AA9家族裂解性多糖单加氧酶的研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2025-1444

摘要/Abstract

摘要：

裂解性多糖单加氧酶（lytic polysaccharide monooxygenase, LPMO）能通过氧化裂解的方式断裂纤维素等多糖链的糖苷键，与糖苷水解酶协同降解木质纤维素时，可以显著提升产糖效率，在木质纤维素生物质转化中具有重要作用。其中，真菌来源的AA9家族LPMO凭借其优异的氧化活性和广泛的底物谱，在食品、能源及材料等领域展现出广阔的应用前景。本文系统综述了AA9家族LPMO的来源及功能的多样性，指出不同来源的LPMO对糖苷水解酶的协同促进效果、底物降解类型存在显著差异；对其结构进行深入分析发现，催化结构域表面的Loop区对底物的识别、结合以及氧化区域的选择起着核心调控作用；除此之外，在双结构域的LPMO中，CBM结构域也会对LPMO结合底物的过程产生重要影响。同时，本文还对MALDI-TOF-MS、HPAEC-PAD及基于显色反应等多种酶活测定方法的原理与适用范围进行分析比较，发现色谱‒质谱联用策略是目前定量分析LPMO氧化产物的最主要的方法之一。最后，本文详细总结了LPMO在食品加工、生物能源及纳米纤维素制备等领域展现出的应用潜力。因此，通过LPMO酶资源的挖掘或酶分子改良获得更多适合工业生产应用的新型酶制剂是目前研究工作的重要方向。深入剖析了当前AA9家族LPMO研究在酶资源功能表征、底物特异性分子机制解析以及实际应用效率提升等方面存在的关键技术问题，并针对性地提出了结合宏基因组学挖掘新酶、融合结构预测与机器学习解析底物选择性、针对不同预处理工艺开展酶分子定向改造等未来研究方向与策略建议。

关键词: 裂解性多糖单加氧酶, AA9家族, 功能调控机制, 氧化区域选择性, 协同催化, 活性测定, 工业应用

Abstract:

Lytic polysaccharide monooxygenases (LPMOs) can cleave glycosidic bonds in polysaccharide chains such as cellulose through oxidative cleavage. When acting synergistically with glycoside hydrolases in lignocellulose degradation, LPMOs significantly enhance sugar release efficiency, playing a crucial role in lignocellulosic biomass conversion. Among them, fungal LPMOs from the AA9 family exhibit broad substrate spectra and excellent oxidative activity, demonstrating great application potential in the food, energy, and materials industries. This review systematically summarizes the diversity of sources and functions of AA9 family LPMOs, highlighting that LPMOs from different origins exhibit marked differences in their promoting effects on glycoside hydrolases and substrate degradation patterns. In-depth structural analysis reveals that the Loop regions on the surface of the catalytic domain play a central regulatory role in substrate recognition, binding, and regioselectivity of oxidation. Additionally, in multi-domain LPMOs, the carbohydrate-binding module (CBM) significantly influences the process of substrate binding. Furthermore, this review compares the principles and applicability of various activity measurement methods, including MALDI-TOF-MS, HPAEC-PAD, and colorimetric assays, indicating that combined chromatography-mass spectrometry strategies are among the most widely used approaches for the quantitative analysis of LPMO oxidation products. Finally, the considerable application potential of LPMOs in food processing, bioenergy, and nanocellulose preparation is discussed in detail. Therefore, the discovery of novel LPMO enzymes or the molecular engineering of existing ones to obtain new enzyme preparations more suitable for industrial applications is an important direction for current research. Moreover, this review deeply analyzes the key technical challenges in current AA9 family LPMO research, including functional characterization of enzyme resources, elucidation of the molecular mechanisms underlying substrate specificity, and improvement of practical application efficiency. Targeted future research directions and strategic suggestions are proposed, such as mining novel enzymes using metagenomics, integrating structural prediction and machine learning to analyze substrate selectivity, and performing targeted enzyme molecular engineering tailored to different pretreatment processes. This review aims to provide a theoretical reference for the development, application, and molecular engineering of AA9 family LPMOs.

