过氧化物酶的重组表达和应用研究进展

doi:10.12211/2096-8280.2024-027

摘要/Abstract

摘要：

过氧化物酶作为一类自然界中广泛分布的酶，参与了如生物体的先天免疫防疫、植物微生物抗氧化应激、真菌木质素降解、植物细胞壁代谢及伤口愈合等重要生命过程。随着测序、基因编辑、重组蛋白表达和高通量筛选技术的飞速发展，越来越多的过氧化物酶被发现、表征和重组表达。这些种类丰富、数量庞大及催化性能卓越的过氧化物酶，在众多领域的应用研究中受到广泛关注。近年来过氧化物酶在重组表达上取得了显著进展，进一步促进了其在应用研究领域的开发。本文从系统进化分类及功能角度上对过氧化物酶进行了简要概述，对近年来过氧化物酶在大肠杆菌、酵母和真菌中重组表达研究进展及其在环境修复、化合物检测的应用研究成果进行系统地综述，重点介绍了过氧化物酶应用于生物合成高附加值化合物方面的最新研究进展，并对其目前在该领域应用研究中存在的底物和产物非专一性问题及辅因子H₂O₂细胞毒性问题进行讨论。过氧化物酶在医学检测、环境保护和生物合成等领域中的应用潜力巨大。然而，当前的技术和应用仍面临一些挑战，比如过氧化物酶在复杂环境中的稳定性和活性差、酶制剂生产成本高及专一性差问题。未来，通过结合蛋白质工程、合成生物学和固定化技术等多学科的最新进展，可以有效解决这些挑战，推动过氧化物酶在各个领域的广泛应用。

关键词: 过氧化物酶, 蛋白重组表达, 污染物降解, 化合物检测, 生物合成

Abstract:

As a class of enzymes widely distributed in nature, peroxidases are involved in important life processes such as innate immunity and epidemic prevention of organisms, anti-oxidative stress of plant microorganisms, fungal lignin degradation, plant cell wall metabolism and wound healing. With the rapid development of sequencing, gene editing, recombinant protein expression and high-throughput screening technologies, more and more peroxidases have been discovered, characterized and recombinantly expressed. These peroxidases, characterized by their species diversity, abundant quantity, and excellent catalytic performance, have attracted extensive attention in many fields of application research. In recent years, remarkable progress has been made in the recombinant expression of peroxidases, which further promotes their development in the field of applied research. Additionally, as we deepen our understanding of the catalytic properties of peroxidases, new opportunities have emerged for their application in the field of biosynthesis. Their high catalytic activity allows for rapid oxidation reactions under mild conditions and enables the construction of multi-enzyme cascade systems in conjunction with other enzymes, thereby facilitating the efficient synthesis of complex compounds. This paper provides a brief overview of peroxidases from the perspective of systematic evolutionary classification and function. It systematically reviews recent progress in the recombinant expression of peroxidases in Escherichia coli, yeast, and fungi, as well as their application research achievements in environmental remediation and compound detection. The focus is on the latest research advances in the application of peroxidases for the biosynthesis of high-value-added compounds. The paper also discusses the current issues in this field, such as substrate and product non-specificity and the cytotoxicity of the cofactor H₂O₂.Peroxidases have enormous potential for applications in medical diagnostics, environmental protection, and biosynthesis. However, current technologies and applications still face several challenges, such as the stability and activity of peroxidases in complex environments, high production costs of enzyme preparations, and poor specificity. In the future, by integrating the latest advances in protein engineering, synthetic biology, and immobilization technology, these challenges can be effectively addressed, promoting the widespread application of peroxidases across various fields.

Key words: peroxidase, Protein recombinant expression, Degradation of pollutants, Detection of compounds, Biological synthesis

中图分类号:

Q812

李庚, 申晓林, 孙新晓, 王佳, 袁其朋. 过氧化物酶的重组表达和应用研究进展[J]. 合成生物学, DOI: 10.12211/2096-8280.2024-027.

Geng Li, Xiaolin Shen, Xinxiao Sun, Jia Wang, Qipeng Yuan. Research progress in recombinant expression and application of peroxidases[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-027.

