仿生分区室固定化多酶体系

doi:10.12211/2096-8280.2024-025

摘要/Abstract

摘要：

仿生分区室固定化多酶耦联是体外合成生物学的前沿技术，目的是实现多酶分区室固定化和反应的时空分离。与简单共固定化不同，仿生分区室固定化技术通过控制酶在载体上的空间分布，形成底物通道促进中间产物传递，并提高串联或耦联反应的系统稳定性、产率和产物纯度。本文综述了近年来仿生分区室固定化多酶体系的进展，包括金属有机框架（MOFs）、聚合物囊泡和聚合物胶囊等固定化策略。MOFs具有结构可控、功能易调控等优点，采用分级多孔、MOF-on-MOF和多种MOF组合等仿生策略构建分区室微反应器，实现高效的体外多酶耦联催化反应。聚合物囊泡的膜结构可模拟天然磷脂双分子层，将多个小囊泡包封到大囊泡形成“囊泡中囊泡”模仿细胞器分区室固定化酶。聚合物胶囊是通过模板法形成核壳纳米球体结构，结构稳定性优异，进一步通过层层自组装能够形成多层核壳结构实现分区室固定化。将来，微流控等技术与仿生分区室固定化多酶技术融合，将促进体外合成生物学和绿色生物制造等领域的发展。

关键词: 多酶耦联, 仿生分区室, 固定化酶, 金属有机框架, 聚合物囊泡, 聚合物胶囊

Abstract:

Biomimetic compartmentalization immobilization of multi-enzyme system is a frontier for in vitro synthetic biology, focusing on the spatial and temporal separation of reactions. Compared with simple co-immobilization, biomimetic compartmentalization immobilization can form substrate channels and promote the transmission of intermediates for sequential or coupling reaction. By controlling the relative positions of the enzymes on carriers, this method improves system stability, productivity, and purity. In this paper, we summarized the recent advances of carriers for biomimetic compartmentalization immobilization of multi-enzyme systems, including metal-organic frameworks (MOFs), polymer vesicles and polymer capsules. Metal-organic frameworks (MOFs) are porous coordination materials which are composed of metal ions as nodes and organic linkers. MOFs possess unique characteristics including high porosity, large specific surface area and tunable structure, which are suitable for multi-enzyme systems. The strategies involving the hierarchically porous MOFs, MOF-on-MOF and multi-MOF combinations construct compartmentalized environments for efficient catalytic reactions in vitro. Polymer vesicles are hollow nanostructures composed of amphiphilic block copolymers. The membrane structure of polymer vesicles, similar to the natural phospholipid bilayers, has good mechanical stability and biocompatibility for protecting enzyme molecules, and provides unique microenvironment for sequential reactions. Multiple small vesicles were encapsulated into the larger vesicles to form a "vesicle-in-vesicle" by mimicking the structure of cellular organelles. Polymer capsules with a core-shell spherical nanostructure are formed by the templating method, and have structural stability and excellent shape controllability. Multilayered core-shell structures created by layer-by-layer self-assembly are applied for compartmentalized immobilization of multi-enzyme. In the future, the integration of microfluidic technologies with biomimetic compartmentalization immobilization of multi-enzyme is expected to provide highly efficient and stable multi-enzyme catalytic systems for in vitro synthetic biology and green biomanufacturing.

Key words: multi-enzyme coupling, compartmentalized biomimetic, immobilized enzyme, metal-organic frameworks, polymer vesicles, polymer capsules

中图分类号:

Q814.3

董玲玲, 李斐煊, 雷航彬, 宋启迪, 王世珍. 仿生分区室固定化多酶体系[J]. 合成生物学, DOI: 10.12211/2096-8280.2024-025.

Lingling DONG, Feixuan LI, Hangbin LEI, Qidi SONG, Shizhen WANG. Biomimetic compartmentalization immobilization of multi-enzyme system[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-025.

