双水相微流控制备复合固定化酶微球及其生物催化应用研究

doi:10.11949/0438-1157.20251118

摘要/Abstract

摘要：

本研究采用双水相微流控技术成功制备了用于固定化酶的海藻酸钙/羧甲基壳聚糖（CA/CMCS）复合微球，并通过中心旋转组合设计（CCRD）系统优化了微球双水相微流控制备过程的处方参数及制备工艺。对微球形貌及固定化酶的酶学性质的研究结果显示，酶被成功负载于微球内部，固定化后其热稳定性与pH稳定性显著提高，重复使用性和储存稳定性也明显优于游离酶，同时在催化动力学方面表现出更优的性能。进一步地，制备的固定化β-半乳糖苷酶（β-Gal）/葡萄糖氧化酶（GOD）/辣根过氧化物酶（HRP）三酶体系的复合微球能够高效催化多酶级联反应，且提高底物浓度可显著提升反应速率；此外，制备的固定化超氧化物歧化酶（SOD）/过氧化氢酶（CAT）双酶体系的复合微球表现出优异的活性氧清除能力，在重复使用8次后清除率仍可维持在60%以上，显示出良好的操作稳定性与应用前景。上述研究表明，利用双水相微流控技术制备的复合固定化酶微球表现出了用于生物催化应用的优势与潜力。

关键词: 双水相, 微流体学, 微球, 生物催化, 酶, 固定化, 微反应器

Abstract:

In this study, calcium alginate/carboxymethyl chitosan (CA/CMCS) composite microspheres for enzyme immobilization were successfully fabricated by microfluidic aqueous two-phase system. The formulation parameters and preparation process were systematically optimized through Central Composite Rotatable Design (CCRD). Characterization of the microsphere morphology and enzymatic properties demonstrated successful enzyme encapsulation within the microspheres. The immobilized enzymes exhibited significantly enhanced thermal stability and pH stability, along with improved reusability and storage stability compared to free enzymes, as well as superior performance in catalytic kinetics. Furthermore, the immobilized β-galactosidase/glucose oxidase/horseradish peroxidase (β-Gal/GOD/HRP) triple-enzyme system effectively catalyzed cascade reactions, with substrate concentration increase significantly enhancing the reaction rate. Additionally, the immobilized superoxide dismutase (SOD)/catalase (CAT) dual-enzyme system displayed excellent reactive oxygen species scavenging capacity, maintaining over 60% clearance after eight reuse cycles, indicating good operational stability and application potential. These findings demonstrate that the composite immobilized enzyme microspheres prepared by aqueous two-phase microfluidic technology possess significant advantages and potential for biocatalytic applications.

Key words: aqueous two-phase system, microfluidics, microsphere, biocatalysis, enzyme, immobilization, microreactor

中图分类号:

TQ 464.8

杜福泰, 辛欢, 郑惠元, 王伟江, 马庆明. 双水相微流控制备复合固定化酶微球及其生物催化应用研究[J]. 化工学报, DOI: 10.11949/0438-1157.20251118.

Futai DU, Huan XIN, Huiyuan ZHENG, Weijiang WANG, Qingming MA. Microfluidic aqueous two-phase system-based fabrication of immobilized enzyme composite microspheres for biocatalytic applications[J]. CIESC Journal, DOI: 10.11949/0438-1157.20251118.