图/表 7

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78	PREETHI P S, VICKRAM S, DAS R, et al. Bioprospecting of novel peroxidase from Streptomyces coelicolor strain SPR7 for carcinogenic azo dyes decolorization [J]. Chemosphere, 2023, 310: 136836.
79	HAMDI S, ALLALA F, MECHRI S, et al. Biochemical and molecular characterization of a new heme peroxidase from Aspergillus niger CTM10002, and its application in textile reactive dye decolorization [J]. Process Biochemistry, 2022, 121: 619-634.
80	KALSOOM U, BHATTI H N, AFTAB K, et al. Biocatalytic potential of Brassica oleracea L. var. botrytis leaves peroxidase for efficient degradation of textile dyes in aqueous medium [J]. Bioprocess and Biosystems Engineering, 2023, 46(3) : 453-465.
81	AI J, ZHANG W, LIAO G, et al. NH₂Fe₃O₄@ SiO₂supported peroxidase catalyzed H₂O₂ for degradation of endocrine disrupter from aqueous solution: Roles of active radicals and NOMs [J]. Chemosphere, 2017, 186: 733-742.
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表达宿主	过氧化物酶	表达方式和策略	表达效果	参考文献
大肠杆菌	HRP	周质表达，通过定向进化提高酶表达活力	表达量110 mg/L 酶活力140 U/L	[18]
大肠杆菌	HRP	胞内表达，纯化的包涵体通过体外重折叠复性	比酶活 10 U/mg	[19]
大肠杆菌	HRP	胞内表达，纯化的包涵体通过体外重折叠复性	表达量 1000 mg/L 比酶活 62.5 U/mg	[20]
大肠杆菌	HRP	周质表达，通过将HRP的N-末端与DsbA蛋白融合，利用SRP途径将融合蛋白转运至周质	表达量 48 mg/L 比酶活 12.7 U/mg	[20]
大肠杆菌	HRP	胞内表达，通过与甘油酸激酶（PGK）融合表达实现可溶性过量表达，重组蛋白与钙离子、氯化血红素和氧化型谷胱甘肽一起简单孵育后激活活性	酶回收量72 mg/L 比酶活为商业酶的60%	[21]
大肠杆菌	MnP	胞内表达，纯化的包涵体通过体外重折叠复性	酶活力 345 U/L 比酶活 3.63 U/mg	[22]
大肠杆菌	MnP	胞内表达，通过在细胞培养物中加入化学物质（0.25% Triton X-100、0.25% Tween-80、0.5%甘油和1%乙醇）改善蛋白溶解度	酶活力 310 U/L 比酶活 0.441 U/mg	[22]
大肠杆菌	MnP	胞内表达，通过密码子优化、冷休克启动子的控制表达、培养过程中连续添加血红素及共表达伴侣蛋白改善蛋白溶解度	表达量30 mg/L k_cat/K_M= 406 s^-1·mM^-1	[23]
大肠杆菌	MnP	胞内表达，通过共表达二硫键异构酶DsbC改善MnP的溶解度	比酶活445 U/mg	[24]
毕赤酵母	MnP	周质表达，与α 因子前导序列的融合表达	酶活力 48 U/L 比酶活 42 U/mg	[25]
酿酒酵母	HRP	周质表达，通过定向进化提高酶表达活力	酶活力1080 U/L	[26]
毕赤酵母	HRP	周质表达，通过定向进化提高酶表达活力	表达量 6 mg/L 比酶活 980 U/mg	[27]
毕赤酵母	HRP	周质表达，通过敲除α-1,6-甘露糖基转移酶（OCH1编码）改善HRP高糖基化问题，通过发酵优化提高表达水平	表达量101 mg/L 比酶活278.1 U/mg	[28]
毕赤酵母	HRP	周质表达，通过与链球菌蛋白G（SpG）融合表达提高表达量和比酶活	表达量 113 mg/L 比酶活 1624 U/mg	[29]
毕赤酵母	HRP	周质表达，OCH1缺陷的毕赤酵母菌株中表达了通过定向进化得到的突变体HRP 13A7-N175S	表达量 132 mg/L 比酶活 2008 U/mg	[30]
黑曲霉	MnP	周质表达，共表达钙连蛋白，培养基添加血红素	表达量70 mg/L	[31]
米曲霉	MnP	周质表达，在Taka淀粉酶启动子下表达融合了分泌信号的MnP	表达效率 0.33 U/min·Lk_cat=132 s^-1	[32]

