Reactor engineering for the enzymatic synthesis of 5-hydroxymethylfurfural stearate in a batch bioreactor and a packed bed flow bioreactor

Abstract

The enzymatic esterification of 5-hydroxymethylfurfural (HMF) with long-chain fatty acids offers a sustainable route for producing biolubricants and other high-value chemicals. This work evaluates the synthesis of 5-hidroxymethylfurfural stearate catalyzed by immobilized lipases in both batch and continuous packed-bed bioreactors, combining molecular dynamics (MD) simulations with experimental validation to identify suitable green solvents. Four solvents were tested: 2-methyl-3-buten-2-ol (2-MB), tert-butanol (TB), 2-methyltetrahydrofuran (2-MeTHF), and cyclopentyl methyl ether (CPME). MD simulations revealed that CPME increased hydrophobic surface exposure and flexibility near the catalytic site, favoring substrate accessibility. In preliminary experimental tests, CPME provided the highest conversion (50%). In batch bioreactor at 40 °C, 30 mM HMF and 250 mM stearic acid achieved 67% conversion with Candida antarctica lipase B (CALB), maintaining full activity (100%) over four reuse cycles. In continuous operation, using a single packed-bed bioreactor at 0.02 mL min⁻¹ yielded conversions above 50% (residence time ≈ 55 min), while connecting two packed-bed bioreactors in series increased conversion to over 90% and productivity to 0.094 h⁻¹, compared with 0.076 h⁻¹ for one column and 0.003 h⁻¹ in batch mode. Deviations from ideal plug flow were observed over time, attributed to substrate or product deposition and in-situ water formation shifting the reaction equilibrium. Overall, CPME proved to be an efficient and sustainable solvent for the enzymatic synthesis of 5-hydroxymethylfurfural stearate, demonstrating the feasibility of continuous operation and highlighting pathways for further optimization through improved immobilization or reactor design.

Graphical Abstract

Keywords

Lipases / Biobased solvents / Flow biocatalysis / 5-hydroxymethylfurfural / Enzymatic esterification

References

[1] Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, Lindah E (2015) Gromacs: high performance molecular simulations through multi-level parallelism fromlaptops to supercomputers. SoftwareX 1–2:19–25. https://doi.org/10.1016/j.softx.2015.06.001

[2] Agger JW, Zeuner B. Bio-based surfactants: enzymatic functionalization and production from renewable resources. Curr Opin Biotechnol. 2022, 78: 102842.

[3] Aguieiras ECG, Papadaki A, Mallouchos Aet al. . Enzymatic synthesis of bio-based wax esters from palm and soybean fatty acids using crude lipases produced on agricultural residues. Ind Crops Prod. 2019, 139111499.

[4] Al-Zuhair S, Almenhali A, Hamad Iet al. . Enzymatic production of biodiesel from used/waste vegetable oils: Design of a pilot plant. Renewable Energy. 2011, 362605-2614.

[5] Ali A, Moradian JM, Naveed Aet al. . To overcome the unconventional polymerization behavior of single-site symmetrical metallocene catalyst in the presence of borate. Appl Catal A. 2024, 683: 119830.

[6] Ali A, Moradian JM, Naveed Aet al. . Capability of isoprene and butadiene as comonomers under metallocene catalysis: Activating active sites with the addition of 1-hexene and propylene. J Catal. 2024, 438115729.

[7] Ali A, Nadeem M, Naveed Aet al. . Copolymerization of ethylene and isoprene initiated by metallocene catalyst. Arab J Chem. 2024, 17105989.

[8] Ali A, Mohammadi Moradian J, Naveed Aet al. . Progress in cathode materials for rechargeable Zinc-Ion batteries: from inorganic and organic systems to hybrid frameworks and biomass-derived innovations. Prog Mater Sci. 2026, 1561-116.

[9] Annunziata F, Contente ML, Anzi Vet al. . Enzymatic continuous-flow preparation of nature-inspired phenolic esters as antiradical and antimicrobial agents. Food Chem. 2022, 390: 133195.

[10] Araya A, Guajardo N, Lienqueo ME. Control of selectivity in the oxidation of 5-hydroxymethylfurfural to 5- formyl-2-furancarboxylic acid catalyzed by laccase in a multiphasic gas-liquid microbioreactor. Bioresour Technol. 2024, 394130154.

