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Synthesis of Short-Chain Alkyl Butyrate through Esterification Reaction Using Immobilized Rhodococcus Cutinase and Analysis of Substrate Specificity through Molecular Docking

  • Seok-Jae Won (Department of Biotechnology, The Catholic University of Korea) ;
  • Joung Han Yim (Korea Polar Research Institute) ;
  • Hyung Kwoun Kim (Department of Biotechnology, The Catholic University of Korea)
  • Received : 2022.11.09
  • Accepted : 2022.11.26
  • Published : 2023.02.28

Abstract

Alkyl butyrate with fruity flavor is known as an important additive in the food industry. We synthesized various alkyl butyrates from various fatty alcohol and butyric acid using immobilized Rhodococcus cutinase (Rcut). Esterification reaction was performed in a non-aqueous system including heptane, isooctane, hexane, and cyclohexane. As a result of performing the alkyl butyrate synthesis reaction using alcohols of various chain lengths, it was found that the preference for the alcohol substrate had the following order: C6 > C4 > C8 > C10 > C2. Through molecular docking analysis, it was found that the greater the hydrophobicity of alcohol, the higher the accessibility to the active site of the enzyme. However, since the number of torsions increased as the chain length increased, it became difficult for the hydroxyl oxygen of the alcohol to access the γO of serine at the enzyme active site. These molecular docking results were consistent with substrate preference results of the Rcut enzyme. The Rcut maintained the synthesis efficiency at least for 5 days in isooctane solvent. We synthesized as much as 452 mM butyl butyrate by adding 100 mM substrate daily for 5 days and performing the reaction. These results show that Rcut is an efficient enzyme for producing alkyl butyrate used in the food industry.

Keywords

Acknowledgement

This research was supported by Korea Polar Research Institute (PE21150) and funded by the Ministry of Oceans and Fisheries.