表达宿主	过氧化物酶	表达方式和策略	表达效果	参考文献
大肠杆菌	HRP	周质表达，通过定向进化提高酶表达活力	表达量110 mg/L 酶活力140 U/L	[18]
大肠杆菌	HRP	胞内表达，纯化的包涵体通过体外重折叠复性	比酶活 10 U/mg	[19]
大肠杆菌	HRP	胞内表达，纯化的包涵体通过体外重折叠复性	表达量 1000 mg/L 比酶活 62.5 U/mg	[20]
大肠杆菌	HRP	周质表达，通过将HRP的N-末端与DsbA蛋白融合，利用SRP途径将融合蛋白转运至周质	表达量 48 mg/L 比酶活 12.7 U/mg	[20]
大肠杆菌	HRP	胞内表达，通过与甘油酸激酶（PGK）融合表达实现可溶性过量表达，重组蛋白与钙离子、氯化血红素和氧化型谷胱甘肽一起简单孵育后激活活性	酶回收量72 mg/L 比酶活为商业酶的60%	[21]
大肠杆菌	MnP	胞内表达，纯化的包涵体通过体外重折叠复性	酶活力 345 U/L 比酶活 3.63 U/mg	[22]
大肠杆菌	MnP	胞内表达，通过在细胞培养物中加入化学物质（0.25% Triton X-100、0.25% Tween-80、0.5%甘油和1%乙醇）改善蛋白溶解度	酶活力 310 U/L 比酶活 0.441 U/mg	[22]
大肠杆菌	MnP	胞内表达，通过密码子优化、冷休克启动子的控制表达、培养过程中连续添加血红素及共表达伴侣蛋白改善蛋白溶解度	表达量30 mg/L k_cat/K_M= 406 s^-1·mM^-1	[23]
大肠杆菌	MnP	胞内表达，通过共表达二硫键异构酶DsbC改善MnP的溶解度	比酶活445 U/mg	[24]
毕赤酵母	MnP	周质表达，与α 因子前导序列的融合表达	酶活力 48 U/L 比酶活 42 U/mg	[25]
酿酒酵母	HRP	周质表达，通过定向进化提高酶表达活力	酶活力1080 U/L	[26]
毕赤酵母	HRP	周质表达，通过定向进化提高酶表达活力	表达量 6 mg/L 比酶活 980 U/mg	[27]
毕赤酵母	HRP	周质表达，通过敲除α-1,6-甘露糖基转移酶（OCH1编码）改善HRP高糖基化问题，通过发酵优化提高表达水平	表达量101 mg/L 比酶活278.1 U/mg	[28]
毕赤酵母	HRP	周质表达，通过与链球菌蛋白G（SpG）融合表达提高表达量和比酶活	表达量 113 mg/L 比酶活 1624 U/mg	[29]
毕赤酵母	HRP	周质表达，OCH1缺陷的毕赤酵母菌株中表达了通过定向进化得到的突变体HRP 13A7-N175S	表达量 132 mg/L 比酶活 2008 U/mg	[30]
黑曲霉	MnP	周质表达，共表达钙连蛋白，培养基添加血红素	表达量70 mg/L	[31]
米曲霉	MnP	周质表达，在Taka淀粉酶启动子下表达融合了分泌信号的MnP	表达效率 0.33 U/min·Lk_cat=132 s^-1	[32]

