Abstract
Ethyl carbamate (EC) is mainly found in fermented foods and fermented alcoholic beverages, which could cause carcinogenic potential to humans. Reducing EC is one of the key research priorities to address security of fermented foods. Enzymatic degradation of EC with EC hydrolase in food is the most reliable and efficient method. However, poor tolerance to ethanol severely hinders application of EC hydrolase. In this study, the mutants of EC hydrolase were screened by diphasic high pressure molecular dynamic simulations (dHP-MD). The best variant with remarkable improvement in specific activity and was H68A/K70R/S325N, whose specific activity was approximately 3.42-fold higher than WT, and relative enzyme activity under 20% (v/v) was 5.02-fold higher than WT. Moreover, the triple mutant increased its stability by acquiring more hydration shell and forming extra hydrogen bonds. Furthermore, the ability of degrading EC of the immobilized triple mutant was both detected in mock wine and under certain reaction conditions. The stability of immobilized triple mutant and WT were both improved, and immobilized triple mutant degraded nearly twice as much EC as that of immobilized WT. Overall, dHP-MD was proved to effectively improve enzyme activity and ethanol tolerance for extent application at industrial scale.
Key points
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1
Diphasic high pressure molecular dynamic simulations were applied for ration engineering of ethyl carbamate hydrolase with improved ethanol tolerance.
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The specific activity of H68A/K70R/S325N was 3.42-fold higher than WT.
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The tolerance of triple mutant H68A/K70R/S325N to 20%(v/v) ethanol was increased to 41.16%, which was 5.02-fold higher than WT.
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Introduction
Ethyl carbamate (EC) is a toxic substance with a potential carcinogenic risk and genotoxicity to human beings (Forkert 2010). It is produced during the storage and transport of a wide range of fermented foods. EC is reported to be carcinogenic (Qin et al. 2021), which could be enhanced by ethanol in alcoholic beverages (Sakano et al. 2002), thus the content of EC in alcoholic beverages such as yellow wine, white wine, wine, sake and brandy has attracted increasing attention. The limit for EC in yellow wine is down to 100 μg/L, while the concentration of EC in yellow wine brewing ranges from 100 to 750 μg/L (Chen et al. 2017), which leads to great food safety and health concerns for long-term drinkers. The main methods used to reduce EC in food are process optimization (Weber and Sharypov 2009), metabolic engineering (Wu et al. The research data generated and/or analyzed during the current study are available upon reasonable request. 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Biol Bull 36(11):94–102. https://doi.org/10.13560/j.cnki.biotech.bull.1985.2020-0220 We are thankful for the support from the Wuxi Supercomputer Platform and Protein purification Platform of Jiangnan University. This work was supported by the National Key Research and Development Program of China (2021YFC2104001, 2022YFC21055001); the China Postdoctoral Science Foundation (2022M711368); the Fundamental Research Funds for the Central Universities (JUSRP122037); and the Natural Science Foundation of Jiangsu Province, Science, and Technology Department of Jiangsu Province, China (BK20221081). All authors contributed to the study conception and design. QJ. Z conducted experiments. QJ. Z and MF. L wrote the manuscript. All authors read and approved the final manuscript. This article does not contain any studies with human participants or animals performed by any of the authors. All authors provided consent for the publication of the manuscript. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Primer pairs used for site-directed mutagenesis of EC hydrolase by PCR. Table S2. Molecular dynamic constants of single-point mutations, double-point mutations and three-point mutations. Table S3. Comparison of mutation and properties of part of EC hydrolase and urethanase. Figure S1. RMSF of protein structure under 1 bar and 10% (v/v), 50% (v/v), 100% (v/v) ethanol. Figure S2. RMSF of protein structure under 500 bar and 10% (v/v), 50% (v/v), 100% (v/v) ethanol. Figure S3. RMSF of protein structure under 1000 bar and 10% (v/v), 50% (v/v), 100% (v/v) ethanol. Figure S4. Optimal temperature of the double mutant proteins. Figure S5. Ethanol tolerance of the double mutant proteins. Figure S6. RMSF of the triple mutant protein structure under 1 bar and 50% (v/v) ethanol. Figure S7. Hydrophilic accessible surface area and hydrophobic accessible surface area of WT and triple mutant enzyme. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Zan, Q., Long, M., Zheng, N. et al. Improving ethanol tolerance of ethyl carbamate hydrolase by diphasic high pressure molecular dynamic simulations.
AMB Expr 13, 32 (2023). https://doi.org/10.1186/s13568-023-01538-7 Received: Accepted: Published: DOI: https://doi.org/10.1186/s13568-023-01538-7Availability of data and materials
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