何长安¹,段金电¹,王志祥²,张全³,杨照²,胡玉静¹

• 1:南京工业大学 生物与制药工程学院，江苏 南京 211816
• 2:中国药科大学 工学院，江苏 南京 211198
• 3:中石化(大连)石油化工研究院有限公司，辽宁 大连 116045

摘要(Abstract)：

连续流动化学是一种在连续流动体系中完成化学反应的关键技术,近年来在制药、化学、能源及材料等领域展现出重要应用价值。其中,酶催化因其高对映选择性,在流动化学模式下的医药合成具有显著优势,但氧化还原酶辅因子NAD(P)⁺的高效、低成本循环再生仍是亟待解决的科学挑战。本文综述了连续流动生物催化体系中辅因子的最新研究进展,系统分析了当前流动体系下辅因子再生技术存在的关键问题,并展望了未来可能的发展方向,为促进流动生物催化技术的工业化应用提供参考。

关键词(KeyWords)： 流动化学;辅因子循环;微反应器;生物催化;电化学;光化学

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(22308158);江苏省高等学校基础科学自然科学研究面上项目(23KJB530008);中国石化重大科技项目(2350639042)。

作者(Author): 何长安¹,段金电¹,王志祥²,张全³,杨照²,胡玉静¹

DOI: 10.3969/j.issn.1003-9015.2025.00.024

参考文献(References)：

• [1] PLUTSCHACK M B, PIEBER B, GILMORE K, et al. The Hitchhiker's guide to flow chemistry [J].Chemical Reviews, 2017, 117(18): 11796-11893.
  [2] SHELDON R A, WOODLEY J M. Role of biocatalysis in sustainable chemistry [J].Chemical Reviews, 2018, 118(2): 801-838.
  [3] HUGHES D L. Applications of flow chemistry in the pharmaceutical industry—highlights of the recent patent literature [J].Organic Process Research & Development, 2020, 24(10): 1850-1860.
  [4] DEVINE P N, HOWARD R M, KUMAR R, et al. Extending the application of biocatalysis to meet the challenges of drug development [J].Nature Reviews Chemistry, 2018, 2(12): 409-421.
  [5] BRITTON J, MAJUMDAR S, WEISS G A. Continuous flow biocatalysis [J].Chemical Society Reviews, 2018, 47(15): 5891-5918.
  [6] TAMBORINI L, FERNANDES P, PARADISI F, et al. Flow bioreactors as complementary tools for biocatalytic process intensification [J].Trends in Biotechnology, 2018, 36(1): 73-88.
  [7] SANTI M, SANCINETO L, NASCIMENTO V, et al. Flow biocatalysis: A challenging alternative for the synthesis of APIs and natural compounds [J].International Journal of Molecular Sciences, 2021, 22(3): 990.
  [8] THOMPSON M P, PEÑAFIEL I, COSGROVE S C, et al. Biocatalysis using immobilized enzymes in continuous flow for the synthesis of fine chemicals [J].Organic Process Research & Development, 2019, 23(1): 9-18.
  [9] KNOWLES J R. Enzyme catalysis: Not different, just better [J].Nature, 1991, 350(6314): 121-124.
  [10] MARTÍ S, ROCA M, ANDRÉS J, et al. Theoretical insights in enzyme catalysis [J].Chemical Society Reviews, 2004, 33(2): 98-107.
  [11] KLIBANOV A M. Immobilized enzymes and cells as practical catalysts [J].Science, 1983, 219(4585): 722-727.
  [12] LIANG W B, WIED P, CARRARO F, et al. Metal-organic framework-based enzyme biocomposites [J].Chemical Reviews, 2021, 121(3): 1077-1129.
  [13] LIESE A, HILTERHAUS L. Evaluation of immobilized enzymes for industrial applications [J].Chemical Society Reviews, 2013, 42(15): 6236-6249.
  [14] DICOSIMO R, MCAULIFFE J, POULOSE A J, et al. Industrial use of immobilized enzymes [J].Chemical Society Reviews, 2013, 42(15): 6437-6474.
  [15] 许丽莉, 赵晴晴, 李青云, 等. 米根霉全细胞脂肪酶在化学-酶法环氧化反应体系中的稳定性[J]. 高校化学工程学报, 2024, 38(3): 449-458. XU L L, ZHAO Q Q, LI Q Y, et al. Stability of Rhizopus oryzae whole-cell lipase in a chemo-enzymatic epoxidation reaction [J].Journal of Chemical Engineering of Chinese Universities, 2024, 38(3): 449-458.
  [16] FASAN R, KAN S B J, ZHAO H M. A continuing career in biocatalysis: Frances H. Arnold [J].ACS Catalysis, 2019, 9(11): 9775-9788.