污染物类型	降解底物	过氧化物酶	研究成果	参考文献
酚类污染物	苯酚	芜菁过氧化物酶（TP）	采用海藻酸盐包埋分离于芜菁根的TP，在最佳条件下对苯酚的平均脱除率达93%。	[59, 60]
	苯酚	辣根过氧化物酶（HRP）	采用一步包埋法将HRP酶固定在半透性海藻酸盐膜，在重复使用5次时酶活下降至50%。	[61]
	苯酚	辣根过氧化物酶（HRP）	在改性丙烯腈共聚物膜上共价固定HRP，对100 mg/L的苯酚溶液脱除率达95.4%，具备高稳定性，在酶操作的20天后酶活下降至50%。	[62]
	苯酚、对甲酚	黑萝卜过氧化物酶（RSVNP）	以交联酶聚集体形式固定化的RSVNP-CLEAs具备高稳定性，储存60天后保持100%的活性，最佳条件下显示出对苯酚和对甲酚92%和98%的高消除率。	[63]
	苯酚、对氯苯酚	辣根过氧化物酶（HRP）	HRP通过戊二醛偶联共价固定在磁珠上，固定化HRP保留了79%游离酶活性，在磁稳定流化床反应器中能够消除100%的苯酚和92%对氯苯酚。	[64]
	苯酚	辣根过氧化物酶（HRP）	物理固定在改性还原氧化石墨烯（RGO·NH2纳米颗粒（NPs）的HRP显示出对高浓度苯酚（2.5 g/L)100%的去除率，重复使用10次后仍能保留60%的初始酶活性。	[65]
	对溴苯酚	苦瓜过氧化物酶（BGP）	Con A层状藻酸钙纤维素珠为载体固定的BGP在填充床反应器中能够清除96%的对溴苯酚，而且连续运行30天后仍能保持75%的清除率。	[66]
	苯酚	辣根过氧化物酶（HRP）	通过戊二醛交联剂固定在生物碳上的HRP具备高稳定性，重复使用10次后仍保持70%的酶活性。	[67]
	苯酚、对氯苯酚	辣根过氧化物酶（HRP）	固定在阳离子微孔淀粉的HRP显示出较游离酶更强的苯酚和对氯苯酚的去除能力，重复使用10次后仍保留66%的初始活性。	[68]
	苯酚	辣根过氧化物酶（HRP）	以氧化海藻酸酪胺微珠为载体固定的HRP在最佳条件下能够去除96%的苯酚，重复使用4次后仍保留61%的初始活性。	[69]
合成染料	甲基橙	辣根过氧化物酶（HRP）	采用聚丙烯酰胺凝胶包埋提取于新鲜辣根的HRP，在填充床生物反应器中能够降解90%的甲基橙。	[70]
	酚红	辣根过氧化物酶（HRP）	采用了包埋和共价键合组合方法将HRP固定在海藻酸钙-淀粉杂化小球上，固定化HRP对酚红的去除率为55.87%，是游离HRP的11.5倍。	[71]
	黑色CKF、蓝色GWF、红色C4BLN	来自裂褶菌IBL-06的木质素过氧化物酶（LiP）	以戊二醛为交联剂将LiP固定化得的交联酶聚集体能够有效降解黑色CKF、蓝色GWF和红色C4BLN 三种活性染料，处理6 h后的降解率分别为89.6%、81.46%和79.6%。	[72]
	晶状丽春红6R（CP6R）	大豆过氧化物酶（SBP）	40 ppm的CP6R染料溶液可以在一分钟内被SBP完全降解，进一步研究结果表明偶氮染料通过对称和不对称偶氮键断裂两种途径降解。	[73]
	结晶紫	来源于解淀粉芽孢杆菌MN-13的过氧化物酶（BaDyP）	BaDyP处理结晶紫（50 mg/L) 72 h后，降解率为63.0 %。	[74]
	偶氮料DY106	西葫芦过氧化物酶（CP）	最佳条件下，海藻酸钙包埋的CP能够在2分钟内使87%以上的染料DY106（50 mg/L)脱色。	[75]
	刚果红、甲基橙、铬黑T	来源于白腐真菌Phanerochaete sp. HSD的锰过氧化物酶（MnP）	菌液中提取的MnP粗酶能有效地降解刚果红、甲基橙和铬黑三种偶氮染料，在数小时内能够将浓度200 mg/L以上的染料降解90%以上。	[76]
	台盼蓝	大豆过氧化物酶（SBP）	聚丙烯酰胺（PA）包埋的SBP具备更好的稳定性和可重复使用性，在30 min内能够使台盼蓝（40 ppm）降解约80%。	[77]
	偶氮染料直红 23 和直蓝15	来源于天蓝色链霉菌SPR7的过氧化物酶POX	在添加10 mM的木质素的条件下，POX经过20小时能够使偶氮染料直红23和直蓝15脱色75.4%和90%。	[78]
	酸性蓝158（AB）	黑曲霉CTM10002来源的锰过氧化物酶MnP AN30	在反应24 h后，纯化的MnP AN30对染料AB的脱色率达到98%。	[79]
	绿松石蓝133G、Drim Red CL4BN	花椰菜过氧化物酶（CFP）	提取自新鲜花椰菜叶的CFP在最佳条件下能够使染料绿松石蓝133G（25 ppm）和Drim Red CL4BN（50 ppm)降解85%和92%。	[80]
药物活性化合物	17β-雌激素（E2）	辣根过氧化物酶（HRP）	以磁性Fe3O4为载体固定的HRP在最佳条件下能够使E2（1000 mg/L)降解80%。	[81]
	双酚a（BPA）、三氯生（TCS）、雌酮（E1）、17β雌二醇（E2）和17α炔雌醇（EE2）	黑管菌来源的多功能过氧化物酶（VP）	分离于黑管菌的VP能够高效去除五种污染物，去除速率达到（2.5-5.0 mg·L^-1·min^-1）。	[82]
	三氯生（TCS）、磺胺甲恶唑（SMX）、雌酮（E1）、17β-雌二醇（E2）和17α-炔雌醇（EE2））	大豆过氧化物酶（SBP）	在优化的条件下，SBP对浓度各10 μM的药物污染物废水降解率在95%以上。	[83]
	磺胺甲恶唑（SMX）	大豆过氧化物酶（SBP）	在78 nM 的SBP和56 μM H₂O₂的条件下反应3小时，浓度为500 ppm的SMX能够降解78%。	[84]
	双氯芬酸、卡马西平和扑热息痛	辣根过氧化物酶（HRP）和木质素过氧化物酶（LiP）	溶胶-凝胶包封的HRP和LiP酶复合物都能够改善酶在酸性介质中的稳定性，而且在反应3天后可以完全降解双氯芬酸、卡马西平和扑热息痛。	[85]
	四环素（TC）和土霉素（OTC）	来源于黄孢原毛平革菌的过氧化物酶（MnP）	添加40 U/L的MnP时，50 mg/L的TC和OTC能够在4 h内降解72.5%和84.3%。	[86]
	磺胺甲恶唑（SMX）和卡马西平（CBZ）	辣根过氧化物酶（HRP）	由聚（氯乙烯）电纺丝纤维封装固定化的HRP显著提升了其稳定性和重复使用性，而且能够在24 h内降解80%以上10 mg/L的SMZ和CBZ。	[87]
	磺胺甲恶唑、萘普生、四环素、雌二醇、酮康唑、酮咯酸和双氯芬酸	来源于烟曲霉的氯过氧化物酶（CPO）	CPO能够氧化磺胺甲恶唑、萘普生、四环素、雌二醇、酮康唑、酮咯酸和双氯芬酸，反应10 min后，所有底物均能转化80%以上。	[88]

