Improving the thermal stability of <italic style="font-style: italic">trans</italic>-epoxysuccinate hydrolase

Wenna BAO; Jinfeng YAO; Haifeng PAN; Ronglin ZHU; Xinying LI; Hongxiu LIAO

doi:10.1631/jzus.B2500069

Your Location：

Home >

Browse articles >

Improving the thermal stability of trans-epoxysuccinate hydrolase

Scan QR Code

Alert me when the article has been cited

Submit

Research Article | Updated：2026-01-27

Improving the thermal stability of trans-epoxysuccinate hydrolase
Improving the thermal stability of trans-epoxysuccinate hydrolase
反式环氧琥珀酸水解酶的热稳定性改造
Journal of Zhejiang University-SCIENCE B Vol. 27, Issue 1, Pages: 89-100(2026)
Affiliations：

1.School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China
2.School of Life Sciences and Health, Huzhou College, Huzhou 313000, China
Funds：
DOI：10.1631/jzus.B2500069
CLC：
CSTR：
Received：09 February 2025，

Revised：2025-05-12，

Published Online：22 December 2025，

Published：27 January 2026
Accepted：

鲍文娜,姚锦锋,潘海峰等.反式环氧琥珀酸水解酶的热稳定性改造[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2026,27(01):89-100.

Wenna BAO, Jinfeng YAO, Haifeng PAN, et al. Improving the thermal stability of trans-epoxysuccinate hydrolase[J]. Journal of Zhejiang University-SCIENCE B, 2026, 27(1): 89-100.
鲍文娜,姚锦锋,潘海峰等.反式环氧琥珀酸水解酶的热稳定性改造[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2026,27(01):89-100. DOI： 10.1631/jzus.B2500069.

Wenna BAO, Jinfeng YAO, Haifeng PAN, et al. Improving the thermal stability of trans-epoxysuccinate hydrolase[J]. Journal of Zhejiang University-SCIENCE B, 2026, 27(1): 89-100. DOI： 10.1631/jzus.B2500069.

摘要

本研究通过运用分子动力学模拟、B因子分析和饱和突变筛选等方法，提高了源自韩国假单胞菌的反式环氧琥珀酸水解酶（TESH）的热稳定性。在筛选出的11个影响该特性的突变体中，4个突变体活性有所提高，其中突变体A142C和S178Q的米氏常数（

）较低，其

cat

（催化常数）/

比值分别比野生型的高3.7倍和0.9倍。上述两个突变体在50 ℃下的半衰期（

https://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=99131305&type=

3.47133350

https://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=99131322&type=

4.14866638

）值相较野生型分别提高了107%和59%。分子对接和分子动力学模拟表明，与野生型相比，上述两个突变体与底物的相互作用更强，结合能更低，均方根偏差更小，且突变位点附近的静电势能更低，疏水性增强。综上，本研究结果可为内消旋酒石酸的工业化生产提供参考，具有现实意义。

Abstract

This study used molecular dynamics simulations

B-factor analysis

and saturation mutagenesis screening to enhance the thermal stability of the

trans

-epoxysuccinate hydrolase (TESH) derived from

Pseudomonas koreensis

. Eleven mutants that influence this characteristic were selected

yielding four mutants with improved activity. Among them

mutants A142C and S178Q exhibited lower Michaelis constant (

) values

and their

cat

ratios (

cat

catalytic constant) were 3.7 and 0.9 times higher than those of the wild type

respectively. The values of half-life at 50 ℃ (

https://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=99131256&type=

3.47133350

https://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=99131258&type=

4.14866638

) of the two mutants were increased by 107% and 59%

respectively

compared to the wild type. Molecular docking and molecular dynamics simulations indicated that the two mutants showed stronger substrate interaction

lower binding energy

and reduced root mean square deviation compared to the wild type

along with decreased electrostatic potential energy and increased hydrophobicity near their mutation sites. The study of protein thermal stability engineering and associated mechanisms provides a valuable reference and holds practical significance for the industrial production of

meso

-tartaric acid.

关键词

Keywords

references

Archelas A , Furstoss R , 1998 . Epoxide hydrolases: new tools for the synthesis of fine organic chemicals . Trends Biotechnol , 16 ( 3 ): 108 - 116 . https://doi.org/10.1016/S0167-7799(97)01161-X https://doi.org/10.1016/S0167-7799(97)01161-X

Ban XF , Xie XF , Li CM , et al. , 2021 . The desirable salt bridges in amylases: distribution, configuration and location . Food Chem , 354 : 129475 . https://doi.org/10.1016/j.foodchem.2021.129475 https://doi.org/10.1016/j.foodchem.2021.129475

Bao WN , Pan HF , Zhang ZH , et al. , 2014 . Analysis of essential amino acid residues for catalytic activity of cis -epoxysuccinate hydrolase from Bordetella sp. BK-52 . Appl Microbiol Biotechnol , 98 ( 4 ): 1641 - 1649 . https://doi.org/10.1007/s00253-013-5019-2 https://doi.org/10.1007/s00253-013-5019-2

