Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China; Ningbo Research Institute, Zhejiang University, Ningbo 315100, PR China.
J Biosci Bioeng. 2021 Sep;132(3):220-225. doi: 10.1016/j.jbiosc.2021.05.003. Epub 2021 Jun 18.
Ethyl carbamate (EC), widely found in alcoholic beverages, has been revealed to be a probable carcinogen in humans. Urethanase (EC 3.5.1.75) is an effective enzyme for the degradation of EC; however, the previously identified urethanases exhibited insufficient acid and alcohol resistance. In this study, an enantioselective amidase (AmdA) screened from Agrobacterium tumefaciens d exhibited urethanase activity with excellent alcohol resistance. AmdA was first overexpressed in Escherichia coli; however, the recombinant protein was primarily located in inclusion bodies, and thus, co-expression of molecular chaperones was used. The activity of AmdA increased 3.1 fold to 307 U/L, and the specific activity of urethanase with C-terminal His-tags reached 0.62 U/mg after purification through a Ni-NTA column. Subsequently, the enzymatic properties and kinetic constants of AmdA were investigated. The optimum temperature for AmdA was 55 °C, it showed the highest activity at pH 7.5, and the K was 0.964 mM. Moreover, after 1 h of heat treatment at 37 °C in a 5-20% (v/v) ethanol solution, the residual urethanase activity was higher than 91%, considerably more than that reported thus far.
氨基甲酸乙酯（EC）广泛存在于酒精饮料中，已被证实是人类的一种可能致癌物。尿烷酶（EC 3.5.1.75）是一种有效降解 EC 的酶；然而，之前鉴定的尿烷酶表现出对酸和酒精的耐受力不足。在这项研究中，从根癌农杆菌 d 中筛选出一种对映体选择性酰胺酶（AmdA），表现出对酒精的优异耐受力的尿烷酶活性。AmdA 首先在大肠杆菌中过表达；然而，重组蛋白主要位于包涵体中，因此，使用共表达分子伴侣。AmdA 的活性增加了 3.1 倍，达到 307 U/L，并且带有 C 末端 His 标签的尿烷酶的比活达到 0.62 U/mg，经过 Ni-NTA 柱纯化后。随后，研究了 AmdA 的酶学性质和动力学常数。AmdA 的最适温度为 55°C，在 pH 7.5 时表现出最高的活性，K 为 0.964 mM。此外，在 37°C 的 5-20%（v/v）乙醇溶液中热处理 1 小时后，残余尿烷酶活性高于 91%，明显高于迄今为止报道的水平。
