Cloning and secretory expression of an amidase from Kluyvera cryocrescens and characterization of its potential in preparation of chiral amino acids. An amidase belonging to the Ntn-hydrolase superfamily was identified from Kluyvera cryocrescens ZJB-17005 (Kc-Ami). The maximum activity of Kc-Ami was observed at pH 8.5 and 55 °C. Remarkably, Kc-Ami showed an excellent enantioselectivity (99% ee) using rac-4-(hydroxy(methyl)phosphoryl)-2-(2-phenylacetamido) butanoic acid as substrate. Kc-Ami remained stable at pH 7.0–9.0 and exhibited prominent thermostability with a half-life time of 59.1, 47.4 and 20.4 h at 50, 55 and 60 °C, respectively. Kc-Ami could be appllied to synthesize chiral amino acids and its derivatives with excellent enantioselectivity (> 99% ee). The synthesized chiral amino acids could contain short or long side chain, and further the side chain could be replaced with –OH, –COOH or benzene ring. Kc-Ami exhibited remarkable thermostability and excellent enantioselectivity for synthesizing chiral amino acids and its derivatives. This specific characteristic provides great potential for industrial application in preparation of chiral amino acids and its derivatives.

中文翻译：

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枯草芽孢杆菌 Kluyvera cryocrescens 新型酰胺酶的分泌表达及表征

来自 Kluyvera cryocrescens 的酰胺酶的克隆和分泌表达及其在制备手性氨基酸中的潜力。一种属于 Ntn-水解酶超家族的酰胺酶是从 Kluyvera cryocrescens ZJB-17005 (Kc-Ami) 中鉴定出来的。在 pH 8.5 和 55 °C 下观察到 Kc-Ami 的最大活性。值得注意的是，使用 rac-4-（羟基（甲基）磷酰基）-2-（2-苯基乙酰胺基）丁酸作为底物时，Kc-Ami 显示出优异的对映选择性（99% ee）。Kc-Ami 在 pH 7.0-9.0 下保持稳定，并表现出突出的热稳定性，在 50、55 和 60°C 下的半衰期分别为 59.1、47.4 和 20.4 小时。Kc-Ami 可用于合成具有优异对映选择性（> 99% ee）的手性氨基酸及其衍生物。合成的手性氨基酸可以含有短或长的侧链，并且进一步的侧链可以用-OH、-COOH或苯环代替。Kc-Ami 在合成手性氨基酸及其衍生物方面表现出显着的热稳定性和优异的对映选择性。这种特殊的特性为制备手性氨基酸及其衍生物的工业应用提供了巨大的潜力。