Discovery and characterization of novel esterase with EC-hydrolyzing activity

Abstract

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

CLC Number:

Q814.9

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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