Enzymatic synthesis of phosphatidyl-EPA/DHA using Candida antarctica lipase B immobilized on mesoporous MIL-88 A

Abstract

Mesoporous MIL-88A (Meso-MIL-88A) shows significant potential as an effective carrier for immobilizing large molecules such as lipases. This study investigates Meso-MIL-88A immobilized lipase for the catalysis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) incorporation into soybean phosphatidylcholine. Phosphatidyl-EPA and phosphatidyl-DHA are known for their superior health benefits and have garnered significant global attention. Firstly, we developed a novel and green water-washing strategy to synthesize Meso-MIL-88A, demonstrating its significant potential as an effective carrier for immobilizing large molecules such as lipases. Nitrogen adsorption/desorption and XPS analyses revealed that water as the eluent yielded larger average pore diameters and lower sulfur residue content compared to ethanol. This optimized Meso-MIL-88A carrier was used to immobilize Candida antarctica lipase B (CalB@Meso-MIL-88A), which was applied to the production of phosphatidyl-EPA and phosphatidyl-DHA. The immobilized CalB@Meso-MIL-88A exhibited exceptional catalytic efficiency, achieving an unprecedented sn-1 positional incorporation rate of 86.8% (44.2% EPA, 42.7% DHA) with 90% EPA/DHA-ethyl ester (EE) donors, while 97% DHA-EE resulted in a record-high 90.1% DHA incorporation at the sn-1 position. Kinetic studies and molecular docking simulations indicated a higher substrate affinity for DHA-EE, attributed to enhanced transfer efficiency of EPA/DHA-EE in organic solvent system. This study presents the first demonstration of the potential of Meso-MIL-88A for industrial lipase immobilization via this optimized route, offering an eco-friendly and highly efficient catalytic application for nutraceutical synthesis.

Keywords

Immobilized lipase / Kinetic study / Mesoporous MIL-88A (Meso-MIL-88A) / Molecular docking simulation / Phosphatidyl EPA/DHA

References

[1] Ahmmed MK, Ahmmed F, Tian H, Carne A, Bekhit AED. Marine omega-3 (n-3) phospholipids: a comprehensive review of their properties, sources, bioavailability, and relation to brain health. Compr Rev Food Sci Food Saf, 2020, 19(1): 64-123

[2] Ahmmed MK, Hachem M, Ahmmed F, Rashidinejad A, Oz F, Bekhit AA, Carne A, Bekhit AEA. Marine fish-derived lysophosphatidylcholine: properties, extraction, quantification, and brain health application. Molecules, 2023, 28(7): 3088

[3] Andrew Lin KY, Chang HA, Hsu CJ. Iron-based metal organic framework, MIL-88A, as a heterogeneous persulfate catalyst for decolorization of Rhodamine B in water. RSC Adv, 2015, 5(4132520-32530

[4] Baeza-Jiménez R, Noriega-Rodríguez JA, García HS, Otero C. Structured phosphatidylcholine with elevated content of conjugated linoleic acid: optimization by response surface methodology. Eur J Lipid Sci Technol, 2012, 114(11): 1261-1267

[5] Cai G, Jiang HL. A modulator‐induced defect‐formation strategy to hierarchically porous metal–organic frameworks with high stability. Angew Chem Int Ed Engl, 2017, 56(2): 563-567

[6] Chen W, Guo W, Gao F, Chen L, Chen S, Li D. Phospholipase A1-catalysed synthesis of docosahexaenoic acid-enriched phosphatidylcholine in reverse micelles system. Appl Biochem Biotechnol, 2017, 182(3): 1037-1052

[7] Chen L, Takatani N, Beppu F, Miyashita K, Hosokawa M. The effect of n-3 PUFA binding phosphatidylglycerol on metabolic syndrome-related parameters and n-3 PUFA accretion in diabetic/obese KK-A(y) mice. Nutrients, 2019, 11(12): 2866

[8] Chen WY, Lin SP, Zheng JW, Ling J, Zhong NJ. Ultrastable hierarchically porous nucleotide-based MOFs and their use for enzyme immobilization and catalysis. Colloids Surf, B, 2025, 245: 114294

[9] Cheng QJ, Chi XY, Liang YC, Li WX, Sun JX, Tao J, Wang Z. Immobilization of lipase in Cu-BTC MOF with enhanced catalytic performance for resolution of N-hydroxymethyl Vince lactam. Appl Biochem Biotechnol, 2023, 195(21216-1230

[10] Chojnacka A, Gładkowski W, Grudniewska A. Lipase-catalyzed transesterification of egg-yolk phophatidylcholine with concentrate of n-3 polyunsaturated fatty acids from cod liver oil. Molecules, 2017, 22(101771

[11] Du L, Yang YH, Wang YM, Xue CH, Kurihara H, Takahashi K. EPA-enriched phospholipids ameliorate cancer-associated cachexia mainly via inhibiting lipolysis. Food Funct, 2015, 6(123652-3662

