降解氨基甲酸乙酯的酯酶挖掘及酶学性质表征

摘要/Abstract

摘要： 氨基甲酸乙酯（EC）水解酶可降解传统发酵食品在酿造或储存过程中形成的2A类致癌物EC。目前所发现的EC水解酶数量有限且存在酸耐受性或乙醇耐受性差等多种缺陷，因此筛选鉴定新型EC水解酶对降解传统发酵食品中的EC至关重要。本研究在NCBI数据库中挖掘出4个在pH 4.5及7.0条件下均具有EC降解能力的酯酶（ES1、ES5、ES8和ES9），实现了它们在大肠杆菌中的可溶性表达，并对其进行了纯化及酶学性质表征。结果表明，ES1、ES5、ES8和ES9最适温度分别为55、55、70及70 oC，ES1、ES5、ES9 最适pH呈碱性，ES8呈中性；4种酶在温度高于45 oC或pH低于5.0条件下稳定性显著降低；4种酶的活性受到Fe3+、Co2+、Cu2+的强烈抑制；4种酶在体积分数10 %乙醇存在条件下，分别可保持50.48 %、48.44 %、42.60 %及33.59 %左右的酶活力。4种酶在质量浓度为5 % NaCl条件下，分可保持24.09 %、20.41 %、16.11 %及10.73 %左右的酶活力。4种酶在pH 4.5条件下对EC的Km值分别为210.7、213.8、256.2及127.2 mmol/L，kcat/Km值分别为214.31、203.20、161.46及257.43 s-1·M-1。结构分析推测，ES9底物通道入口较窄，可能影响了EC的进入，从而影响了ES9对EC的最大反应速率；而ES9具有相对较高的EC亲和力，可能归因于ES9与EC之间较强的氢键作用力。本研究获得了具有良好乙醇耐受性及EC水解性能的酯酶，丰富了EC水解酶酶库，为未来控制低酒精度的传统发酵食品中EC含量提供了新方法。

关键词: 氨基甲酸乙酯, 氨基甲酸乙酯水解酶, 酯酶, 传统发酵食品, 食品安全

Abstract: Ethyl carbamate (EC) hydrolase can degrade EC, a class 2A carcinogen formed during brewing or storage of traditional fermented foods. The number of EC hydrolases identified so far is limited and there are some prob-lems with their biochemical properties such as low acid tolerance or ethanol tolerance, so screening and identi-fication of novel EC hydrolases is crucial for the degradation of EC in traditional fermented foods. In this study, four esterases (ES1, ES5, ES8 and ES9) with relatively high EC-hydrolyzing activity were mined from the NCBI database, and their soluble expression in recombinant Escherichia coli were achieved. Then, the enzymes were purified and characterized for their enzymatic properties. The results showed that the optimal temperatures for the four enzymes (ES1, ES5, ES8 and ES9) are 55, 55, 70 and 70 oC, respectively. The optimum pH of ES1, ES5 and ES9 is alkaline and ES8 is neutral. The stability of all four enzymes were significantly reduced at temper-atures higher than 45 oC or at pH lower than 5. The activities of all 4 enzymes were strongly inhibited by Fe3+, Co2+, and Cu2+. In the presence of 10 % (v/v) ethanol，the four enzymes maintained 50.48 %, 48.44 %, 42.60 %, and 33.59 % activity, respectively. The four enzymes were able to maintain about 24.09 %, 20.41 %, 16.11 % and 10.73 % enzyme activity at a mass concentration of 5 % NaCl. The Km values of the four enzymes against EC at pH 4.5 were 210.7, 213.8, 256.2, 127.2 mmol/L, and the kcat/Km values were 214.31, 203.20, 161.46 and 257.43 s-1·M-1, respectively. Structural analysis indicated that the narrow substrate channel of ES9 may have affected the entry of EC, thus affecting its Vmax towards EC; while the relatively high EC affinity of ES9 may be at-tributed to the strong hydrogen bonding force between ES9 with EC. The esterases with EC degradation ob-tained in this study have good ethanol tolerance, which not only enriched the EC enzyme library, but also provided a new method to control the EC content in traditional fermented foods with low alcohol content in the future.

Key words: ethyl carbamate, urethanase, esterase, traditional fermented food, food safety

中图分类号:

Q814.9

刘庆涛朱司宝王天文李闯钱森和张温清程凡田淑芳. 降解氨基甲酸乙酯的酯酶挖掘及酶学性质表征[J]. 食品科学.

Qing-Tao LIU Si-Bao ZHU Chuang Li. Discovery and characterization of novel esterase with EC-hydrolyzing activity[J]. FOOD SCIENCE.

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