[11] Balboa C, Schrebler RA, Lienqueo ME, Guajardo N. Effect of Operational Variables on the Yield of Chemoenzymatic Oxidation of 2,5-Furandicarboxaldehyde to 2,5-Furandicarboxylic Acid in Fed-Batch and Continuous Packed-Bed Millibioreactor. Processes. 2022, 102-13.

[12] Benyahia F, O’Neill KE. Enhanced voidage correlations for packed beds of various particle shapes and sizes. Part Sci Technol. 2005, 23: 169-177.

[13] Berendsen HJC, van der Spoel D, van Drunen R (1995) GROMACS: A message-passing parallel molecular dynamics implementation. Comput Phys Commun 91:43–56. https://doi.org/10.1016/0010-4655(95)00042-E

[14] Bolaño Losada C, Slaný O, Byrtusová D et al (2025) Compatible traits of oleaginous Mucoromycota fungi for lignocellulose-based simultaneous saccharification and fermentation. Biotechnol Biofuels Bioprod 18. https://doi.org/10.1186/s13068-025-02621-w

[15] Brzozowski AM, Savage H, Verma CSet al. . Structural origins of the interfacial activation in Thermomyces (Humicola) lanuginosa lipase. Biochemistry. 2000, 39: 15071-15082.

[16] Bussi G, Donadio D, Parrinello M. Canonical sampling through velocity rescaling. J Chem Phys. 2007.

[17] Cao J, Wu R, Zhu Fet al. . Enzymes in nearly anhydrous deep eutectic solvents : Insight into the biocompatibility and thermal stability. Enzym Microb Technol. 2022, 157: 110022.

[18] Castro GAD, Fernandes SA. Furans derived from lignocellulosic biomass as renewable substrates in multicomponent reactions. Sustainable Chem Pharm. 2025, 44101948.

[19] Chen JW, Wu WT. Regeneration of immobilized Candida antarctica lipase for transesterification. J Biosci Bioeng. 2003, 95466-469.

[20] Costa IO, Morais JRF, de Medeiros Dantas JM et al (2023) Enzyme immobilization technology as a tool to innovate in the production of biofuels: A special review of the Cross-Linked Enzyme Aggregates (CLEAs) strategy. Enzym Microb Technol 170

[21] Darden T, York D, Pedersen L (1993) in Large Systems. 10089–10092

[22] de Paula MS, de Oliveira LFC, Cunha FTet al. . Evaluation of continuous ethyl ester synthesis from acid soybean oil using a dual immobilized enzyme system. Biomass Bioenergy. 2021, 144: 105898.

[23] Domínguez de María P, Guajardo N. Biocatalytic Valorization of Furans: Opportunities for Inherently Unstable Substrates. Chemsuschem. 2017, 104123-4134.

[24] Duan ZQ, Du W, Liu DH. The mechanism of solvent effect on the positional selectivity of Candida antarctica lipase B during 1,3-diolein synthesis by esterification. Bioresour Technol. 2011, 102: 11048-11050.

[25] Duque TS, Barroso GM, Borges CEet al. . Current and future development of Acrocomia aculeata focused on biofuel potential and climate change challenges. Sci Rep. 2025, 15: 1-12.

[26] Eid N, Ameduri B, Boutevin B. Synthesis and Properties of Furan Derivatives for Epoxy Resins. ACS Sustainable Chem Eng. 2021, 9: 8018-8031.

[27] Fenton HJH, Gostling M (1901) Derivatives of methylfurfural. J Chem Soc 807–816

[28] Galmés M, García-Junceda E, Świderek K, Moliner V. Exploring the Origin of Amidase Substrate Promiscuity in CALB by a Computational Approach. ACS Catal. 2020, 10: 1938-1946.

