Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, P. R. China.
ZJU-Hangzhou Global Scientific and Technological Innovation Center, No. 733 Jianshe San Road, Xiaoshan District, Hangzhou 311200, Zhejiang, P. R. China.
J Agric Food Chem. 2022 Jun 15;70(23):7267-7278. doi: 10.1021/acs.jafc.2c01406. Epub 2022 Jun 2.
The amidase from d3 (AmdA) degrades the carcinogenic ethyl carbamate (EC) in alcoholic beverages. However, its limited catalytic activity hinders practical applications. Here, multiple sequence alignment was first used to predict single variants with improved activity. Afterward, AlphaFold 2 was applied to predict the three-dimensional structure of AmdA and 21 amino acids near the catalytic triad were randomized by saturation mutagenesis. Each of the mutation libraries was then screened, and the improved single variants were combined to obtain the best double variant I97L/G195A that showed a 3.1-fold increase in the urethanase activity and a 1.5-fold increase in ethanol tolerance. MD simulations revealed that the mutations shortened the distance between catalytic residues and the substrate and enhanced the occurrence of a critical hydrogen bond in the catalytic pocket. This study displayed a useful strategy to engineer an amidase for the improvement of urethanase activity, and the variant obtained provided a good candidate for applications in the food industry.
d3 中的 amidase（AmdA）能够降解酒精饮料中的致癌物质乙基氨基甲酸酯（EC）。然而，其有限的催化活性限制了其实际应用。在这里，首先使用多重序列比对来预测具有改善活性的单一变体。随后，应用 AlphaFold 2 预测 AmdA 的三维结构，并通过饱和诱变随机化靠近催化三联体的 21 个氨基酸。然后筛选每个突变文库，并将改进的单一变体组合以获得最佳的双变体 I97L/G195A，其脲酶活性提高了 3.1 倍，乙醇耐受性提高了 1.5 倍。MD 模拟表明，突变缩短了催化残基与底物之间的距离，并增强了催化口袋中关键氢键的发生。本研究展示了一种用于工程化酰胺酶以提高脲酶活性的有用策略，所获得的变体为在食品工业中的应用提供了一个很好的候选者。
