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Alteration of substrate specificity of aspartase by directed evolution

机译：通过定向进化改变天冬氨酸酶的底物特异性

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Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1), which catalyzes the reversible deamination Of L-aspartic acid to yield fumaric acid and ammonia, is highly selective towards L-aspartic acid. We screened for enzyme variants with altered substrate specificity by a directed evolution method. Random mutagenesis was performed on all Escherichia coli aspartase gene (aspA) by error-prone PCR to construct a mutant library. The mutant library was introduced to E. coli and the transformants were screened for production of fumaric acid-mono amide front L-aspartic acid-alpha-amide. Through the screening, one mutant, MA2100, catalyzing deamination Of L-aspartic acid-alpha-amide was achieved. Gene analysis of the MA2100 mutant indicated that the mutated enzyme had a K327N mutation. The characteristics of the mutated enzyme were examined. The optimum pH values for the L-aspartic acid and L-aspartic acid-alpha-amide of the mutated enzyme were pH 8.5 and 6.0, respectively. The K-m value and V-max value for the L-aspartic acid of the mutated enzyme were 28.3 mM and 0.26 U/mg, respectively. The K-m value and V-max value for the L-aspartic acid-alpha-amide of the mutated enzyme were 1450 mM and 0.47 U/mg, respectively. This is the first report describing the alteration of the substrate specificity of aspartase, an industrially important enzyme. (C) 2005 Elsevier B.V. All rights reserved.
机译：天冬氨酸酶（L-天冬氨酸氨裂合酶，EC 4.3.1.1），催化L-天冬氨酸的可逆脱氨，生成富马酸和氨，对L-天冬氨酸具有高度选择性。我们通过定向进化方法筛选了具有改变的底物特异性的酶变体。通过易错PCR对所有大肠杆菌天冬氨酸酶基因（aspA）进行随机诱变，以构建突变体文库。将突变体文库引入大肠杆菌，并筛选转化子以生产富马酸-单酰胺-前L-天冬氨酸-α-酰胺。通过筛选，获得了一种催化L-天冬氨酸-α-酰胺脱氨基的突变体MA2100。 MA2100突变体的基因分析表明，突变的酶具有K327N突变。检查了突变酶的特性。突变酶的L-天冬氨酸和L-天冬氨酸-α-酰胺的最佳pH值分别为pH 8.5和6.0。突变酶的L-天冬氨酸的K-m值和V-max值分别为28.3mM和0.26U / mg。突变酶的L-天冬氨酸-α-酰胺的K-m值和V-max值分别为1450mM和0.47U / mg。这是描述工业上重要酶天冬氨酸酶底物特异性改变的第一份报告。 （C）2005 Elsevier B.V.保留所有权利。

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