[29] Gómez-Meyer D, Guajardo N, Lienqueo ME (2023) Improvement of the operational stability of the Novozym 435^® biocatalyst in the oxidation of 2,5-furandicarboxaldehyde to 2,5-furandicarboxylic acid in a continuous packed-bed microbioreactor. Tetrahedron Lett 121. https://doi.org/10.1016/j.tetlet.2023.154475

[30] González J, Guajardo N (2023) Flow enzymatic esterification of 5-hydroxymethylfurfural and liquid–liquid extraction of 5-acetyl-hydroxymethylfurfural using deep eutectic solvents in semicontinuous mode. 409–418

[31] Guajardo N. Immobilization of Lipases Using Poly(vinyl) Alcohol. Polymers. 2023, 15: 2021.

[32] Guajardo N, Domínguez de María P (2019) Continuous Biocatalysis in Environmentally-Friendly Media: A Triple Synergy for Future Sustainable Processes. ChemCatChem 11. https://doi.org/10.1002/cctc.201900773

[33] Guajardo N, Schrebler RA. Upstream and Downstream Bioprocessing in Enzyme Technology. Pharmaceutics. 2024, 1638.

[34] Guajardo N, Domínguez de María HP, Ahumada Ket al. . Water as Cosolvent: Nonviscous Deep Eutectic Solvents for Efficient Lipase-Catalyzed Esterifications. ChemCatChem. 2017, 91393-1396.

[35] Guajardo N, Ahumada K, Domínguez de María P. Immobilization of Pseudomonas stutzeri lipase through Cross-linking Aggregates (CLEA) for reactions in Deep Eutectic Solvents. J Biotechnol. 2021, 337: 18-23.

[36] Guo W, Kortenbach T, Qi Wet al. . Selective tandem catalysis for the synthesis of 5-hydroxymethylfurfural from glucose over in-situ phosphated titania catalysts: Insights into structure, bi-functionality and performance in flow microreactors. Appl Catal B. 2022, 301: 120800.

[37] Hess B (2008) P-LINCS : A Parallel Linear Constraint Solver for Molecular Simulation. 116–122

[38] Hu L, He A, Liu Xet al. . Biocatalytic Transformation of 5-Hydroxymethylfurfural into High-Value Derivatives: Recent Advances and Future Aspects. ACS Sustainable Chem Eng. 2018, 615915-15935.

[39] Hu D, Zhang M, Xu H et al (2021) Recent advance on the catalytic system for efficient production of biomass-derived 5-hydroxymethylfurfural. Renew Sustain Energy Rev 147. https://doi.org/10.1016/j.rser.2021.111253

[40] Huang J, Rauscher S, Nawrocki Get al. . CHARMM36m: An improved force field for folded and intrinsically disordered proteins. Nat Methods. 2016, 14: 71-73.

[41] Ioannou I, D’Angelo SC, Galán-Martín Áet al. . Process modelling and life cycle assessment coupled with experimental work to shape the future sustainable production of chemicals and fuels. Reaction Chem Eng. 2021, 6: 1179-1194.

[42] Jo S, Kim T, Iyer VG, Im W (2008) Software News and Updates CHARMM-GUI : A Web-Based Graphical User Interface for CHARMM. https://doi.org/10.1002/jcc

[43] Klibanov AM (1989) Enzymatic catalysis in anhydrous organic solvents. 141–144

[44] Klibanov AM (2001) Improving enzymes by using them in organic solvents. 409

[45] Krystof M, Pérez-Sánchez M, de Domínguez P. Lipase-Mediated Selective Oxidation of Furfural and 5-Hydroxymethylfurfural. Chemsuschem. 2013, 6: 826-830.

[46] Kumar A, Verma V, Dubey VK et al (2023) Industrial applications of fungal lipases: a review. Front Microbiol 14. https://doi.org/10.3389/fmicb.2023.1142536

[47] Lee YJ, Kim HW, Lee Jet al. . Synthesis of structured lipids using microalgae oil and capric acid via a two-step enzyme reaction. Process Biochem. 2025, 150: 213-220.

[48] Li C, Tan T, Zhang H, Feng W. Analysis of the conformational stability and activity of Candida antarctica lipase B in organic solvents: Insight from molecular dynamics and quantum mechanics/simulations. J Biol Chem. 2010, 285: 28434-28441.

[49] Madsen JK, Sørensen TR, Kaspersen JDet al. . Promoting protein self-association in non-glycosylated Thermomyces lanuginosus lipase based on crystal lattice contacts. Biochim et Biophys Acta - Proteins Proteom. 2015, 1854: 1914-1921.

