基于组合设计策略提高卤醇脱卤酶HheC降解2，3-DCP的活性
发布时间：2018-10-20 08:42
【摘要】：1,2,3-三氯丙烷(TCP)作为表氯醇工业生产中的主要副产物,已经成为一种持久性的地下水污染物,对人体具有潜在致癌性,急需建立一种廉价高效的技术将TCP从污染源中清除。相比传统的手段,生物降解环境污染物的方法更加环保高效,同时也能节约成本带来更多的效益。卤醇脱卤酶(HHDHs)是一种能够催化卤代醇的碳-卤键断裂的裂解酶,因此可以降解环境中的TCP。同时,它们也可以在亲核试剂存在的情况下催化逆向的环氧化物开环反应,可以用来制备多种具有重要应用价值的手性卤代醇、环氧化物和β-取代醇。改造卤醇脱卤酶的脱卤活性和立体选择性是本论文研究的两个重要方面。最新研究显示,在生物降解1,2,3-三氯丙烷(TCP)的多酶体系中,HheC酶催化中间产物2,3-二氯-1-丙醇(2,3-DCP)发生脱卤反应的活性极低,严重制约了TCP的生物转化。为此,本论文借助组合式改造策略来提高HheC酶对2,3-DCP的催化活性。首先运用分子动力学模拟获得HheC在对2,3-DCP进行脱卤时相关的氨基酸残基信息,预测筛选出得到可能发挥作用的10个关键位点;然后借助半理性设计的策略,将这10个位点进行合理组合构建了6个饱和突变文库;筛选约8200个克隆子后,最终获得了30个针对底物2,3-DCP脱卤活性提高的突变体,其中突变体P84A活性提高了21.8倍,F86I提高16.7倍,F12Q/F186L和W249P分别提高17.4倍和16.9倍。筛选得到对映体选择性提高的突变体F86S/L142Y(由ER=7.6提高到ER=200),W139A(由野生态的ER=7.6提高到ER=184),P84A也从ER=7.6提高到ER=72。通过分子对接和MD分析进一步探究上述突变体催化特性大幅改进的结构基础。结果表明:P84、F86、F12、F186和W249这些位点对提高HheC降解2,3-DCP的活性至关重要,且这些位点突变为小侧链氨基酸时活性更高;氨基酸P84,F86,L142,T134,N176和W139在调控HheC酶对底物的立体选择性方面也起着关键作用,P84,F86和W139突变成小侧链氨基酸时,卤醇脱卤酶HheC对(R)-2,3-DCP有很强的偏好性,而当T134和N176发生突变时,HheC酶对(R)-2,3-DCP的偏好性全部丧失,有逆转HheC偏好性的趋势。综上所述,卤醇脱卤酶HheC的脱卤活性和立体选择性的调控是由多个氨基酸共同完成的。因此,组合式改造策略对HheC相关催化特性的改造具有很大的优势。
[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.
【学位授予单位】：电子科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X592;Q814
本文编号：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.
【学位授予单位】：电子科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X592;Q814
【参考文献】
相关期刊论文 前1条
1 李玉平,曹宏斌,张懿;难降解有机污染物的生物处理[J];现代化工;2004年04期
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