  [17] CHEN K, ARNOLD F H. Engineering new catalytic activities in enzymes [J].Nature Catalysis, 2020, 3(3): 203-213.
  [18] ARNOLD F H. Innovation by evolution: Bringing new chemistry to life (Nobel lecture) [J].Angewandte Chemie International Edition, 2019, 58(41): 14420-14426.
  [19] ARNOLD F H. Combinatorial and computational challenges for biocatalyst design [J].Nature, 2001, 409(6817): 253-257.
  [20] YANG Y, ARNOLD F H. Navigating the unnatural reaction space: Directed evolution of heme proteins for selective carbene and nitrene transfer [J].Accounts of Chemical Research, 2021, 54(5): 1209-1225.
  [21] WANG X D, SABA T, YIU H H P, et al. Cofactor NAD(P)H regeneration inspired by heterogeneous pathways [J].Chem, 2017, 2(5): 621-654.
  [22] MARSHALL J R, YAO P Y, MONTGOMERY S L, et al. Screening and characterization of a diverse panel of metagenomic imine reductases for biocatalytic reductive amination [J].Nature Chemistry, 2021, 13(2): 140-148.
  [23] LIANG Y J, WEI J L, QIU X, et al. Homogeneous oxygenase catalysis [J].Chemical Reviews, 2018, 118(10): 4912-4945.
  [24] REED N L, YOON T P. Oxidase reactions in photoredox catalysis [J].Chemical Society Reviews, 2021, 50(5): 2954-2967.
  [25] BORNSCHEUER U T, HUISMAN G W, KAZLAUSKAS R J, et al. Engineering the third wave of biocatalysis [J].Nature, 2012, 485(7397): 185-194.
  [26] WU H, TIAN C Y, SONG X K, et al. Methods for the regeneration of nicotinamidecoenzymes [J].Green Chemistry, 2013, 15(7): 1773-1789.
  [27] HOLLMANN F, ARENDS I W C E, BUEHLER K. Biocatalytic redox reactions for organic synthesis: Nonconventional regeneration methods [J].ChemCatChem, 2010, 2(7): 762-782.
  [28] TAYLOR K E, JONES J B. Nicotinamide coenzyme regeneration by dihydropyridine and pyridinium compounds [J].Journal of the American Chemical Society, 1976, 98(18): 5689-5694.
  [29] HOLLMANN F, ARENDS I W C E, HOLTMANN D. Enzymatic reductions for the chemist [J].Green Chemistry, 2011, 13(9): 2285-2314.
  [30] WITTMANN C, KRULL R.Biosystems engineering I:Creatingsuperior biocatalysts[M]. Berlin: Springer, 2010.
  [31] QUINTO T, KÖHLER V, WARD T R. Recent trends in biomimetic NADH regeneration [J].Topics in Catalysis, 2014, 57(5): 321-331.
  [32] HU Y J, CHEN J, WANG Y Q, et al. Biocatalysts used for multi-step reactions in continuous flow [J].Chemical Engineering Journal, 2022, 437: 135400.
  [33] ELSHERBINI M, WIRTH T. Electroorganic synthesis under flow conditions [J].Accounts of Chemical Research, 2019, 52(12): 3287-3296.
  [34] FRONTANA-URIBE B A, DANIEL LITTLE R, IBANEZ J G, et al. Organic electrosynthesis: A promising green methodology in organic chemistry [J].Green Chemistry, 2010, 12(12): 2099-2119.
  [35] YOSHIDA J I. Flash chemistry using electrochemical method and microsystems [J].Chemical Communications, 2005(36): 4509-4516.
  [36] SIU E, WON K, PARK C B. Electrochemical regeneration of NADH using conductive vanadia-silica xerogels [J].Biotechnology Progress, 2007, 23(1): 293-296.