[12] Estiasih T, Marianty R, Ahmadi K. Characteristics and emulsifying properties of structured phospholipids from palm pressed fiber and omega-3 fatty acid concentrates from by-products of fish processing by enzymatic acidolysis. J Food Sci Technol, 2020, 58(10): 3689-3700

[13] Fu HF, Song XX, Wu L, Zhao C, Wang P, Wang CC. Room-temperature preparation of MIL-88A as a heterogeneous photo-fenton catalyst for degradation of Rhodamine B and Bisphenol A under visible light. Mater Res Bull, 2020, 125: 110806

[14] Garcia-Quinto E, Garcia-Garcia P, Guisan JM, Fernandez-Lorente G. Enzymatic synthesis of mono- and disubstituted phospholipids by direct condensation of Oleic acid and Glycerophosphocholine with immobilized lipases and phospholipase. Food Chem, 2023, 401: 134109

[15] Gazquez A, Larque E. Towards an optimized fetal DHA accretion: differences on maternal DHA supplementation using phospholipids vs. triglycerides during pregnancy in different models. Nutrients, 2021, 13(2): 511

[16] Hachem M, Nacir H. Emerging role of phospholipids and lysophospholipids for improving brain docosahexaenoic acid as potential preventive and therapeutic strategies for neurological diseases. Int J Mol Sci, 2022, 23(73969

[17] He WS, Sun Y, Li Z, Yang H, Li J, Wang Q, Tan C, Zou B. Enhanced antioxidant capacity of lipoic acid in different food systems through lipase-mediated esterification with phytosterols. J Sci Food Agric, 2022, 102(157115-7125

[18] Hmoudah M, El-Qanni A, Tesser R, Esposito R, Petrone A, Jung O-S, Salmi T, Russo V, Di Serio M. Assessment of the robustness of MIL-88A in an Aqueous solution: experimental and DFT investigations. Mater Sci Eng B Solid State Mater Adv Appl, 2023, 288: 116179

[19] Hu YL, Dai LM, Liu DH, Du W. Hydrophobic pore space constituted in macroporous ZIF-8 for lipase immobilization greatly improving lipase catalytic performance in biodiesel preparation. Biotechnol Biofuels, 2020, 13(186

[20] Hu YL, Zhou H, Dai LM, Liu DH, Al-Zuhair S, Du W. Lipase immobilization on macroporous ZIF-8 for enhanced enzymatic biodiesel production. ACS Omega, 2021, 6(32143-2148

[21] Jang WY, Sohn JH, Chang JH. Thermally stable and reusable silica and nano-fructosome encapsulated CalB enzyme particles for rapid enzymatic hydrolysis and acylation. Int J Mol Sci, 2023, 24(129838

[22] Kim J, Chen BL, Reineke TM, Li HL, Eddaoudi M, Moler DB, O'Keeffe M, Yaghi OM. Assembly of metal-organic frameworks from large organic and inorganic secondary building units: new examples and simplifying principles for complex structures. J Am Chem Soc, 2001, 123(348239-8247

[23] Li D, Qin X, Wang W, Li Z, Yang B, Wang Y. Synthesis of DHA/EPA-rich phosphatidylcholine by immobilized phospholipase A1: effect of water addition and vacuum condition. Bioprocess Biosyst Eng, 2016, 39(8): 1305-1314

[24] Li Y, Liu Z, Dai L, Liu D, Du W. Synthesis and application of mesoporous MIL-88A for enhanced lipase immobilization and high value oil conversion. Chem Eng J, 2024, 496: 153802

[25] Lian XZ, Fang Y, Joseph E, Wang Q, Li JL, Banerjee S, Lollar C, Wang X, Zhou HC. Enzyme-MOF (metal-organic framework) composites. Chem Soc Rev, 2017, 46(11): 3386-3401

[26] Marsaoui N, Laplante S, Raies A, Naghmouchi K. Incorporation of omega-3 polyunsaturated fatty acids into soybean lecithin: effect of amines and divalent cations on transesterification by lipases. World J Microbiol Biotechnol, 2013, 29(122233-2238

[27] Marsaoui N, Naghmouchi K, Baah J, Raies A, Laplante S. Incorportation of ethyl esters of EPA and DHA in soybean lecithin using Rhizomucor miehei lipase: effect of additives and solvent-free conditions. Appl Biochem Biotechnol, 2015, 176(3): 938-946

[28] Mellot-Draznieks C, Serre C, Surblé S, Audebrand N, Férey G. Very large swelling in hybrid frameworks: a combined computational and powder diffraction study. J Am Chem Soc, 2005, 127(46): 16273-16278

[29] Ning X, Yasuda T, Kitaguchi T, Ueda H. Construction of fluorescent immunosensor quenchbody to detect His-tagged recombinant proteins produced in bioprocess. Sensors (Basel), 2021, 21(15): 4993

[30] Oger C, Marton Z, Brinkmann Y, Bultel-Poncé V, Durand T, Graber M, Galano J-M. Lipase-catalyzed regioselective monoacetylation of unsymmetrical 1,5-primary diols. J Org Chem, 2010, 75(61892-1897