图/表 20

参考文献 46

[1]	Xiao Y P, Wu J, Chen P H, et al. Biocatalytic cascade reactions for management of diseases[J]. Chemical Society Reviews, 2025, 54(7): 3247-3271.
[2]	宋伟, 王金辉, 胡贵鹏, 等. 多酶级联催化合成(R)-β-酪氨酸[J]. 化工学报, 2022, 73(1): 352-361.
	Song W, Wang J H, Hu G P, et al. Cascade catalysis for the synthesis of (R)-β-tyrosine[J]. CIESC Journal, 2022, 73(1): 352-361.
[3]	Wang Y X, Zhao Q C, Haag R, et al. Biocatalytic synthesis using self-assembled polymeric nano- and microreactors[J]. Angewandte Chemie International Edition, 2022, 61(52): e202213974.
[4]	Li X Y, Zhang Y, He A, et al. Biohybrid catalysis in biomedicine[J]. Coordination Chemistry Reviews, 2025, 545: 217003.
[5]	Pyser J B, Chakrabarty S, Romero E O, et al. State-of-the-art biocatalysis[J]. ACS Central Science, 2021, 7(7): 1105-1116.
[6]	Vasudhevan P, Zhang R Y, Ma H, et al. Biocatalytic enzymes in food packaging, biomedical, and biotechnological applications: a comprehensive review[J]. International Journal of Biological Macromolecules, 2025, 300: 140069.
[7]	Liu X R, Qiu M Y, Zhang Y Y, et al. Enzyme immobilization based on reticular framework materials: Strategy, food applications, and prospect[J]. Advances in Colloid and Interface Science, 2025, 344: 103589.
[8]	Ran L, Lu Y, Chen L, et al. Design, synthesis, and application of immobilized enzymes on artificial porous materials[J]. Advanced Science, 2025, 12(20): 2500345.
[9]	Xu C Z, Tong S S, Sun L Q, et al. Cellulase immobilization to enhance enzymatic hydrolysis of lignocellulosic biomass: an all-inclusive review[J]. Carbohydrate Polymers, 2023, 321: 121319.
[10]	Zdarta J, Meyer A S, Jesionowski T, et al. Developments in support materials for immobilization of oxidoreductases: a comprehensive review[J]. Advances in Colloid and Interface Science, 2018, 258: 1-20.
[11]	Xing M Y, Chen Y, Li B Q, et al. Highly efficient removal of patulin using immobilized enzymes of Pseudomonas aeruginosa TF-06 entrapped in calcium alginate beads[J]. Food Chemistry, 2022, 377: 131973.
[12]	Ouyang J, Pu S J, Wang J Z, et al. Enzymatic hydrolysate of geniposide directly acts as cross-linking agent for enzyme immobilization[J]. Process Biochemistry, 2020, 99: 187-195.
[13]	Li C, Zhang G F, Liu N, et al. Preparation and properties of Rhizopus oryzae lipase immobilized using an adsorption-crosslinking method[J]. International Journal of Food Properties, 2016, 19(8): 1776-1785.
[14]	Wang F, Xu H, Wang M M, et al. Application of immobilized enzymes in juice clarification[J]. Foods, 2023, 12(23): 4258.
[15]	Guo S, Wang S, Meng J, et al. Immobilized enzyme for screening and identification of anti-diabetic components from natural products by ligand fishing[J]. Critical Reviews in Biotechnology, 2023, 43(2): 242-257.
[16]	严昕怡, 大村拓, 朱江煜. 酶固定化新策略及其应用研究进展[J]. 化学通报(中英文), 2025, 88(7): 754-760.
	Yan X Y, Omura T, Zhu J Y. Research progress in new strategies and applications of enzyme immobilization[J]. Chemistry, 2025, 88(7): 754-760.
[17]	Lu J W, Nie M F, Li Y R, et al. Design of composite nanosupports and applications thereof in enzyme immobilization: a review[J]. Colloids and Surfaces B: Biointerfaces, 2022, 217: 112602.
[18]	Du F T, Xin H, Zheng H Y, et al. Aqueous liquid–liquid phase separation (AqLLPS) droplet microreactors for biocatalysis[J]. Green Chemistry, 2025, 27(28): 8448-8466.
[19]	Zhang J H, Chen J L, Sha Y, et al. Water-mediated active conformational transitions of lipase on organic solvent interfaces[J]. International Journal of Biological Macromolecules, 2024, 277: 134056.
[20]	Zhang N N, Bittner J P, Fiedler M, et al. Unraveling alcohol dehydrogenase catalysis in organic–aqueous biphasic systems combining experiments and molecular dynamics simulations[J]. ACS Catalysis, 2022, 12(15): 9171-9180.
[21]	Yuan H, Li F, Jia L F, et al. Bacteria-inspired aqueous-in-aqueous compartmentalization by in situ interfacial biomineralization[J]. Small Methods, 2023, 7(2): 2201309.
[22]	贾露凡, 王艺颖, 董钰漫, 等. 微流控双水相贴壁液滴流动强化酶促反应研究[J]. 化工学报, 2023, 74(3): 1239-1246.
	Jia L F, Wang Y Y, Dong Y M, et al. Aqueous two-phase system based adherent droplet microfluidics for enhanced enzymatic reaction[J]. CIESC Journal, 2023, 74(3): 1239-1246.
[23]	[23] He D Q, Cao D Z, Ben C X, et al. Ionic liquid gel microspheres as an emerging platform for constructing liquid compartment microreactors[J]. Green Chemistry, 2022, 24(15): 5952-5964.
[24]	马敬, 李磊, 邓小康, 等. 双水相微流控技术研究进展[J]. 科学通报, 2025, 70(12): 1799-1818.
	Ma J, Li L, Deng X K, et al. Advances in aqueous two-phase microfluidic technology[J]. Chinese Science Bulletin, 2025, 70(12): 1799-1818.
[25]	潘大伟, 汪伟, 谢锐, 等. 微流控乳液模板法构建功能微颗粒过程中介尺度结构定向调控的研究进展[J]. 化工学报, 2022, 73(6): 2306-2317.