污染物类型	降解底物	过氧化物酶	研究成果	参考文献
酚类污染物	苯酚	芜菁过氧化物酶（TP）	采用海藻酸盐包埋分离于芜菁根的TP，在最佳条件下对苯酚的平均脱除率达93%。	[59, 60]
	苯酚	辣根过氧化物酶（HRP）	采用一步包埋法将HRP酶固定在半透性海藻酸盐膜，在重复使用5次时酶活下降至50%。	[61]
	苯酚	辣根过氧化物酶（HRP）	在改性丙烯腈共聚物膜上共价固定HRP，对100 mg/L的苯酚溶液脱除率达95.4%，具备高稳定性，在酶操作的20天后酶活下降至50%。	[62]
	苯酚、对甲酚	黑萝卜过氧化物酶（RSVNP）	以交联酶聚集体形式固定化的RSVNP-CLEAs具备高稳定性，储存60天后保持100%的活性，最佳条件下显示出对苯酚和对甲酚92%和98%的高消除率。	[63]
	苯酚、对氯苯酚	辣根过氧化物酶（HRP）	HRP通过戊二醛偶联共价固定在磁珠上，固定化HRP保留了79%游离酶活性，在磁稳定流化床反应器中能够消除100%的苯酚和92%对氯苯酚。	[64]
	苯酚	辣根过氧化物酶（HRP）	物理固定在改性还原氧化石墨烯（RGO·NH2纳米颗粒（NPs）的HRP显示出对高浓度苯酚（2.5 g/L)100%的去除率，重复使用10次后仍能保留60%的初始酶活性。	[65]
	对溴苯酚	苦瓜过氧化物酶（BGP）	Con A层状藻酸钙纤维素珠为载体固定的BGP在填充床反应器中能够清除96%的对溴苯酚，而且连续运行30天后仍能保持75%的清除率。	[66]
	苯酚	辣根过氧化物酶（HRP）	通过戊二醛交联剂固定在生物碳上的HRP具备高稳定性，重复使用10次后仍保持70%的酶活性。	[67]
	苯酚、对氯苯酚	辣根过氧化物酶（HRP）	固定在阳离子微孔淀粉的HRP显示出较游离酶更强的苯酚和对氯苯酚的去除能力，重复使用10次后仍保留66%的初始活性。	[68]
	苯酚	辣根过氧化物酶（HRP）	以氧化海藻酸酪胺微珠为载体固定的HRP在最佳条件下能够去除96%的苯酚，重复使用4次后仍保留61%的初始活性。	[69]
合成染料	甲基橙	辣根过氧化物酶（HRP）	采用聚丙烯酰胺凝胶包埋提取于新鲜辣根的HRP，在填充床生物反应器中能够降解90%的甲基橙。	[70]
	酚红	辣根过氧化物酶（HRP）	采用了包埋和共价键合组合方法将HRP固定在海藻酸钙-淀粉杂化小球上，固定化HRP对酚红的去除率为55.87%，是游离HRP的11.5倍。	[71]
	黑色CKF、蓝色GWF、红色C4BLN	来自裂褶菌IBL-06的木质素过氧化物酶（LiP）	以戊二醛为交联剂将LiP固定化得的交联酶聚集体能够有效降解黑色CKF、蓝色GWF和红色C4BLN 三种活性染料，处理6 h后的降解率分别为89.6%、81.46%和79.6%。	[72]
	晶状丽春红6R（CP6R）	大豆过氧化物酶（SBP）	40 ppm的CP6R染料溶液可以在一分钟内被SBP完全降解，进一步研究结果表明偶氮染料通过对称和不对称偶氮键断裂两种途径降解。	[73]
	结晶紫	来源于解淀粉芽孢杆菌MN-13的过氧化物酶（BaDyP）	BaDyP处理结晶紫（50 mg/L) 72 h后，降解率为63.0 %。	[74]
