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Protein Engineering of Toluene-o-Xylene Monooxygenase from Pseudomonas stutzeri OX1 for Synthesizing 4-Methylresorcinol, Methylhydroquinone, and Pyrogallol

机译:斯氏假单胞菌OX1中甲苯-邻二甲苯单加氧酶的蛋白质工程,用于合成4-甲基间苯二酚,甲基氢醌和邻苯三酚

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摘要

Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 oxidizes toluene to 3- and 4-methylcatechol and oxidizes benzene to form phenol; in this study ToMO was found to also form catechol and 1,2,3-trihydroxybenzene (1,2,3-THB) from phenol. To synthesize novel dihydroxy and trihydroxy derivatives of benzene and toluene, DNA shuffling of the alpha-hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, and F205 were used to generate random mutants. The mutants were initially identified by screening with a rapid agar plate assay and then were examined further by high-performance liquid chromatography and gas chromatography. Several regiospecific mutants with high rates of activity were identified; for example, Escherichia coli TG1/pBS(Kan)ToMO expressing the F205G TouA saturation mutagenesis variant formed 4-methylresorcinol (0.78 nmol/min/mg of protein), 3-methylcatechol (0.25 nmol/min/mg of protein), and methylhydroquinone (0.088 nmol/min/mg of protein) from o-cresol, whereas wild-type ToMO formed only 3-methylcatechol (1.1 nmol/min/mg of protein). From o-cresol, the I100Q saturation mutagenesis mutant and the M180T/E284G DNA shuffling mutant formed methylhydroquinone (0.50 and 0.19 nmol/min/mg of protein, respectively) and 3-methylcatechol (0.49 and 1.5 nmol/min/mg of protein, respectively). The F205G mutant formed catechol (0.52 nmol/min/mg of protein), resorcinol (0.090 nmol/min/mg of protein), and hydroquinone (0.070 nmol/min/mg of protein) from phenol, whereas wild-type ToMO formed only catechol (1.5 nmol/min/mg of protein). Both the I100Q mutant and the M180T/E284G mutant formed hydroquinone (1.2 and 0.040 nmol/min/mg of protein, respectively) and catechol (0.28 and 2.0 nmol/min/mg of protein, respectively) from phenol. Dihydroxybenzenes were further oxidized to trihydroxybenzenes with different regiospecificities; for example, the I100Q mutant formed 1,2,4-THB from catechol, whereas wild-type ToMO formed 1,2,3-THB (pyrogallol). Regiospecific oxidation of the natural substrate toluene was also checked; for example, the I100Q mutant formed 22% o-cresol, 44% m-cresol, and 34% p-cresol, whereas wild-type ToMO formed 32% o-cresol, 21% m-cresol, and 47% p-cresol.
机译:来自斯氏假单胞菌OX1的甲苯-邻二甲苯单加氧酶(ToMO)将甲苯氧化为3-和4-甲基邻苯二酚,并氧化苯形成苯酚;在这项研究中,发现ToMO还可以从苯酚中形成邻苯二酚和1,2,3-三羟基苯(1,2,3-THB)。为了合成苯和甲苯的新型二羟基和三羟基衍生物,ToMO(TouA)的α-羟化酶片段的DNA改组和TouA活性位点残基I100,Q141,T201和F205的饱和诱变被用于生成随机突变体。最初通过快速琼脂平板分析法筛选突变体,然后通过高效液相色谱和气相色谱进一步检查。鉴定了几种具有高活性的区域特异性突变体。例如,表达F205G TouA饱和诱变变体的大肠杆菌TG1 / pBS(Kan)ToMO形成了4-甲基间苯二酚(0.78 nmol / min / mg的蛋白质),3-甲基邻苯二酚(0.25 nmol / min / mg的蛋白质)和甲基氢醌(0.088 nmol / min / mg蛋白质)来自邻甲酚,而野生型ToMO仅形成3-甲基邻苯二酚(1.1 nmol / min / mg蛋白质)。从邻甲酚中,I100Q饱和诱变突变体和M180T / E284G DNA改组突变体形成了甲基氢醌(分别为0.50和0.19 nmol / min / mg的蛋白质)和3-甲基邻苯二酚(0.49和1.5 nmol / min / mg的蛋白质,分别)。 F205G突变体由苯酚形成儿茶酚(0.52 nmol / min / mg蛋白质),间苯二酚(0.090 nmol / min / mg蛋白质)和对苯二酚(0.070 nmol / min / mg蛋白质),而野生型ToMO仅形成儿茶酚(1.5 nmol / min / mg蛋白质)。 I100Q突变体和M180T / E284G突变体均由苯酚形成对苯二酚(分别为1.2和0.040 nmol / min / mg的蛋白质)和邻苯二酚(分别为0.28和2.0 nmol / min / mg的蛋白质)。二羟基苯被进一步氧化为具有不同区域特异性的三羟基苯;例如,I100Q突变体由邻苯二酚形成1,2,4-THB,而野生型ToMO形成1,2,3-THB(邻苯三酚)。还检查了天然底物甲苯的区域特异性氧化。例如,I100Q突变体形成了22%的邻甲酚,44%的间甲酚和34%的对甲酚,而野生型ToMO形成了32%的邻甲酚,21%的间甲酚和47%的对甲酚。 。

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