Bao WN , Pan HF , Zhang ZH , et al. , 2015 . Isolation of the stable strain Labrys sp. BK-8 for l (+)-tartaric acid production . J Biosci Bioeng , 119 ( 5 ): 538 - 542 . https://doi.org/10.1016/j.jbiosc.2014.10.013 https://doi.org/10.1016/j.jbiosc.2014.10.013

Bhonsle JB , Venugopal D , Huddler DP , et al. , 2007 . Application of 3D-QSAR for identification of descriptors defining bioactivity of antimicrobial peptides . J Med Chem , 50 ( 26 ): 6545 - 6553 . https://doi.org/10.1021/jm070884y https://doi.org/10.1021/jm070884y

Carugo O , 2022 . B -factor accuracy in protein crystal structures . Acta Crystallogr Sect D Struct Biol , 78 ( 1 ): 69 - 74 . https://doi.org/10.1107/S2059798321011736 https://doi.org/10.1107/S2059798321011736

Cheng YQ , Wang L , Pan HF , et al. , 2014 . Purification and characterization of a novel cis -epoxysuccinate hydrolase from Klebsiella sp. that produces l (+)-tartaric acid . Biotechnol Lett , 36 ( 11 ): 2325 - 2330 . https://doi.org/10.1007/s10529-014-1614-2 https://doi.org/10.1007/s10529-014-1614-2

da Fonseca AM , Caluaco BJ , Madureira JMC , et al. , 2024 . Screening of potential inhibitors targeting the main protease structure of SARS-CoV-2 via molecular docking, and approach with molecular dynamics, RMSD, RMSF, H-bond, SASA and MMGBSA . Mol Biotechnol , 66 ( 8 ): 1919 - 1933 . https://doi.org/10.1007/s12033-023-00831-x https://doi.org/10.1007/s12033-023-00831-x

Han JY , Ding YJ , Wei QN , et al. , 2024 . Expression and characterization of a bifunctional glycoside hydrolase IDSGH5-23 from Ruminococcus albus . J Zhejiang Univ (Agric Life Sci) , 50 ( 6 ): 963 - 972 . https://doi.org/10.3785/j.issn.1008-9209.2023.09.281 https://doi.org/10.3785/j.issn.1008-9209.2023.09.281

Han NY , Ma Y , Mu YL , et al. , 2019 . Enhancing thermal tolerance of a fungal GH11 xylanase guided by B-factor ana lysis and multiple sequence alignment. Enzyme Microb Technol , 131 : 109422 . https://doi.org/10.1016/j.enzmictec.2019.109422 https://doi.org/10.1016/j.enzmictec.2019.109422

Hendsch ZS , Tidor B , 1994 . Do salt bridges stabilize proteins? A continuum electrostatic analysis . Protein Sci , 3 ( 2 ): 211 - 226 . https://doi.org/10.1002/pro.5560030206 https://doi.org/10.1002/pro.5560030206

Kamatani Y , Okazaki H , Imai K , et al. , 1977 . Production of l (+)-tartaric acid . US Patent 4011135 A.

Liao HX , Pan HF , Yao JF , et al. , 2024 . Essential amino acid residues and catalytic mechanism of trans -epoxysuccinate hydrolase for production of meso -tartaric acid . Biotechnol Lett , 46 ( 5 ): 739 - 749 . https://doi.org/10.1007/s10529-024-03490-3 https://doi.org/10.1007/s10529-024-03490-3

Prescher G , Schreyer G , 1979 . Process for the production of pure racemic acid and mesotartaric acid and separation of maleic acid from synthetic tartaric acid . US Patent 4150241 A.

Rosenberg M , Miková H , Krištofíková L , 1999 . Production of l -tartaric acid by immobilized bacterial cells Nocardia Tartaricans . Biotechnol Lett , 21 ( 6 ): 491 - 495 . https://doi.org/10.1023/A:1005592104426 https://doi.org/10.1023/A:1005592104426

Sato E , Yanai A , 1976 . Method for preparing d -tartaric acid . US Patent 3957579 A.

Sakura K , Esther W , Darshan S , et al. , 2023 . Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 24 ( 12 ): 1069 - 1092 . https://doi.org/10.1631/jzus.B2200552 https://doi.org/10.1631/jzus.B2200552

Sinha S , Tam B , Wang SM , 2022 . Applications of molecular dynamics simulation in protein study . Membranes , 12 ( 9 ): 844 . https://doi.org/10.3390/membranes12090844 https://doi.org/10.3390/membranes12090844

Steinreiber A , Faber K , 2001 . Microbial epoxide hydrolases for preparative biotransformations . Curr Opin Biotechnol , 12 ( 6 ): 552 - 558 . https://doi.org/10.1016/S0958-1669(01)00262-2 https://doi.org/10.1016/S0958-1669(01)00262-2

