发现甲苯邻二甲苯单加氧酶(ToMO)氧化氯苯形成2-氯苯酚(2-CP,4%),3-CP(12%),和4-CP(84%),总产物形成率为1.2±0.17nmol/min/mg蛋白质。还发现ToMO从3-CP和4-CP形成4-氯邻苯二酚(4-CC),初始速率为0.54±0.10和0.40±0.04nmol/min/mg蛋白质,分别,和氯氢醌(CHQ,13%),4-氯间苯二酚(4-CR,3%),和2-CP的3-CC(84%),初始产物形成率为1.1±0.32nmol/min/mg蛋白质。为了提高氧化速率和改变氯芳烃的氧化区域特异性,以及研究ToMO(TouA)α羟化酶片段的活性位点残基L192和A107的作用,我们使用了蛋白质工程的饱和诱变方法。分离出13种TouA变体,其中一些在这里发现的最好的替代品以前从未被研究过。具体来说,鉴定出TouA变体L192V具有1.8-,1.4-,2.4-,对氯苯的羟基化活性快4.8倍,2-CP,3-CP,4-CP,分别,与原生ToMO相比。L192V变体还具有从4%到13%2-CP的氯苯区域特异性,并从3-CP产生新产物3-CC(4%)。大多数分离的变体被鉴定为改变氧化的区域特异性。例如,与原生ToMO相比,变体A107T,A107N,和A107M生产6.3-,7.0-,4-CR比2-CP多7.3倍,分别,和变体A107G和A107G/L192V产生3-CC(33%和39%,分别)来自3-CP,而天然ToMO则没有。IMPORTANCE氯苯是一种常用的有毒溶剂,被美国环境保护局列为重点环境污染物。这里,我们报道了表达甲苯邻二甲苯单加氧酶(ToMO)的大肠杆菌TG1细胞可以成功地氧化氯苯形成二羟基氯芳烃,是有价值的工业化合物。ToMO在室温下在水中仅使用分子氧和细胞提供的辅因子进行此操作。利用蛋白质工程技术,我们还分离了具有增强的氧化活性以及微调的区域特异性的ToMO变体,这使得直接微生物氧合更具吸引力。这项工作的意义在于能够降解环境污染物,同时使用环境友好的生物方法生产有价值的化学品,而不是昂贵的,复杂的化学过程。
Toluene o-xylene monooxygenase (ToMO) was found to oxidize chlorobenzene to form 2-chlorophenol (2-CP, 4%), 3-CP (12%), and 4-CP (84%) with a total product formation rate of 1.2 ± 0.17 nmol/min/mg protein. It was also discovered that ToMO forms 4-chlorocatechol (4-CC) from 3-CP and 4-CP with initial rates of 0.54 ± 0.10 and 0.40 ± 0.04 nmol/min/mg protein, respectively, and chlorohydroquinone (CHQ, 13%), 4-chlororesorcinol (4-CR, 3%), and 3-CC (84%) from 2-CP with an initial product formation rate of 1.1 ± 0.32 nmol/min/mg protein. To increase the oxidation rate and alter the oxidation regiospecificity of chloroaromatics, as well as to study the roles of active site residues L192 and A107 of the alpha hydroxylase fragment of ToMO (TouA), we used the saturation mutagenesis approach of protein engineering. Thirteen TouA variants were isolated, among which some of the best substitutions uncovered here have never been studied before. Specifically, TouA variant L192V was identified which had 1.8-, 1.4-, 2.4-, and 4.8-fold faster hydroxylation activity toward chlorobenzene, 2-CP, 3-CP, and 4-CP, respectively, compared to the native ToMO. The L192V variant also had the regiospecificity of chlorobenzene changed from 4% to 13% 2-CP and produced the novel product 3-CC (4%) from 3-CP. Most of the isolated variants were identified to change the regiospecificity of oxidation. For example, compared to the native ToMO, variants A107T, A107N, and A107M produced 6.3-, 7.0-, and 7.3-fold more 4-CR from 2-CP, respectively, and variants A107G and A107G/L192V produced 3-CC (33 and 39%, respectively) from 3-CP whereas native ToMO did not. IMPORTANCE Chlorobenzene is a commonly used toxic solvent and listed as a priority environmental pollutant by the US Environmental Protection Agency. Here, we report that Escherichia coli TG1 cells expressing toluene o-xylene monooxygenase (ToMO) can successfully oxidize chlorobenzene to form dihydroxy chloroaromatics, which are valuable industrial compounds. ToMO performs this at room temperature in water using only molecular oxygen and a cofactor supplied by the cells. Using protein engineering techniques, we also isolated ToMO variants with enhanced oxidation activity as well as fine-tuned regiospecificities which make direct microbial oxygenations even more attractive. The significance of this work lies in the ability to degrade environmental pollutants while at the same time producing valuable chemicals using environmentally benign biological methods rather than expensive, complex chemical processes.