Conversion of Sterically Demanding α,α-Disubstituted Phenylacetonitriles by the Arylacetonitrilase from Pseudomonas fluorescens EBC191
Conversion of Sterically Demanding α,α-Disubstituted Phenylacetonitriles by the Arylacetonitrilase from Pseudomonas fluorescens EBC191
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DOI：
10.1128/aem.05570-11
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发表时间：
2012-01-01
影响因子：
4.4
通讯作者：
Stolz, Andreas
中科院分区：
文献类型：
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作者：
Baum, Stefanie;Williamson, Dael S.;Stolz, Andreas
荧光假单胞菌EBC191的丁腈酶可转化为2-甲基-2-苯基丙腈，该酶在α-位上含有一个朝向氰基的季碳原子，也可转化为类似的对无菌要求很高的底物，如2-羟基-2-苯基丙腈(苯乙酮氰醇)或2-乙酰氧基-2-甲基苯乙腈。2-甲基-2-苯基丙腈被水解到几乎化学计量比的相应酸量。苯乙酮氰醇以3.4：1左右的比例转化为相应的酸和酰胺。苯乙酮氰醇优先生成(R)-酸和(S)-酰胺。该酶的同源模型表明，与氨基酸残基Tyr54的空间位阻可以损害对空间要求很高的底物的结合或转化。因此，产生了几个在各自残基中携带突变的酶变体，并随后分析了反应的底物特异性和对映体选择性。确定了显示苯乙酮氰醇转化率相对活性增加的酶变体。这些反应的手性分析显示了特殊的反应动力学，这表明这些酶变体转化苯乙酮氰醇的非首选(S)对映体的反应速度比(首选)(R)对映体的反应速度快。荧光假单胞菌EBC191和木薯(Manihotesculenta)植物源(S)氧化腈酶的重组全细胞催化剂在pH值为4.5的条件下将苯乙酮和氰化物转化为(S)-异丙基乳酸酯和(S)-异丙内酰胺。这些重组细胞是由酮合成稳定的手性季碳中心的有前景的催化剂。
The nitrilase from Pseudomonas fluorescens EBC191 converted 2-methyl-2-phenylpropionitrile, which contains a quaternary carbon atom in the alpha-position toward the nitrile group, and also similar sterically demanding substrates, such as 2-hydroxy-2-phenylpropionitrile (acetophenone cyanohydrin) or 2-acetyloxy-2-methylphenylacetonitrile. 2-Methyl-2-phenylpropionitrile was hydrolyzed to almost stoichiometric amounts of the corresponding acid. Acetophenone cyanohydrin was transformed to the corresponding acid (atrolactate) and amide (atrolactamide) at a ratio of about 3.4:1. The (R)-acid and the (S)-amide were formed preferentially from acetophenone cyanohydrin. A homology model of the nitrilase suggested that steric hindrance with amino acid residue Tyr54 could impair the binding or conversion of sterically demanding substrates. Therefore, several enzyme variants that carried mutations in the respective residues were generated and subsequently analyzed for the substrate specificity and enantioselectivity of the reactions. Enzyme variants that demonstrated increased relative activities for the conversion of acetophenone cyanohydrin were identified. The chiral analysis of these reactions demonstrated peculiar reaction kinetics, which suggested that the enzyme variants converted the nonpreferred (S)-enantiomer of acetophenone cyanohydrin with a higher reaction rate than that of the (preferred) (R)-enantiomer. Recombinant whole-cell catalysts that simultaneously produced the nitrilase from P. fluorescens EBC191 and a plant-derived (S)-oxynitrilase from cassava (Manihot esculenta) converted acetophenone plus cyanide at pH 4.5 to (S)-atrolactate and (S)-atrolactamide. These recombinant cells are promising catalysts for the synthesis of stable chiral quaternary carbon centers from ketones.