, F86 and W139 mutated to small side chain amino acids, HheC had a strong preference for (R) 2N 3-DCP. However, when T134 and N176 mutated, the preference of HheC to (R) 23-DCP was all lost, which reversed the tendency of HheC preference. In conclusion, the dehalogenation activity and stereoselectivity of halogenated alcohol dehalogenase HheC were regulated by multiple amino acids. Therefore, the combined modification strategy has great advantages in the modification of HheC related catalytic properties.
【學(xué)位授予單位】：電子科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：X592;Q814
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 李玉平,曹宏斌,張懿;難降解有機(jī)污染物的生物處理[J];現(xiàn)代化工;2004年04期
，本文編號(hào)：2282636
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