[50] Mahfoudhi A, Benmabrouk S, Fendri A, Sayari A. Fungal lipases as biocatalysts: A promising platform in several industrial biotechnology applications. Biotechnol Bioeng. 2022, 119: 3370-3392.

[51] Mäki-Arvela P, Salminen E, Riittonen Tet al. . The challenge of efficient synthesis of biofuels from lignocellulose for future renewable transportation fuels. Int J Chem Eng 2012. 2012.

[52] Martínez L. ComplexMixtures.jl: Investigating the structure of solutions of complex-shaped molecules from a solvent-shell perspective. J Mol Liq. 2022, 347: 12-17.

[53] Matte CR, Bordinhaõ C, Poppe JKet al. . Synthesis of butyl butyrate in batch and continuous enzymatic reactors using Thermomyces lanuginosus lipase immobilized in Immobead 150. J Mol Catal B: Enzymatic. 2016, 127: 67-75.

[54] Mine Y, Zhang L, Fukunaga K, Sugimura Y. Enhancement of enzyme activity and enantioselectivity by cyclopentyl methyl ether in the transesterification catalyzed by Pseudomonas cepacia lipase co-lyophilized with cyclodextrins. Biotechnol Lett. 2005, 27: 383-388.

[55] Mohtashami M, Fooladi J, Haddad-Mashadrizeh Aet al. . Molecular mechanism of enzyme tolerance against organic solvents: Insights from molecular dynamics simulation. Int J Biol Macromol. 2019, 122: 914-923.

[56] Nazarian Z, Arab SS. Solvent-dependent activity of Candida antarctica lipase B and its correlation with a regioselective mono aza-Michael addition - experimental and molecular dynamics simulation studies. Heliyon. 2022, 8: e10336.

[57] Nie T, Bai X, Liao Zet al. . Engineering Patatin for enhanced lipase activity and long-chain fatty acid specificity via rational design. Food Chem. 2025, 482144155.

[58] Ognjanovic N, Bezbradica D, Knezevic-Jugovic Z. Enzymatic conversion of sunflower oil to biodiesel in a solvent-free system: Process optimization and the immobilized system stability. Bioresour Technol. 2009, 1005146-5154.

[59] Outeiriño D, Costa-Trigo I, Ochogavias Aet al. . Biorefinery of brewery spent grain to obtain bioproducts with high value-added in the market. New Biotechnol. 2024, 79: 111-119.

[60] Parrinello M, Rahman A (1981) Polymorphic transitions in single crystals: A new molecular dynamics method. 7190:7182–7190

[61] Pedro KCNR, da Silva GAR, da Silva MAP et al (2025) Immobilization of lipase on zeolite, silica, and silica-aluminas and its use in hydrolysis, esterification, and transesterification reactions. https://doi.org/10.1016/j.cattod.2024.115141. Catalysis Today 447:

[62] Petrenz A, María PD, De, Ramanathan Aet al. . Medium and reaction engineering for the establishment of achemo-enzymatic dynamic kinetic resolution of rac-benzoin in batchand continuous mode. J Mol Catal B: Enzymatic. 2015, 114: 42-49.

[63] Pollardo AA, Lee Hshik, Lee Det al. . Solvent effect on the enzymatic production of biodiesel from waste animal fat. J Clean Prod. 2018, 185: 382-388.

[64] Qin Y, Zong M, Lou W, Li N (2016a) Biocatalytic Upgrading of 5 – Hydroxymethylfurfural (HMF) with Levulinic Acid to HMF Levulinate in Biomass-Derived Solvents. 4–8. https://doi.org/10.1021/acssuschemeng.6b00996

[65] Qin YZ, Zong MH, Lou WY, Li N. Biocatalytic upgrading of 5-hydroxymethylfurfural (HMF) with levulinic acid to HMF levulinate in biomass-derived solvents. ACS Sustainable Chem Eng. 2016, 4: 4050-4054.

[66] Rehm S, Trodler P, rgen J P (2001) Solvent-induced lid opening in lipases: a molecular dynamics study. Protein Sci 19:2122–130

[67] Remonatto D, Miotti RH, Monti Ret al. . Applications of immobilized lipases in enzymatic reactors: A review. Process Biochem. 2022, 114: 1-20.

