Improving the thermostability of N-carbamyl-d-amino acid amidohydrolase by error-prone PCR
Improving the thermostability of N-carbamyl-d-amino acid amidohydrolase by error-prone PCR
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DOI：
10.1007/s00253-008-1748-z
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发表时间：
2009-02
影响因子：
5
通讯作者：
Hong Yu;J. Li;Dalong Zhang;Yunliu Yang;Weihong Jiang;Sheng Yang
中科院分区：
文献类型：
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作者：
Hong Yu;J. Li;Dalong Zhang;Yunliu Yang;Weihong Jiang;Sheng Yang
为了使固定化的N-氨甲酰-D-氨基酸酰胺水解酶（DCase）更容易生产D-氨基酸，我们采用定向诱变的方法提高了高可溶性Ralstonia pickettii的DCase-M3的酶热稳定性。除了先前报道的几个与热稳定性相关的氨基酸位点外，本研究还发现了六个新的突变位点。最耐热的突变体，其中第12个氨基酸已从谷氨酰胺变为亮氨酸，显示出7 °C的热稳定性增加。亲本和突变体DCases的比较表征表明，虽然突变体的氧化稳定性略有降低，但其动力学性质和高溶解度不受影响。该突变酶有望应用于D-氨基酸工业化生产的全酶法工艺的开发。
To facilitate the easier production ofd-amino acids usingN-carbamyl-d-amino acid amidohydrolase (DCase) in an immobilized form, we improved the enzymatic thermostability of highly soluble DCase-M3 ofRalstonia pickettiiusing directed mutagenesis. Six novel mutation sites were identified in this study, apart from several thermostability-related amino acid sites reported previously. The most thermostable mutant, in which the 12th amino acid had been changed from glutamine to leucine, showed a 7 °C increase in thermostability. Comparative characterization of the parental and mutant DCases showed that although there was a slight reduction in the oxidative stability of the mutants, their kinetic properties and high solubility were not affected. The mutated enzymes are expected to be applied to the development of a fully enzymatic process for the industrial production ofd-amino acids.