	偶氮料DY106	西葫芦过氧化物酶（CP）	最佳条件下，海藻酸钙包埋的CP能够在2分钟内使87%以上的染料DY106（50 mg/L)脱色。	[75]
	刚果红、甲基橙、铬黑T	来源于白腐真菌Phanerochaete sp. HSD的锰过氧化物酶（MnP）	菌液中提取的MnP粗酶能有效地降解刚果红、甲基橙和铬黑三种偶氮染料，在数小时内能够将浓度200 mg/L以上的染料降解90%以上。	[76]
	台盼蓝	大豆过氧化物酶（SBP）	聚丙烯酰胺（PA）包埋的SBP具备更好的稳定性和可重复使用性，在30 min内能够使台盼蓝（40 ppm）降解约80%。	[77]
	偶氮染料直红 23 和直蓝15	来源于天蓝色链霉菌SPR7的过氧化物酶POX	在添加10 mM的木质素的条件下，POX经过20小时能够使偶氮染料直红23和直蓝15脱色75.4%和90%。	[78]
	酸性蓝158（AB）	黑曲霉CTM10002来源的锰过氧化物酶MnP AN30	在反应24 h后，纯化的MnP AN30对染料AB的脱色率达到98%。	[79]
	绿松石蓝133G、Drim Red CL4BN	花椰菜过氧化物酶（CFP）	提取自新鲜花椰菜叶的CFP在最佳条件下能够使染料绿松石蓝133G（25 ppm）和Drim Red CL4BN（50 ppm)降解85%和92%。	[80]
药物活性化合物	17β-雌激素（E2）	辣根过氧化物酶（HRP）	以磁性Fe3O4为载体固定的HRP在最佳条件下能够使E2（1000 mg/L)降解80%。	[81]
	双酚a（BPA）、三氯生（TCS）、雌酮（E1）、17β雌二醇（E2）和17α炔雌醇（EE2）	黑管菌来源的多功能过氧化物酶（VP）	分离于黑管菌的VP能够高效去除五种污染物，去除速率达到（2.5-5.0 mg·L^-1·min^-1）。	[82]
	三氯生（TCS）、磺胺甲恶唑（SMX）、雌酮（E1）、17β-雌二醇（E2）和17α-炔雌醇（EE2））	大豆过氧化物酶（SBP）	在优化的条件下，SBP对浓度各10 μM的药物污染物废水降解率在95%以上。	[83]
	磺胺甲恶唑（SMX）	大豆过氧化物酶（SBP）	在78 nM 的SBP和56 μM H₂O₂的条件下反应3小时，浓度为500 ppm的SMX能够降解78%。	[84]
	双氯芬酸、卡马西平和扑热息痛	辣根过氧化物酶（HRP）和木质素过氧化物酶（LiP）	溶胶-凝胶包封的HRP和LiP酶复合物都能够改善酶在酸性介质中的稳定性，而且在反应3天后可以完全降解双氯芬酸、卡马西平和扑热息痛。	[85]
	四环素（TC）和土霉素（OTC）	来源于黄孢原毛平革菌的过氧化物酶（MnP）	添加40 U/L的MnP时，50 mg/L的TC和OTC能够在4 h内降解72.5%和84.3%。	[86]
	磺胺甲恶唑（SMX）和卡马西平（CBZ）	辣根过氧化物酶（HRP）	由聚（氯乙烯）电纺丝纤维封装固定化的HRP显著提升了其稳定性和重复使用性，而且能够在24 h内降解80%以上10 mg/L的SMZ和CBZ。	[87]
	磺胺甲恶唑、萘普生、四环素、雌二醇、酮康唑、酮咯酸和双氯芬酸	来源于烟曲霉的氯过氧化物酶（CPO）	CPO能够氧化磺胺甲恶唑、萘普生、四环素、雌二醇、酮康唑、酮咯酸和双氯芬酸，反应10 min后，所有底物均能转化80%以上。	[88]

