SpyCatcher/SpyTag介导的酶免预纯化共价固定化研究进展

doi:10.11949/0438-1157.20251050

摘要/Abstract

摘要：

分子粘合剂SpyCatcher/SpyTag的发现为酶免预纯化共价固定化的研究提供了契机。传统方法常通过有机试剂将SpyCatcher修饰于载体上，功能化的载体能自发识别并特异共价结合含SpyTag的目标酶，从而在无需预纯化的前提下实现酶共价固定。为了减少使用有机试剂，开发更温和、高效且酶空间取向更可控的新策略，基于仿生矿化制备SpyCatcher功能化的无机载体和利用酚类化合物涂层制备SpyCatcher功能化的无机或有机载体应运而生。本文综述了SpyCatcher/SpyTag介导的酶免预纯化共价固定化常规方法，同时结合本课题组的研究成果，重点介绍了仿生矿化和酚类化合物介导的SpyCatcher/SpyTag功能化策略，并将其用于酶免预纯化共价固定化。最后，对今后固定化策略的可能研究方向进行了展望，以期为酶共价固定化新技术平台的研发提供参考。

关键词: 分子粘合剂, 酶分离纯化, 共价固定化, 仿生矿化, 酚类化合物

Abstract:

The discovery of the SpyCatcher/SpyTag has provided an opportunity for enzyme covalent immobilization-free of pre-purification. Usually SpyCatcher was modified on the carrier by organic reagents, which could spontaneously recognize and specifically covalently bind to target SpyTag-enzymes, thus enabling covalent immobilization of target enzymes without pre-purification. To reduce the utilization of organic reagents and develop novel immobilization strategies with better spatial orientation, the SpyCatcher functionalized inorganic carriers based on the biomimetic mineralization and the SpyCatcher functionalized inorganic or organic carriers based on the polyphenol have emerged. Thus, the traditional SpyCatcher/SpyTag-mediated covalent immobilization free of pre-purification are firstly reviewed, then, the biomimetic mineralization and polyphenol-mediated SpyCatcher/SpyTag functionalized strategies for enzyme covalent immobilization free of pre-purification are highlighted. Finally, the future research directions of this field are envisioned, with a view to providing a new technological platform for enzymes covalent immobilization.

Key words: molecular adhesives, enzyme separation and purification, covalent immobilization, biomimetic mineralization, phenolic compounds

中图分类号:

Q 814

周雁红, 杨子蛟, 陈雅鑫, 邓洲, 冯雨, 伍子呈, 卢诗雨, 张光亚. SpyCatcher/SpyTag介导的酶免预纯化共价固定化研究进展[J]. 化工学报, DOI: 10.11949/0438-1157.20251050.

Yanhong ZHOU, Zijiao YANG, Yaxin CHEN, Zhou DENG, Yu FENG, Zicheng WU, Shiyu LU, Guangya ZHANG. Advances in SpyCatcher/SpyTag-mediated covalent immobilization of enzymes free pre-purification[J]. CIESC Journal, DOI: 10.11949/0438-1157.20251050.

图/表 6

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表面偶联基团及其化学结构式	使用到的典型有机试剂	参考文献
环氧基	环氧氯丙烷（Epichlorohydrin, ECH），二甲亚砜，3-（三甲氧基甲硅烷基）丙酯（3-(Trimethoxysilyl) propyl ester, TMSPMA）和乙酸乙酯	^[24-26]
醛基	戊二醛	^[27-29]
羧基	EDC和NHS	^[30-32]
羟基	1,1 ' -羰基二咪唑（1,1'-Carbonyldiimidazole, CDI）	^[33]
溴化氢	EDC和NHS	^[34-35]

表面偶联基团及其化学结构式	使用到的典型有机试剂	参考文献
环氧基	环氧氯丙烷（Epichlorohydrin, ECH），二甲亚砜，3-（三甲氧基甲硅烷基）丙酯（3-(Trimethoxysilyl) propyl ester, TMSPMA）和乙酸乙酯	^[24-26]
醛基	戊二醛	^[27-29]
羧基	EDC和NHS	^[30-32]
羟基	1,1 ' -羰基二咪唑（1,1'-Carbonyldiimidazole, CDI）	^[33]
溴化氢	EDC和NHS	^[34-35]

生物大分子类型	矿化材料	关键生物物质
生物矿化相关蛋白	四氧化三铁纳米颗粒	Mms家族^[37]
生物矿化相关蛋白	碳酸钙颗粒	碳酸酐酶 ^[38]、尿素酶^[39]和蛋清蛋白^[40]
氨基酸	铁纳米颗粒	丝氨酸^[41]
	碳酸钙	天冬氨酸^[42]
	金纳米颗粒	半胱氨酸^[43]
	银纳米颗粒	谷氨酸^[44]
自组装肽	Cu纳米簇	CCYGGPKKKRKVG^[45]
	金纳米颗粒	YYAYY^[46]
	SiO₂纳米管	Ac-FFFFTTTTE-COOH^[47]
人工设计蛋白	磁性纳米颗粒	ELPs^[15]
人工设计蛋白	SiO₂纳米颗粒	ELPs^[48]，R5肽^[49]

生物大分子类型	矿化材料	关键生物物质
生物矿化相关蛋白	四氧化三铁纳米颗粒	Mms家族^[37]
生物矿化相关蛋白	碳酸钙颗粒	碳酸酐酶 ^[38]、尿素酶^[39]和蛋清蛋白^[40]
氨基酸	铁纳米颗粒	丝氨酸^[41]
	碳酸钙	天冬氨酸^[42]
	金纳米颗粒	半胱氨酸^[43]
	银纳米颗粒	谷氨酸^[44]
自组装肽	Cu纳米簇	CCYGGPKKKRKVG^[45]
	金纳米颗粒	YYAYY^[46]
	SiO₂纳米管	Ac-FFFFTTTTE-COOH^[47]
人工设计蛋白	磁性纳米颗粒	ELPs^[15]
人工设计蛋白	SiO₂纳米颗粒	ELPs^[48]，R5肽^[49]

固定化策略	有机试剂用量	重复使用性	载体类型	反应时间	参考文献
常规化学修饰	10% 琥珀酸酐的 DMF	PNGase F活性大约为初始值的78.5% （重复使用5次）	磁性纳米粒子	27 h	^[12]
	0.025 %戊二醛	甲酸脱氢酶活性大约为初始值的70% （重复使用10次）	细菌纤维素	10 h	^[21]
	0.05 %戊二醛	甲酸氢化酶活性为初始值的51.7% （重复使用8次）	二氧化硅	3 h	^[23]
仿生矿化介导	0	地衣多糖酶活性为初始值的85.7% （重复使用10次）	二氧化硅	5 min	^[57]
仿生矿化介导	0	地衣多糖酶活性为初始值的72.7% （重复使用10次）	磁性纳米粒子	12 min	^[16]
酚类化合物介导	0	地衣多糖酶活性为初始值的77.5% （重复使用8次）	尼龙膜	1 h	^[17]
酚类化合物介导	0	地衣多糖酶活性为初始值的78.81% （重复使用8次）	磁性粒子	1 h	^[17]