References

  1. Chen S, Tong X, Woodard RW, Du G, Wu J, Chen J. 2008. Identification and characterization of bacterial cutinase. J. Biol. Chem. 283: 25854-25862. https://doi.org/10.1074/jbc.M800848200
  2. Vazquez-Alcantara L, Oliart-Ros RM, Garcia-Borquez A, Pena-Montes C. 2021. Expression of a cutinase of Moniliophthora roreri with polyester and PET-plastic residues degradation activity. Microbiol. Spectr. 9: 976.
  3. Yan Z, Wang L, Xia W, Liu Z, Gu L, Wu J. 2021. Synergistic biodegradation of poly (ethylene terephthalate) using Microbacterium oleivorans and Thermobifida fusca cutinase. Appl. Microbiol. Biotechnol. 105: 4551-4560. https://doi.org/10.1007/s00253-020-11067-z
  4. Yang S, Xu H, Yan Q, Liu Y, Zhou P, Jiang Z. 2013. A low molecular mass cutinase of Thielavia terrestris efficiently hydrolyzes poly (esters). J. Ind. Microbiol. Biotechnol. 40: 217-226. https://doi.org/10.1007/s10295-012-1222-x
  5. de Marco A. 2009. Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microb. Cell Fact. 8: 26.
  6. Gaciarz A, Khatri NK, Velez-Suberbie ML, Saaranen MJ, Uchida Y, Keshavarz-Moore E, et al. 2017. Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media. Microb. Cell Fact. 16: 108.
  7. Su L, Hong R, Guo X, Wu J, Xia Y. 2016. Short-chain aliphatic ester synthesis using Thermobifida fusca cutinase. Food Chem. 206: 131-136. https://doi.org/10.1016/j.foodchem.2016.03.051
  8. Dutta K, Dasu VV. 2011. Synthesis of short chain alkyl esters using cutinase from Burkholderia cepacia NRRL B2320. J. Molec. Catal. B. 72: 150-156. https://doi.org/10.1016/j.molcatb.2011.05.013
  9. Xu Y, Wang X, Liu X, Li X, Zhang C, Li W, et al. 2021. Discovery and development of a novel short-chain fatty acid ester synthetic biocatalyst under aqueous phase from Monascus purpureus isolated from Baijiu. Food Chem. 338: 128025.
  10. Mahapatra P, Kumari A, Garlapati VK, Banerjee R, Nag A. 2009. Enzymatic synthesis of fruit flavor esters by immobilized lipase from Rhizopus oligosporus optimized with response surface methodology. J. Molec. Catal. B. 60: 57-63. https://doi.org/10.1016/j.molcatb.2009.03.010
  11. Cvjetko M, Vorkapic-Furac J, Znidarsic-Plazl P. 2012. Isoamyl acetate synthesis in imidazolium-based ionic liquids using packed bed enzyme microreactor. Process Biochem. 47: 1344-1350. https://doi.org/10.1016/j.procbio.2012.04.028
  12. Jaiswal KS, Rathod VK. 2022. Process intensification of enzymatic synthesis of flavor esters: a review. Chem. Rec. 22: e202100213.
  13. Friedrich JL, Pena FP, Garcia-Galan C, Fernandez-Lafuente R, Ayub MA, Rodrigues RC. 2013. Effect of immobilization protocol on optimal conditions of ethyl butyrate synthesis catalyzed by lipase B from Candida antarctica. J. Chem. Technol. Biotechnol. 88: 1089-1095. https://doi.org/10.1002/jctb.3945
  14. Elias N, Wahab RA, Chandren S, Razak FIA, Jamalis J. 2019. Effect of operative variables and kinetic study of butyl butyrate synthesis by Candida rugosa lipase activated by chitosan-reinforced nanocellulose derived from raw oil palm leaves. Enzyme Microb. Technol. 130: 109367.
  15. Dos Santos MMO, Gama RS, de Carvalho Tavares IM, Santos PH, Goncalves MS, de Carvalho MS, et al. 2021. Application of lipase immobilized on a hydrophobic support for the synthesis of aromatic esters. Biotechnol. Appl. Biochem. 68: 538-546. https://doi.org/10.1002/bab.1959
  16. Vandamme EJ, Soetaert W. 2002. Bioflavours and fragrances via fermentation and biocatalysis. J. Chem. Technol. Biotechnol. 77: 1323-1332. https://doi.org/10.1002/jctb.722
  17. Khan NR, Rathod VK. 2015. Enzyme catalyzed synthesis of cosmetic esters and its intensification: A review. Process Biochem. 50: 1793-1806. https://doi.org/10.1016/j.procbio.2015.07.014
  18. Martinez A, Maicas S. 2021. Cutinases: characteristics and insights in industrial production. Catalyst 11: 1194.
  19. de Barros DP, Azevedo AM, Cabral JM, Fonseca LP. 2012. Optimization of flavor esters synthesis by Fusarium solani pisi cutinase. J. Food Biochem. 36: 275-284. https://doi.org/10.1111/j.1745-4514.2010.00535.x
  20. Duan X, Liu Y, You X, Jiang Z, Yang S, Yang S. 2017. High-level expression and characterization of a novel cutinase from Malbranchea cinnamomea suitable for butyl butyrate production. Biotechnol. Biofuels 10: 223.
  21. Nikolaivits E, Makris G, Topakas E. 2017. Immobilization of a cutinase from Fusarium oxysporum and application in pineapple flavor synthesis. J. Agric. Food Chem. 65: 3505-3511. https://doi.org/10.1021/acs.jafc.7b00659
  22. Xu H, Yan Q, Duan X, Yang S, Jiang Z. 2015. Characterization of an acidic cold-adapted cutinase from Thielavia terrestris and its application in flavor ester synthesis. Food Chem. 188: 439-445. https://doi.org/10.1016/j.foodchem.2015.05.026
  23. Won S, Yim JH, Kim H. 2022. Functional production, characterization, and immobilization of a cold-adapted cutinase from Antarctic Rhodococcus sp. Protein Expr. Purif. 195: 106077.
  24. Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, et al. 2018. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46: W296-W303. https://doi.org/10.1093/nar/gky427
  25. Basso A, Serban S. 2019. Industrial applications of immobilized enzymes-a review. Mol. Catal. 479: 110607.
  26. Martins AB, Friedrich JL, Rodrigues RC, Garcia-Galan C, Fernandez-Lafuente R, Ayub MA. 2013. Optimized butyl butyrate synthesis catalyzed by Thermomyces lanuginosus lipase. Biotechnol. Prog. 29: 1416-1421. https://doi.org/10.1002/btpr.1793
  27. Ahmed EH, Raghavendra T, Madamwar D. 2010. An alkaline lipase from organic solvent tolerant Acinetobacter sp. EH28: application for ethyl caprylate synthesis. Bioresour. Technol. 101: 3628-3634. https://doi.org/10.1016/j.biortech.2009.12.107
  28. Kim S, Chen J, Cheng T, Gindulyte A, He J, He S, et al. 2021. PubChem in 2021: new data content and improved web interfaces. Nucleic Acids Res. 49(D1): D1388-D1395. https://doi.org/10.1093/nar/gkaa971
  29. Su A, Shirke A, J Baik, Y Zou, R Gross. 2018. Immobilized cutinases: preparation, solvent tolerance and thermal stability. Enzyme Microb. Technol. 116: 33-40. https://doi.org/10.1016/j.enzmictec.2018.05.010
  30. Wang Z, Su T, Zhao J. 2021. Immobilization of Fusarium solani cutinase onto magnetic genipin-crosslinked chitosan beads. Catalysts 11: 1158.
  31. Duan X, Jiang Z, Liu Y, Yan Q, Xiang M, Yang S. 2019. High-level expression of codon-optimized Thielavia terrestris cutinase suitable for ester biosynthesis and biodegradation. Int. J. Biol. Macromol. 135: 768-775. https://doi.org/10.1016/j.ijbiomac.2019.05.173
  32. Shen J, Cai X, Dou B, Qi F, Zhang X, Liu Z, Zheng Y. 2020. Expression and characterization of a CALB-type lipase from Sporisorium reilianum SRZ2 and its potential in short-chain flavor ester synthesis. Front. Chem. Sci. Eng. 14: 868-879. https://doi.org/10.1007/s11705-019-1889-x
  33. Foukis A, Gkini OA, Stergiou P, Papamichael EM. 2018. New insights and tools for the elucidation of lipase catalyzed esterification reaction mechanism in n-hexane: The synthesis of ethyl butyrate. Mol. Catal. 455: 159-163. https://doi.org/10.1016/j.mcat.2018.06.004
  34. Cai Y, Xing S, Zhang Q, Zhu R, Cheng K, Li C, et al. 2021. Expression, purification, properties, and substrate specificity analysis of Aspergillus niger GZUF36 lipase in Escherichia coli. Process Biochem. 111: 118-127. https://doi.org/10.1016/j.procbio.2021.09.002