Stigter D , Alonso DO , Dill KA , 1991 . Protein stability: electrostatics and compact denatured states . Proc Natl Acad Sci USA , 88 ( 10 ): 4176 - 4180 . https://doi.org/10.1073/pnas.88.10.4176 https://doi.org/10.1073/pnas.88.10.4176

Sumbalova L , Stourac J , Martinek T , et al. , 2018 . HotsPot Wizard 3.0: web server for automated design of mutations and smart libraries based on sequence input information . Nucleic Acids Res , 46 ( W1 ): W356 - W362 . https://doi.org/10.1093/nar/gky417 https://doi.org/10.1093/nar/gky417

Sun ZT , Liu Q , Qu G , et al. , 2019 . Utility of B-factors in protein science: interpreting rigidity, flexibility, and internal motion and engineering thermostability . Chem Rev , 119 ( 3 ): 1626 - 1665 . https://doi.org/10.1021/acs.chemrev.8b00290 https://doi.org/10.1021/acs.chemrev.8b00290

Tang H , Shi K , Shi C , et al. , 2019 . Enhancing subtilisin thermostability through a modified normalized B-factor analysis and loop-grafting strategy . J Biol Chem , 294 ( 48 ): 18398 - 18407 . https://doi.org/10.1074/jbc.RA119.010658 https://doi.org/10.1074/jbc.RA119.010658

Taylor WR , 1999 . Protein structural domain identification . Protein Eng Des Sel , 12 ( 3 ): 203 - 216 . https://doi.org/10.1093/protein/12.3.203 https://doi.org/10.1093/protein/12.3.203

Wang ZQ , Wang YS , Su ZG , 2013 . Purification and characterization of a cis -epoxysuccinic acid hydrolase from Nocardia tartaricans CAS-52, and expression in Escherichia coli . Appl Microbiol Biotechnol , 97 ( 6 ): 2433 - 2441 . https://doi.org/10.1007/s00253-012-4102-4 https://doi.org/10.1007/s00253-012-4102-4

Waterhouse A , Bertoni M , Bienert S , et al. , 2018 . SWISS-MODEL: homology modelling of protein structures and complexes . Nucleic Acids Res , 46 ( W1 ): W296 - W303 . https://doi.org/10.1093/nar/gky427 https://doi.org/10.1093/nar/gky427

Weng JR , Yang S , Shen JK , et al. , 2023 . Molecular dynamics simulation reveals DNA-specific recognition mechanism via c-Myb in pseudo-palindromic consensus of mim-1 promoter . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 24 ( 10 ): 883 - 895 . https://doi.org/10.1631/jzus.B2200634 https://doi.org/10.1631/jzus.B2200634

Zhang Y , Geary T , Simpson BK , 2019 . Genetically modified food enzymes: a review . Curr Opin Food Sci , 25 : 14 - 18 . https://doi.org/10.1016/j.cofs.2019.01.002 https://doi.org/10.1016/j.cofs.2019.01.002

Zhu WL , Sun HM , Jiang QX , et al. , 2022 . Enhancing the thermal stability of glutathione bifunctional synthase by B-factor strategy and un/folding free energy calculation . Catalysts , 12 ( 12 ): 1649 . https://doi.org/10.3390/catal12121649 https://doi.org/10.3390/catal12121649

Views

Downloads

CSCD

Tools

Publicity Resources

Molecular dynamics simulation reveals DNA-specific recognition mechanism via c-Myb in pseudo-palindromic consensus of mim-1 promoter

Molecular dynamics simulation of the interactions between EHD1 EH domain and multiple peptides#

Microstructural characteristic and martensite-austenite constituent of X70 pipeline steel under ultra-fast cooling process

Research on the turbulent particle dispersion model in baffle demisters

Multi-resolution analysis of wind loads characteristics on wind barrier caused by CRH380 train based on field measurement data

Related Author

Shan WANG

Dongyun HAO

Yuzi XU

Hongsen LIU

Jinkang SHEN

Shuo YANG

Jinru WENG

Mao-Jun Wang

Related Institution

Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Engineering Research Center of Oral Biomaterials and Devices of Zhejiang Province

Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University

Institute of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences (JAAS)

Department of Chemistry, Zhejiang University

Address：Journal of Zhejiang University-SCIENCE, 38 Zheda Road, Hangzhou, China Postal code：310027
Tel：+86-571-87952783 Email：cjzhang@zju.edu.cn
Technical support is provided by Beijing Founder electronics co., LTD 京ICP备09064830号-19 京公网安备11010802024621
It is recommended to read the content of this site in Chrome&IE9+. Please switch to extreme mode in browser 360.
Cookies We use cookies to help provide and enhance our service and tailor content. By continuing, you agree to the use of cookies.

⁰

Improving the thermal stability of trans-epoxysuccinate hydrolase

Improving the thermal stability of trans-epoxysuccinate hydrolase

DOI：10.1631/jzus.B2500069

摘要

Abstract

关键词

Keywords

references