检测目标物	H₂O₂ 生成酶	检测原理	参考文献
乳糖	β-半乳糖苷酶 + 半乳糖氧化酶	表面共固定β-半乳糖苷酶、半乳糖氧化酶和POD的石墨电极电流传感器	[91]
乳糖	β-半乳糖苷酶 + 葡萄糖氧化酶	POD催化ABTS显色	[92]
尿酸	尿酸氧化酶	POD催化TMB显色	[93]
尿酸	尿酸氧化酶	表面固定HRP的聚四氟乙烯膜电流传感器	[94]
葡萄糖	葡萄糖氧化酶	HRP刻蚀金纳米棒（AuNR）显色	[95]
葡萄糖	葡萄糖氧化酶	表面固定HRP的碳纤维电流传感器	[96]
甘油三酯	脂肪酶 + 甘油激酶 + 3-磷酸甘油氧化酶	HRP催化3,5-二氯-2-羟基苯甲酸和4-氨基苯乙酮聚合显色	[97]
甘油三酯	脂肪酶 + 甘油激酶 + 3-磷酸甘油氧化酶	表面固定功能化酶聚集体的多晶金电极电流传感器	[98]
胆固醇	胆固醇氧化酶	HRP催化ABTS显色	[99]
胆固醇	胆固醇氧化酶 + 胆固醇酯酶	表面共固定胆固醇氧化酶、胆固醇酯酶和POD的聚苯胺膜电流传感器	[100]
酚类化合物	H₂O₂直接添加	表面固定POD的石墨电极电流传感器	[90]