	Pan D W, Wang W, Xie R, et al. Progress on regulation of meso-scale structures for microfluidic emulsion-template synthesis of functional microparticles[J]. CIESC Journal, 2022, 73(6): 2306-2317.
[26]	黄心童, 耿宇昊, 刘恒源, 等. 微流控制备新型功能纳米粒子研究进展[J]. 化工学报, 2023, 74(1): 355-364.
	Huang X T, Geng Y H, Liu H Y, et al. Research progress on new functional nanoparticles prepared by microfluidic technology[J]. CIESC Journal, 2023, 74(1): 355-364.
[27]	鲍博, 赵双良, 徐建鸿. 基于微纳流控技术的流体相态特性研究进展[J]. 化工学报, 2018, 69(11): 4530-4541.
	Bao B, Zhao S L, Xu J H. Progress in studying fluid phase behaviours with micro-and nano-fluidic technology[J]. CIESC Journal, 2018, 69(11): 4530-4541.
[28]	Ben C X, Zhao S J, Wu Q, et al. Hydrophobic ionic liquid gel microspheres as bi-component carriers with a liquid phase to immobilize enzymes for enhanced performance[J]. Advanced Functional Materials, 2024, 34(46): 2407913.
[29]	Tang Q M, Deng N J, Chen J L, et al. One-step fabrication of coconut-like capsules via competitive reactions at an all-aqueous interface for enzyme immobilization[J]. ACS Applied Materials & Interfaces, 2023, 15(8): 10621-10628.
[30]	Xu F L, Wang W J, Zhao W B, et al. All-aqueous microfluidic fabrication of calcium alginate/alkylated chitosan core-shell microparticles with time-sequential functions for promoting whole-stage wound healing[J]. International Journal of Biological Macromolecules, 2024, 282: 136685.
[31]	Zhang X Y, Zhu Y J, Xiong Z J, et al. Broad-spectrum ROS/RNS scavenging catalase-loaded microreactors for effective oral treatment of inflammatory bowel diseases[J]. Small, 2025, 21(23): 2501341.
[32]	Miyagawa A, Nakatani K. Kinetic detection of hydrogen peroxide in single horseradish peroxidase-concentrated silica particle using confocal fluorescence microspectroscopic measurement[J]. Talanta, 2024, 273: 125925.
[33]	Sun M M, Wang L L, Zhuo Y, et al. Multi-enzyme activity of MIL-101 (Fe)-derived cascade nano-enzymes for antitumor and antimicrobial therapy[J]. Small, 2024, 20(17): 2309593.
[34]	Kwon K, Jung J, Sahu A, et al. Nanoreactor for cascade reaction between SOD and CAT and its tissue regeneration effect[J]. Journal of Controlled Release, 2022, 344: 160-172.
[35]	Yasar Mahlicli F, ?en Y, Mutlu M, et al. Immobilization of superoxide dismutase/catalase onto polysulfone membranes to suppress hemodialysis-induced oxidative stress: a comparison of two immobilization methods[J]. Journal of Membrane Science, 2015, 479: 175-189.
[36]	Yang M, Jiang W, Pan Z Q, et al. Synthesis, characterization and SOD-like activity of histidine immobilized silica nanoparticles[J]. Journal of Inorganic and Organometallic Polymers and Materials, 2015, 25(5): 1289-1297.
[37]	Yan H D, Hou W J, Lei B L, et al. Ultrarobust stable ABTS radical cation prepared using Spore@Cu-TMA biocomposites for antioxidant capacity assay[J]. Talanta, 2024, 276: 126282.
[38]	Savéant J M. Electrochemical concerted proton and electron transfers. further insights in the reduction mechanism of superoxide ion in the presence of water and other weak acids[J]. The Journal of Physical Chemistry C, 2007, 111(7): 2819-2822.
[39]	Yao Y Y, Chen S X, Li H. An improved system to evaluate superoxide-scavenging effects of bioflavonoids[J]. ChemistryOpen, 2021, 10(4): 503-514.
[40]	Hackenhaar C R, Rosa C F, Flores E E, et al. Development of a biocomposite based on alginate/gelatin crosslinked with genipin for β-galactosidase immobilization: Performance and characteristics[J]. Carbohydrate Polymers, 2022, 291: 119483.
[41]	Gao Y, Sun W T, Zhang Y G, et al. All-aqueous microfluidics fabrication of multifunctional bioactive microcapsules promotes wound healing[J]. ACS Applied Materials & Interfaces, 2022, 14(43): 48426-48437.
[42]	Smidsr?d O, Skja?k-Br?k G. Alginate as immobilization matrix for cells[J]. Trends in Biotechnology, 1990, 8: 71-78.
[43]	Egbeyemi O I, Hatem W A, Kober U A, et al. Transforming the stability, encapsulation, and sustained release properties of calcium alginate beads through gel-confined coacervation[J]. Langmuir, 2024, 40(23): 11947-11958.
[44]	Mafakher L, Ahmadi Y, Khalili Fard J, et al. Alpha-amylase immobilization: methods and challenges[J]. Pharmaceutical Sciences, 2023, 29(2): 144-155.
[45]	Wang W, Liu J Y, Khan M J, et al. Magnetic macroporous chitin microsphere as a support for covalent enzyme immobilization[J]. International Journal of Biological Macromolecules, 2024, 256: 128214.
[46]	Naskar P, Chakraborty D, Mondal A, et al. Immobilization of α-amylase in calcium alginate-gum odina (CA-GO) beads: an easily recoverable and reusable support[J]. International Journal of Biological Macromolecules, 2024, 258: 129062.