Key words: lytic polysaccharide monooxygenase, AA9 family, functional regulation mechanism, oxidative regioselectivity, synergistic catalysis, activity assay, industrial application

李聪, 于濡沄, 郑菲, 郭玉杰. AA9家族裂解性多糖单加氧酶的研究进展[J]. 生物技术通报, 2026, 42(11): 45-57.

LI Cong, YU Ru-yun, ZHENG Fei, GUO Yu-jie. Research Progress of AA9 Family Lytic Polysaccharide Monooxygenases[J]. Biotechnology Bulletin, 2026, 42(11): 45-57.

图/表 7

图1 AA9家族代表性LPMO的系统进化树分析红色五角星为含CBM1的成员；蓝色五角星为已解析晶体结构的成员；扇形颜色代表不同物种来源，相同颜色代表该物种包含多条序列，未着色区域为仅含单条序列的物种

Fig. 1 Phylogenetic tree analysis of representative AA9 LPMORed pentagrams represent members containing the CBM1 domain. Blue pentagrams indicate members with resolved crystal structures. Fan segment colors distinguish species sources, with the same color indicating one species with multiple sequences, and uncolored segments representing species with only a single sequence

表1 不同来源的AA9家族LPMO及其功能

Table 1 AA9 family LPMO from different sources and their functions

名称 Name	来源 Source	功能 Function	参考文献 Reference
AfAA9B	Aspergillus fumigatus	与纤维素酶协同作用，使汽爆甘蔗渣的糖化效率提升20%	［21］
MsLPMO2	Morchella sextelata	与纤维素酶联用，使稻草和小麦秸秆的还原糖产量分别提升126.4%和133.5%	［7］
MtLPMO9A	Thermothelomyces thermophilus	突变体与木聚糖酶协同作用，使木聚糖的还原糖产量提高57%	［22］
MtLPMO9G	Thermothelomyces thermophilus	与纤维素酶联用，使玉米秸秆的还原糖产量提升242%	［23］
MtLPMO9V	Thermothelomyces thermophilus	与内切葡聚糖酶协同作用，对微晶纤维素的协同效率可达120%	［24］
NcLPMO9C	Neurospora crassa	与纤维二糖水解酶联用，使高结晶纤维素的还原糖产量提升169%	［25］
PaLPMO9B	Podospora anserina	与纤维素酶协同，使酸和碱预处理甘蔗渣的还原糖产量分别提升了30%和20%	［26］
PaLPMO9H	Podospora anserina	与纤维二糖水解酶协同，使PASC的还原糖产量提升60%	［27］
PchAA9C	Phanerochaete chrysosporium	与纤维素酶联用，使玉米秸秆的还原糖产量提升1.34倍	［12］
PchAA9F	Phanerochaete chrysosporium	与纤维素酶联用，使玉米秸秆的还原糖产量提升1.33倍	［12］
PdLPMO9A	Pleurotus djamor	与纤维素酶共同作用，使玉米秸秆还原糖产量提高63.5%	［28］
SbLPMO9A	Sordaria brevicollis	与商业纤维素酶协同，使去木质素小麦秸秆的总还原糖产量提升65%	［29］
SbLPMO9B	Sordaria brevicollis	与商业纤维素酶协同，使无定形纤维素的总还原糖产量提升101%	［29］
TaLPMO9A	Thermoascus aurantiacus	与纤维素酶协同，使微晶纤维素的还原糖产量提升了1.2倍	［30］
TtLPMO9E	Thermothielavioides terrestris	与纤维素酶协同，使微晶纤维素的还原糖产量提升了1.9倍	［30］
TtLPMO9G	Thermothielavioides terrestris	与木聚糖酶联用，使木聚糖的释放量增加42%	［31］

