Yu Hong, Li Jian, Zhang Dalong, Yang Yunliu, Jiang Weihong, Yang Sheng
Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Appl Microbiol Biotechnol. 2009 Feb;82(2):279-85. doi: 10.1007/s00253-008-1748-z. Epub 2008 Nov 5.
To facilitate the easier production of D-amino acids using N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, we improved the enzymatic thermostability of highly soluble DCase-M3 of Ralstonia pickettii using 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 degrees 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 of D-amino acids.
为了便于使用固定化形式的N-氨甲酰-D-氨基酸酰胺水解酶（DCase）更轻松地生产D-氨基酸，我们通过定向诱变提高了皮氏罗尔斯顿氏菌高度可溶的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，本研究还鉴定出六个新的突变位点。最具热稳定性的突变体，其第12位氨基酸已从谷氨酰胺变为亮氨酸，热稳定性提高了7摄氏度。亲本和突变体DCase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性并未受到影响。预计这些突变酶将应用于D-氨基酸工业化生产的全酶法工艺开发。