[68] Sayed ET, Wilberforce T, Elsaid Ket al. . A critical review on environmental impacts of renewable energy systems and mitigation strategies: Wind, hydro, biomass and geothermal. Sci Total Environ. 2021, 766: 144505.

[69] Shimizu S, Smith DJ. Preferential hydration and the exclusion of cosolvents from protein surfaces. J Chem Phys. 2004, 1211148-1154.

[70] Stepankova V, Bidmanova S, Koudelakova Tet al. . Strategies for stabilization of enzymes in organic solvents. ACS Catal. 2013, 3: 2823-2836.

[71] Stergiou PY, Foukis A, Filippou Met al. . Advances in lipase-catalyzed esterification reactions. Biotechnol Adv. 2013, 31: 1846-1859.

[72] Taylor P, Nosé S, Klein ML (2006) Molecular Physics : An International Journal at the Interface Between Chemistry and Physics Constant pressure molecular dynamics for molecular systems. 37–41

[73] Tong X, Busk PK, Lange L, Pang J. New insights into the molecular mechanism of methanol-induced inactivation of Thermomyces lanuginosus lipase: A molecular dynamics simulation study. Mol Simul. 2016, 42: 434-445.

[74] Uppenberg J, Hansen MT, Patkar S, Jones TA (1994) The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica

[75] Uribe J, Lienqueo ME, Guajardo N (2023) Optimization and Determination of Kinetic Parameters of the Synthesis of 5-Lauryl-hydroxymethylfurfural Catalyzed by Lipases. Catalysts 13

[76] Valotta A, Stelzer D, Reiter Tet al. . A multistep (semi)-continuous biocatalytic setup for the production of polycaprolactone. Reaction Chem Eng. 2023, 9: 713-727.

[77] Vanommeslaeghe K, MacKerell AD. Automation of the CHARMM general force field (CGenFF) I: Bond perception and atom typing. J Chem Inf Model. 2012, 52: 3144-3154.

[78] Vanommeslaeghe K, Raman EP, Mackerell AD (2012) Automation of the CHARMM General Force Field (CGenFF) II. Assignment of Bonded Parameters and Partial Atomic Charges

[79] Vardar-Yel N, Tütüncü HE, Sürmeli Y. Lipases for targeted industrial applications, focusing on the development of biotechnologically significant aspects: A comprehensive review of recent trends in protein engineering. Int J Biol Macromol. 2024, 273: 132853.

[80] Vernet G, Ma Y, Serban Set al. . Metal Affinity Fusion Enzyme Immobilization: Batch Process Showcase for Cascading Alcohol Oxidation and Bayer-Villiger Oxidation in Microaqueous Media. ACS Sustainable Chem Eng. 2024, 1210820-10830.

[81] Villeneuve P (2007) Lipases in lipophilization reactions. 25:515–536. https://doi.org/10.1016/j.biotechadv.2007.06.001

[82] Weerathunga H, Sarina S, Zhu HY, Waclawik ER. Oxidative Esterification of 5-Hydroxymethylfurfural into Dimethyl 2,5-Furandicarboxylate Using Gamma Alumina-Supported Gold Nanoparticles. ACS Omega. 2021, 6: 4740-4748.

[83] Xie Q, Zhu H, Xu Pet al. . Transesterification of methyl oleate for sustainable production of biolubricant: Process optimization and kinetic study. Ind Crops Prod. 2022, 182114879.

[84] Zou S, Zhang Z, Lee YYet al. . Lipase-catalyzed esterification for efficient acylglycerols synthesis in a solvent-free system: Process optimization, mass transfer insights and kinetic modeling. Food Chem. 2025, 474143151.

[85] Zuo M, Zheng X, Che Wet al. . Insight into 5-hydroxymethylfurfural synthesis and separation from the aqueous solvent with ion exchange resin catalysts, mechanisms, and theoretical calculations. Sep Purif Technol. 2025, 362131904.

Funding

Fondecyt(1240318)

Fondecyt Postdoctoral(3250050)

RIGHTS & PERMISSIONS

The Author(s)