  [37] ALI I, GILL A, OMANOVIC S. Direct electrochemical regeneration of the enzymatic cofactor 1,4-NADH employing nano-patterned glassy carbon/Pt and glassy carbon/Ni electrodes [J].Chemical Engineering Journal, 2012, 188: 173-180.
  [38] YOON S K, CHOBAN E R, KANE C, et al. Laminar flow-based electrochemical microreactor for efficient regeneration of nicotinamide cofactors for biocatalysis [J].Journal of the American Chemical Society, 2005, 127(30): 10466-10467.
  [39] CANTET J, BERGEL A, COMTAT M. Bioelectrocatalysis of NAD⁺ reduction [J].Bioelectrochemistry and Bioenergetics, 1992, 27(3): 475-486.
  [40] NGAMSOM B, HICKEY A M, GREENWAY G M, et al. The development and evaluation of a conducting matrix for the electrochemical regeneration of the immobilised co-factor NAD(H) under continuous flow [J].Organic & Biomolecular Chemistry, 2010, 8(10): 2419-2424.
  [41] KOCHIUS S, PARK J B, LEY C, et al. Electrochemical regeneration of oxidised nicotinamide cofactors in a scalable reactor [J].Journal of Molecular Catalysis B: Enzymatic, 2014, 103: 94-99.
  [42] YI D, BAYER T, BADENHORST C P S, et al. Recent trends in biocatalysis [J].Chemical Society Reviews, 2021, 50(14): 8003-8049.
  [43] SHELDON R A, PEREIRA P C. Biocatalysis engineering: The big picture [J].Chemical Society Reviews, 2017, 46(10): 2678-2691.
  [44] SHI J F, WU Y Z, ZHANG S H, et al. Bioinspired construction of multi-enzyme catalytic systems [J].Chemical Society Reviews, 2018, 47(12): 4295-4313.
  [45] SHELDON R A, BRADY D. Streamlining design, engineering, and applications of enzymes for sustainable biocatalysis [J].ACS Sustainable Chemistry & Engineering, 2021, 9(24): 8032-8052.
  [46] SCHRITTWIESER J H, VELIKOGNE S, HALL M, et al. Artificial biocatalytic linear cascades for preparation of organic molecules [J].Chemical Reviews, 2018, 118(1): 270-348.
  [47] LANCASTER L, ABDALLAH W, BANTA S, et al. Engineering enzyme microenvironments for enhanced biocatalysis [J].Chemical Society Reviews, 2018, 47(14): 5177-5186.
  [48] ZOR C, REEVE H A, QUINSON J, et al. H₂-Driven biocatalytic hydrogenation in continuous flow using enzyme-modified carbon nanotube columns [J].Chemical Communications, 2017, 53(71): 9839-9841.
  [49] ROMERO-FERNANDEZ M, PARADISI F. Biocatalytic access to betazole using a one-pot multienzymatic system in continuous flow [J].Green Chemistry, 2021, 23(12): 4594-4603.
  [50] FRANKLIN R D, WHITLEY J A, CAPARCO A A, et al. Continuous production of a chiral amine in a packed bed reactor with co-immobilized amine dehydrogenase and formate dehydrogenase [J].Chemical Engineering Journal, 2021, 407: 127065.
  [51] DALL’OGLIO F, CONTENTE M L, CONTI P, et al. Flow-based stereoselective reduction of ketones using an immobilized ketoreductase/glucose dehydrogenase mixed bed system [J].Catalysis Communications, 2017, 93: 29-32.
  [52] POZNANSKY B, THOMPSON L A, WARREN S A, et al. Carbon as a simple support for redox biocatalysis in continuous flow [J].Organic Process Research & Development, 2020, 24(10): 2281-2287.
  [53] YUAN P Y, WANG Q, DENG X L, et al. Coimmobilized dual enzymes in a continuous flow reactor for the efficient synthesis of optically pureγ/δ-lactones [J].ACS Applied Materials & Interfaces,2025, 17(1): 867-879.