表1 不同来源的AA9家族LPMO及其功能

Table 1 AA9 family LPMO from different sources and their functions

名称 Name	来源 Source	功能 Function	参考文献 Reference
AfAA9B	Aspergillus fumigatus	与纤维素酶协同作用，使汽爆甘蔗渣的糖化效率提升20%	［21］
MsLPMO2	Morchella sextelata	与纤维素酶联用，使稻草和小麦秸秆的还原糖产量分别提升126.4%和133.5%	［7］
MtLPMO9A	Thermothelomyces thermophilus	突变体与木聚糖酶协同作用，使木聚糖的还原糖产量提高57%	［22］
MtLPMO9G	Thermothelomyces thermophilus	与纤维素酶联用，使玉米秸秆的还原糖产量提升242%	［23］
MtLPMO9V	Thermothelomyces thermophilus	与内切葡聚糖酶协同作用，对微晶纤维素的协同效率可达120%	［24］
NcLPMO9C	Neurospora crassa	与纤维二糖水解酶联用，使高结晶纤维素的还原糖产量提升169%	［25］
PaLPMO9B	Podospora anserina	与纤维素酶协同，使酸和碱预处理甘蔗渣的还原糖产量分别提升了30%和20%	［26］
PaLPMO9H	Podospora anserina	与纤维二糖水解酶协同，使PASC的还原糖产量提升60%	［27］
PchAA9C	Phanerochaete chrysosporium	与纤维素酶联用，使玉米秸秆的还原糖产量提升1.34倍	［12］
PchAA9F	Phanerochaete chrysosporium	与纤维素酶联用，使玉米秸秆的还原糖产量提升1.33倍	［12］
PdLPMO9A	Pleurotus djamor	与纤维素酶共同作用，使玉米秸秆还原糖产量提高63.5%	［28］
SbLPMO9A	Sordaria brevicollis	与商业纤维素酶协同，使去木质素小麦秸秆的总还原糖产量提升65%	［29］
SbLPMO9B	Sordaria brevicollis	与商业纤维素酶协同，使无定形纤维素的总还原糖产量提升101%	［29］
TaLPMO9A	Thermoascus aurantiacus	与纤维素酶协同，使微晶纤维素的还原糖产量提升了1.2倍	［30］
TtLPMO9E	Thermothielavioides terrestris	与纤维素酶协同，使微晶纤维素的还原糖产量提升了1.9倍	［30］
TtLPMO9G	Thermothielavioides terrestris	与木聚糖酶联用，使木聚糖的释放量增加42%	［31］

图2 AA9家族LPMO的典型三维结构AA9家族LPMO的结构示意图（PDB ID：4D7U）

Fig. 2 The typical three-dimensional structure of AA9 LPMOStructural schematic diagram of the AA9 family LPMO (PDB ID: 4D7U)

图3 LsAA9A-纤维五糖复合物整体及局部结构图

Fig. 3 Overall and local structural diagrams of the LsAA9A-cellopentaose complex

图4 AA9家族LPMO催化结构域序列比对蓝线、绿线、黄线和红线分别对应L2、L3、LS和LC Loop区；红色五角星为文中重点讨论的关键功能残基

Fig. 4 Sequence alignment of catalytic domains of AA9 family LPMOBlue, green, yellow and red lines correspond to the L2, L3, LS, and LC Loop regions, respectively. Red five-pointed stars indicate key functional residues discussed in the text

图5 糖苷水解酶与AA9家族LPMO CBM1序列比对红色五角星为芳香族残基；黄色五角星为极性氨基酸残基；蓝色五角星为半胱氨酸残基

Fig. 5 Sequence alignment of CBM1 from glycoside hydrolases and AA9 family LPMORed five-pointed stars represent aromatic residues; yellow five-pointed stars represent polar amino acid residues; blue five-pointed stars represent cysteine residues

图6 AA9家族LPMO应用示意图

Fig. 6 Schematic diagram of the applications of AA9 family LPMO

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