图/表 6

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固定化策略	MOFs	酶	稳定性	参考文献
孔道分区	PCN-888	GOx和HRP	4次循环后，GOx&HRP@PCN-888活性保持不变	[36]
孔道分区	PCN-333(Al)	HRP 和胆固醇氧化酶（ChOx）	4℃下保存20天后，GOx&HRP@PCN-333仍可检测到50%酶活	[37]
孔道分区	PCN-333(Al)	超氧化物歧化酶（SOD）和过氧化氢酶（CAT）	SOD&CAT@FNPCN-333在储存7天后仍可检测到酶活	[38]
MOF-on-MOF	ZIF-8	GOx和 HRP	HRP@H-ZIF-8-GOx 储存7天后仍可检测到70%酶活	[39]
MOF-on-MOF	ZIF-8	GOx和HRP	GOx@ZIF-8@HRP@ZIF-8 在4℃储存10天后仍可检测到93.96%酶活	[40]
MOF-on-MOF	ZIF-8	GOx 和HRP	/	[41]
MOF-on-MOF	Amine-MIL-101(Cr) and HKUST-1	CA，FDH和GDH	10个循环后，产率达到1077.7%	[42]
MOF-on-MOF	HKUST-1	GOx 和HRP	HRP@GOx@HKUST-1@Fe₃O₄重复使用10次后仍可检测到80.6%酶活	[43]
MOF-on-MOF	MOF-74	脂肪酶（CALB）and GOx	5个循环后仍可检测到79.3%酶活	[44]
多MOFs组合	ZIF-90	N-乙酰己糖胺-1-激酶（NahK），尿苷二磷酸-N-乙酰半乳糖胺焦磷酸酶（GlmU）和多磷酸激酶（PPK）	/	[45]
多MOFs组合	ZIF-8	GOx，HRP 和β-Gal	/	[46]
多MOFs组合	ZIF-8	FDH，GDH 和NADH	FDH&GDH&NADH/ZIF-8使用12小时后保留50%酶活	[47]
多MOFs组合	UiO-66	HRP和GOx	GOX@MOF-Cs和 HRP@MOF-Cs 3个循环后酶活性不变	[48]
多MOFs组合	ZIF- L and MPN	GOx和HRP	GOx–ZIF-L和HRP-ZIF–L在4℃保存30天仍可检测到酶活	[49]

固定化策略	MOFs	酶	稳定性	参考文献
孔道分区	PCN-888	GOx和HRP	4次循环后，GOx&HRP@PCN-888活性保持不变	[36]
孔道分区	PCN-333(Al)	HRP 和胆固醇氧化酶（ChOx）	4℃下保存20天后，GOx&HRP@PCN-333仍可检测到50%酶活	[37]
孔道分区	PCN-333(Al)	超氧化物歧化酶（SOD）和过氧化氢酶（CAT）	SOD&CAT@FNPCN-333在储存7天后仍可检测到酶活	[38]
MOF-on-MOF	ZIF-8	GOx和 HRP	HRP@H-ZIF-8-GOx 储存7天后仍可检测到70%酶活	[39]
MOF-on-MOF	ZIF-8	GOx和HRP	GOx@ZIF-8@HRP@ZIF-8 在4℃储存10天后仍可检测到93.96%酶活	[40]
MOF-on-MOF	ZIF-8	GOx 和HRP	/	[41]
MOF-on-MOF	Amine-MIL-101(Cr) and HKUST-1	CA，FDH和GDH	10个循环后，产率达到1077.7%	[42]
MOF-on-MOF	HKUST-1	GOx 和HRP	HRP@GOx@HKUST-1@Fe₃O₄重复使用10次后仍可检测到80.6%酶活	[43]
MOF-on-MOF	MOF-74	脂肪酶（CALB）and GOx	5个循环后仍可检测到79.3%酶活	[44]
多MOFs组合	ZIF-90	N-乙酰己糖胺-1-激酶（NahK），尿苷二磷酸-N-乙酰半乳糖胺焦磷酸酶（GlmU）和多磷酸激酶（PPK）	/	[45]
多MOFs组合	ZIF-8	GOx，HRP 和β-Gal	/	[46]
多MOFs组合	ZIF-8	FDH，GDH 和NADH	FDH&GDH&NADH/ZIF-8使用12小时后保留50%酶活	[47]
多MOFs组合	UiO-66	HRP和GOx	GOX@MOF-Cs和 HRP@MOF-Cs 3个循环后酶活性不变	[48]
多MOFs组合	ZIF- L and MPN	GOx和HRP	GOx–ZIF-L和HRP-ZIF–L在4℃保存30天仍可检测到酶活	[49]