[31] Prabhavathi Devi BLA, Guo Z, Xu X. Characterization of cross-linked lipase aggregates. J Am Oil Chem Soc, 2009, 86(7): 637-642

[32] Ren JJ, Li TR, Zhou XP, Dong X, Shorokhov AV, Semenov MB, Krevchik VD, Wang YH. Encapsulating all-inorganic perovskite quantum dots into mesoporous metal organic frameworks with significantly enhanced stability for optoelectronic applications. Chem Eng J, 2019, 358: 30-39

[33] Ren GL, Zhao K, Zhao L. A fenton-like method using ZnO doped MIL-88A for degradation of methylene blue dyes. RSC Adv, 2020, 10(66): 39973-39980

[34] Shu LW, Zheng X, Qi SH, Lin SZ, Lu YH, Yao CY, Ling XP. Transesterification of phosphatidylcholine with DHA-rich algal oil using immobilized Candida antarctica lipase B to produce DHA-phosphatidylcholine. Enzyme Microb Technol, 2023, 169: 110266

[35] Sugasini D, Yalagala PCR, Subbaiah PV. Efficient enrichment of retinal DHA with dietary lysophosphatidylcholine-DHA: potential application for retinopathies. Nutrients, 2020, 12(10): 3114

[36] Vikbjerg AF, Mu H, Xu X. Parameters affecting incorporation and by-product formation during the production of structured phospholipids by lipase-catalyzed acidolysis in solvent-free system. J Mol Catal B:enzym, 2005, 36(1–614-21

[37] Wang XM, Qin XL, Li XT, Zhao ZX, Yang B, Wang YH. Insight into the modification of phosphatidylcholine with n-3 polyunsaturated fatty acids-rich ethyl esters by immobilized MAS1 lipase. Mol, 2019, 24(193528

[38] Wang S, Wang TQ, Zheng HY, Fan FQ, Gu ZD, He WX, Zhang B, Shao L, Chen H, Li YN, et al.. Fabrication of mesoporous MOF nanosheets via surfactant-template method for C-S coupling reactions. Microporous Mesoporous Mater, 2020, 303: 110254

[39] Xi X, Feng X, Shi N, Ma X, Lin H, Han Y. Immobilized phospholipase A1-catalyzed acidolysis of phosphatidylcholine from Antarctic krill (Euphausia superba) for docosahexaenoic acid enrichment under supercritical conditions. J Mol Catal B:enzym, 2016, 126: 46-55

[40] Yuan SA, Feng L, Wang KC, Pang JD, Bosch M, Lollar C, Sun YJ, Qin JS, Yang XY, Zhang P, et al.. Stable metal-organic frameworks: design, synthesis, and applications. Adv Mater, 2018, 30(371704303

[41] Zhang WN, Liu YY, Lu G, Wang Y, Li SZ, Cui CL, Wu J, Xu ZL, Tian DB, Huang W, et al.. Mesoporous metal-organic frameworks with size-, shape-, and space-distribution-controlled pore structure. Adv Mater, 2015, 27(182923-2929

[42] Zhang R, Liu Y, Huang X, Xu M, Liu R, Zong W. Interaction of a digestive protease, candida rugosa lipase, with three surfactants investigated by spectroscopy, molecular docking and enzyme activity assay. Sci Total Environ, 2018, 622–623: 306-315

[43] Zhang M, Wang P, Jin D, Jian S, Wu J, Huang M, Xie H, Zhao Q, Yang H, Luo P, et al.. Chain-locked precursor ion scanning based HPLC-MS/MS for in-depth molecular analysis of lipase-catalyzed transesterification of structured phospholipids containing omega-3 fatty acyl chains. Food Chem, 2023, 399: 133982

[44] Zhang T, Li B, Wang Z, Hu D, Zhang X, Zhao B, Wang J. Green biosynthesis of rare DHA-phospholipids by lipase-catalyzed transesterification with edible algal oil in solvent-free system and catalytic mechanism study. Front Bioeng Biotechnol, 2023, 11: 1158348

[45] Zhang TT, Wang J, Zhao YK, Wang ZL, Hu D, Liu YY, Zhang XL, Li HN, Zhao BX, Li BL. Green biosynthesis of DHA-phospholipids in tailor-made supersaturated DHA aqueous solution and catalytic mechanism study. Food Chem, 2024, 431: 137164

[46] Zhao TT, No DS, Kim BH, Garcia HS, Kim YH, Kim IH. Immobilized phospholipase A1-catalyzed modification of phosphatidylcholine with n-3 polyunsaturated fatty acid. Food Chem, 2014, 157: 132-140

[47] Zhou H, Zou ZQ, Dai LM, Liu DH, Du W. Ordered macro-microporous ZIF-8 with different macropore sizes and their stable derivatives for lipase immobilization in biodiesel production. ACS Sustainable Chem Eng, 2022, 10(44): 14503-14514

Funding

National Natural Science Foundation of China(22178198)

National Key Research and Development Program of China(2023YFA0914400)

RIGHTS & PERMISSIONS

The Author(s)