试剂/处理	空白对照	1	2	3	4	5
丙酮酸标准溶液/mL	0	0.1	0.2	0.3	0.4	0.5
PBS（0.2 M，pH 7.4）	0.6	0.5	0.4	0.3	0.2	0.1
各试管摇匀后，置于37℃恒温磁力搅拌器，保温10 min
2，4-二硝基苯肼/mL	0.5	0.5	0.5	0.5	0.5	0.5
各试管摇匀后，置于37℃恒温磁力搅拌器，保温20 min
0.4 M NaOH/mL	5.0	5.0	5.0	5.0	5.0	5.0
充分摇匀后，立即在520 nm波长下比色

试剂/处理	空白对照	1	2	3	4	5
丙酮酸标准溶液/mL	0	0.1	0.2	0.3	0.4	0.5
PBS（0.2 M，pH 7.4）	0.6	0.5	0.4	0.3	0.2	0.1
各试管摇匀后，置于37℃恒温磁力搅拌器，保温10 min
2，4-二硝基苯肼/mL	0.5	0.5	0.5	0.5	0.5	0.5
各试管摇匀后，置于37℃恒温磁力搅拌器，保温20 min
0.4 M NaOH/mL	5.0	5.0	5.0	5.0	5.0	5.0
充分摇匀后，立即在520 nm波长下比色

水平	实验因素
水平	海藻酸钠浓度/%	羧甲基壳聚糖浓度/%	氯化钙浓度/%	引发时间/min	戊二醛浓度/%	交联时间/h
1	0.6	0.2	0.5	10	0.1	1
2	0.9	0.3	1.0	20	0.3	2
3	1.2	0.4	1.5	30	0.5	4
4	1.5	0.5	2.0	40	0.75	6
5	1.8	0.6	2.5	50	1.0	8

水平	实验因素
水平	海藻酸钠浓度/%	羧甲基壳聚糖浓度/%	氯化钙浓度/%	引发时间/min	戊二醛浓度/%	交联时间/h
1	0.6	0.2	0.5	10	0.1	1
2	0.9	0.3	1.0	20	0.3	2
3	1.2	0.4	1.5	30	0.5	4
4	1.5	0.5	2.0	40	0.75	6
5	1.8	0.6	2.5	50	1.0	8

离心管编号	1	2	3	4
PBS（0.05M，pH7.8）	2.0	2.0	2.0	2.0
EDTA-Na₂ （1 μmol/mL）	0.2	0.2	0.2	0.2
L-甲硫氨酸（0.13 mmol/mL）	0.2	0.2	0.2	0.2
NBT（0.75 μmol/mL）	0.2	0.2	0.2	0.2
纯水	0.1	0.1	0.08	0.08
核黄素（2 μmol/mL）	0.3	0.3	0.3	0.3
酶液	0	0	0.02	0.02
总体积	3.0	3.0	3.0	3.0