材料	酶	稳定性	参考文献
聚合物囊泡（聚甲基丙烯酸酯PMA、聚赖氨酸PLL）	GOx和HRP	4℃储存2天后，仍能检测双酶级联到活性	[52]
聚合物囊泡（异氰肽与苯乙烯的嵌段共聚物）	GOx，HRP和CALB	/	[53]
聚合物囊泡（聚(2-甲基恶唑啉)-嵌段-聚(二甲基硅氧烷)-嵌段-聚(2-甲基恶唑啉)PMOXA-PDMS-PMOXA）	GOx，HRP和β-Gal	/	[54]
聚合物囊泡（聚苯乙烯-b-聚(3-(异氰基-丙氨酰-氨基乙基)噻吩PS-b-PIAT）	N-酰基-D葡萄糖胺-2-烯丙基酶（AGE），N-乙酰神经氨酸醛缩酶（NAL）和CMP-唾液酸合成酶（CSS）	/	[55]
聚合物囊泡（聚苯乙烯-b-聚(3-(异氰基-丙氨酰-氨基乙基)噻吩PS-b-PIAT）	PAMO，CALB和ADH	/	[56]
聚合物胶囊（天然多糖）	黄嘌呤氧化酶，尿酸酶和过氧化物酶	7个循环后产率是游离体系的2倍以上	[57]
聚合物胶囊（生物聚合物和碳酸钙）	β-葡萄糖苷酶（β-Glu），GOx和HRP	在4℃保存一个月以上仍可以检测到活性	[58]
聚合物胶囊（藻酸盐和鱼精蛋白）	FDH和FalDH	可循环使用8次以上	[59]
聚合物胶囊（核酸功能化羧甲基纤维素水凝胶）	GOx和β-Gal	在4℃下保存三天仍可检测到活性	[60]
聚合物胶囊（聚苯乙烯磺酸盐(PSS)和聚烯丙胺盐酸盐(PAH)）	人血清蛋白（HSA）	/	[61]
聚合物胶囊（聚烯丙胺盐酸盐和碳酸钙微粒）	(S)-3-羟丁酰辅酶A脱氢酶（DH）和黄素依赖型NADH氧化酶（NOx）	催化反应可进行72小时	[62]

材料	酶	稳定性	参考文献
聚合物囊泡（聚甲基丙烯酸酯PMA、聚赖氨酸PLL）	GOx和HRP	4℃储存2天后，仍能检测双酶级联到活性	[52]
聚合物囊泡（异氰肽与苯乙烯的嵌段共聚物）	GOx，HRP和CALB	/	[53]
聚合物囊泡（聚(2-甲基恶唑啉)-嵌段-聚(二甲基硅氧烷)-嵌段-聚(2-甲基恶唑啉)PMOXA-PDMS-PMOXA）	GOx，HRP和β-Gal	/	[54]
聚合物囊泡（聚苯乙烯-b-聚(3-(异氰基-丙氨酰-氨基乙基)噻吩PS-b-PIAT）	N-酰基-D葡萄糖胺-2-烯丙基酶（AGE），N-乙酰神经氨酸醛缩酶（NAL）和CMP-唾液酸合成酶（CSS）	/	[55]
聚合物囊泡（聚苯乙烯-b-聚(3-(异氰基-丙氨酰-氨基乙基)噻吩PS-b-PIAT）	PAMO，CALB和ADH	/	[56]
聚合物胶囊（天然多糖）	黄嘌呤氧化酶，尿酸酶和过氧化物酶	7个循环后产率是游离体系的2倍以上	[57]
聚合物胶囊（生物聚合物和碳酸钙）	β-葡萄糖苷酶（β-Glu），GOx和HRP	在4℃保存一个月以上仍可以检测到活性	[58]
聚合物胶囊（藻酸盐和鱼精蛋白）	FDH和FalDH	可循环使用8次以上	[59]
聚合物胶囊（核酸功能化羧甲基纤维素水凝胶）	GOx和β-Gal	在4℃下保存三天仍可检测到活性	[60]
聚合物胶囊（聚苯乙烯磺酸盐(PSS)和聚烯丙胺盐酸盐(PAH)）	人血清蛋白（HSA）	/	[61]
聚合物胶囊（聚烯丙胺盐酸盐和碳酸钙微粒）	(S)-3-羟丁酰辅酶A脱氢酶（DH）和黄素依赖型NADH氧化酶（NOx）	催化反应可进行72小时	[62]