Similar articles

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 (K_m) values, and their k_cat/K_m ratios (k_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 °C (T1/250) 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.

反式环氧琥珀酸水解酶的热稳定性改造

[1]ArchelasA, FurstossR, 1998. Epoxide hydrolases: new tools for the synthesis of fine organic chemicals. Trends Biotechnol, 16(3):108-116.

[2]BanXF, XieXF, LiCM, et al., 2021. The desirable salt bridges in amylases: distribution, configuration and location. Food Chem, 354:129475.

[3]BaoWN, PanHF, ZhangZH, 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.

[4]BaoWN, PanHF, ZhangZH, et al., 2015. Isolation of the stable strain Labrys sp. BK-8 for l(+)-tartaric acid production. J Biosci Bioeng, 119(5):538-542.

[5]BhonsleJB, VenugopalD, HuddlerDP, et al., 2007. Application of 3D-QSAR for identification of descriptors defining bioactivity of antimicrobial peptides. J Med Chem, 50(26):6545-6553.

[6]CarugoO, 2022. B-factor accuracy in protein crystal structures. Acta Crystallogr Sect D Struct Biol, 78(1):69-74.

[7]ChengYQ, WangL, PanHF, 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.

[8]da FonsecaAM, CaluacoBJ, MadureiraJMC, 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.

[9]HanJY, DingYJ, WeiQN, 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.

[10]HanNY, MaY, MuYL, et al., 2019. Enhancing thermal tolerance of a fungal GH11 xylanase guided by B-factor analysis and multiple sequence alignment. Enzyme Microb Technol, 131:109422.

[11]HendschZS, TidorB, 1994. Do salt bridges stabilize proteins? A continuum electrostatic analysis. Protein Sci, 3(2):211-226.

[12]KamataniY, OkazakiH, ImaiK, et al., 1977. Production of l(+)-tartaric acid. US Patent 4011135A.

[13]LiaoHX, PanHF, YaoJF, 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.

[14]PrescherG, SchreyerG, 1979. Process for the production of pure racemic acid and mesotartaric acid and separation of maleic acid from synthetic tartaric acid. US Patent 4150241A.

[15]RosenbergM, MikováH, KrištofíkováL, 1999. Production of l-tartaric acid by immobilized bacterial cells Nocardia Tartaricans. Biotechnol Lett, 21(6):491-495.

[16]SatoE, YanaiA, 1976. Method for preparing d-tartaric acid. US Patent 3957579A.

[17]SakuraK, EstherW, DarshanS, 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.

[18]SinhaS, TamB, WangSM, 2022. Applications of molecular dynamics simulation in protein study. Membranes, 12(9):844.

[19]SteinreiberA, FaberK, 2001. Microbial epoxide hydrolases for preparative biotransformations. Curr Opin Biotechnol, 12(6):552-558.

[20]StigterD, AlonsoDO, DillKA, 1991. Protein stability: electrostatics and compact denatured states. Proc Natl Acad Sci USA, 88(10):4176-4180.

[21]SumbalovaL, StouracJ, MartinekT, 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.

[22]SunZT, LiuQ, QuG, 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.

[23]TangH, ShiK, ShiC, et al., 2019. Enhancing subtilisin thermostability through a modified normalized B-factor analysis and loop-grafting strategy. J Biol Chem, 294(48):18398-18407.

[24]TaylorWR, 1999. Protein structural domain identification. Protein Eng Des Sel, 12(3):203-216.

[25]WangZQ, WangYS, SuZG, 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.

[26]WaterhouseA, BertoniM, BienertS, et al., 2018. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res, 46(W1):W296-W303.

[27]WengJR, YangS, ShenJK, 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.

[28]ZhangY, GearyT, SimpsonBK, 2019. Genetically modified food enzymes: a review. Curr Opin Food Sci, 25:14-18.

[29]ZhuWL, SunHM, JiangQX, 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.