  [54] GUAN S L, ZHOU W F, YUE Y T, et al. Efficient synthesis of (S)-nornicotine using co-immobilized IRED and GDH in batch and continuous flow reaction systems [J].Organic Process Research & Development,2024, 28(5): 2050-2060.
  [55] BENÍTEZ-MATEOS A I, LIM D, ROURA PADROSA D, et al. Biocatalytic reduction of heterocyclic imines in continuous flow with immobilized enzymes [J].ACS Sustainable Chemistry & Engineering, 2025, 13(13): 5009-5018.
  [56] WANG H Y, WANG M L, HUANG G X, et al. Four-step continuous-flow biosynthesis of a chiral precursor for angiotensin-converting enzyme inhibitors [J].Chemical Engineering Journal, 2025, 503: 158331.
  [57] PESCHKE T, BITTERWOLF P, GALLUS S, et al. Self-assembling all-enzyme hydrogels for flow biocatalysis [J].Angewandte Chemie International Edition, 2018, 57(52): 17028-17032.
  [58] KEEBLE A H, HOWARTH M. Power to the protein: Enhancing and combining activities using the spy toolbox [J].Chemical Science, 2020, 11(28): 7281-7291.
  [59] BITTERWOLF P, OTT F, RABE K S, et al. Imine reductase based all-enzyme hydrogel with intrinsic cofactor regeneration for flow biocatalysis [J].Micromachines, 2019, 10(11): 783.
  [60] BITTERWOLF P, GALLUS S, PESCHKE T, et al. Valency engineering of monomeric enzymes for self-assembling biocatalytic hydrogels [J].Chemical Science, 2019, 10(42): 9752-9757.
  [61] HARTLEY C J, WILLIAMS C C, SCOBLE J A, et al. Engineered enzymes that retain and regenerate their cofactors enable continuous-flow biocatalysis [J].Nature Catalysis, 2019, 2(11): 1006-1015.
  [62] PADROSA D R, BENÍTEZ-MATEOS A I, CALVEY L, et al. Cell-free biocatalytic syntheses of _L-pipecolic acid: A dual strategy approach and process intensification in flow [J].Green Chemistry, 2020, 22(16): 5310-5316.
  [63] KOKKONEN P, BEDNAR D, PINTO G, et al. Engineering enzyme access tunnels [J].Biotechnology Advances, 2019, 37(6): 107386.
  [64] BERARDI S, DROUET S, FRANCAS L, et al. Molecular artificial photosynthesis [J].Chemical Society Reviews, 2014, 43(22): 7501-7519.
  [65] SEEL C J, GULDER T. Biocatalysis fueled by light: On the versatile combination of photocatalysis and enzymes [J].ChemBioChem, 2019, 20(15): 1871-1897.
  [66] ASSIL-COMPANIONI L, BVCHSENSCHVTZ H C, SOLYMOSI D, et al. Engineering of NADPH supply boosts photosynthesis-driven biotransformations [J].ACS Catalysis, 2020, 10(20): 11864-11877.
  [67] YU Q, CHEN X Y. Insight into the dynamic mixing of droplets and plugs in droplet micro-mixer with Cantor fractal structure [J].Journal of the Brazilian Society of Mechanical Sciences and Engineering, 2023, 45(11): 571.
  [68] HUANG X W, LIU J, YANG Q J, et al. Microfluidic chip-based one-step fabrication of an artificial photosystem I for photocatalytic cofactor regeneration [J].RSC Advances, 2016, 6(104): 101974-101980.
  [69] LIN H C, LIU Y, YANG C H, et al. Microfluidic artificial photosynthetic system for continuous NADH regeneration and _L-glutamate synthesis [J].Catalysis Science & Technology, 2022, 12(12): 4057-4065.
  [70] BURNETT J W H, HOWE R F, WANG X D. Cofactor NAD(P)H regeneration: How selective are the reactions? [J].Trends in Chemistry, 2020, 2(6): 488-492.