浙江农业学报 ›› 2026, Vol. 38 ›› Issue (2): 405-416.DOI: 10.3969/j.issn.1004-1524.20250179
• 综述 • 上一篇
裂解多糖单加氧酶的生物学机制及其在木质纤维素降解中的应用
朱夏菁1,2(
), 孙宏1, 周航海1, 王新1, 皮二旭2,*(), 汤江武1,3,*()
1.浙江省农业科学院 环境资源与土壤肥料研究所 浙江 杭州 310021 2.杭州师范大学 生命与环境科学学院 浙江 杭州 311121 3.浙江省农业科学院 园艺研究所 浙江 杭州 310021
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收稿日期:2025-03-12出版日期:2026-02-25发布日期:2026-03-24 -
作者简介:朱夏菁,研究方向为真菌来源LPMO降解难分解天然生物质。E-mail:zhuxjchn@163.com -
通讯作者:*皮二旭,E-mail:erxupi@hznu.edu.cn
汤江武,E-mail:angjiangwu@sina.com -
基金资助:浙江省自然科学基金杭州区域创新发展联合基金(LHZSZ24C010001);浙江省农业科学院院地科技合作项目(ZJTY2024-A-55)
Biological mechanism of lytic polysaccharide monooxygenase and its applications in lignocellulose degradation
ZHU Xiajing1,2(), SUN Hong1, ZHOU Hanghai1, WANG Xin1, PI Erxu2,*(), TANG Jiangwu1,3,*()
1. Institute of Environmental Resources ,Soil and Fertilizer, Zhejiang Academy of Agricultural Sciences Hangzhou 310021, China 2. College of Life and Environmental Sciences ,Hangzhou Normal University Hangzhou 311121, China 3. Institute of Horticulture ,Zhejiang Academy of Agricultural Sciences Hangzhou 310021, China
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Received:2025-03-12Online:2026-02-25Published:2026-03-24
摘要：
裂解多糖单加氧酶(LPMO)是一种氧化酶,其生化特性、在木质纤维素降解中的作用、蛋白质工程改造与优化,以及在工业生物技术中的应用受到广泛关注。木质纤维素因其复杂的结构与化学成分在天然条件下难以高效降解,其有效降解与转化已成为一项重要挑战。LPMO能够通过氧化作用切断多糖的结晶表面,协同糖苷水解酶高效降解木质纤维素,从而在秸秆等天然难降解生物质的转化中发挥重要作用。采用异源表达在其他宿主中生产LPMO是一种提升LPMO产量并推动其产业化的有效策略,当前已有诸多成功案例通过基因工程手段提升LPMO的稳定性、活性、底物特异性与催化效率。目前,LPMO已在植物抗病、食品工业与畜牧工业等工业生物技术领域展现出巨大的应用潜力。未来研究可进一步深入解析LPMO的催化降解机制,拓展其高效异源表达与蛋白质工程改造策略,以推动LPMO的工业化应用。
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引用本文
朱夏菁, 孙宏, 周航海, 王新, 皮二旭, 汤江武. 裂解多糖单加氧酶的生物学机制及其在木质纤维素降解中的应用[J]. 浙江农业学报, 2026, 38(2): 405-416.
ZHU Xiajing, SUN Hong, ZHOU Hanghai, WANG Xin, PI Erxu, TANG Jiangwu. Biological mechanism of lytic polysaccharide monooxygenase and its applications in lignocellulose degradation[J]. Acta Agriculturae Zhejiangensis, 2026, 38(2): 405-416.
图1 LPMO及其活性中心的三维结构 A,LPMO三维结构示意图;B,4个家族LPMO活性中心三维结构叠加示意图。棕色球体,铜原子;洋红色结构,AA9(PDB:3ZUD);橙色结构,AA10(PDB:2YOY);紫色结构,AA11(PDB:4MAI);绿色结构,AA13(PDB:4OPB)。His,组氨酸;Tyr,酪氨酸;Me-His 1,甲基化组氨酸。
Fig.1 Typical three-dimensional structure of LPMO and its active center A, Three-dimensional structure schematic of LPMO; B, Superposition of three-dimensional structures of active centers from four LPMO families. Brown sphere, Copper atom; magenta structure, AA9 (PDB:3ZUD); Orange structure, AA10 (PDB:2YOY); Purple structure, AA11 (PDB:4MAI); Green structure, AA13 (PDB:4OPB). His, Histidine; Tyr, Tyrosine; Me-His 1, Methylated histidine.
图1 LPMO及其活性中心的三维结构 A,LPMO三维结构示意图;B,4个家族LPMO活性中心三维结构叠加示意图。棕色球体,铜原子;洋红色结构,AA9(PDB:3ZUD);橙色结构,AA10(PDB:2YOY);紫色结构,AA11(PDB:4MAI);绿色结构,AA13(PDB:4OPB)。His,组氨酸;Tyr,酪氨酸;Me-His 1,甲基化组氨酸。
Fig.1 Typical three-dimensional structure of LPMO and its active center A, Three-dimensional structure schematic of LPMO; B, Superposition of three-dimensional structures of active centers from four LPMO families. Brown sphere, Copper atom; magenta structure, AA9 (PDB:3ZUD); Orange structure, AA10 (PDB:2YOY); Purple structure, AA11 (PDB:4MAI); Green structure, AA13 (PDB:4OPB). His, Histidine; Tyr, Tyrosine; Me-His 1, Methylated histidine.
表1 LPMO的基因工程表达系统
Table 1 Expression system of LPMO genetic engineering
| LPMO名称 LPMO name | 表达系统 Expression system | 参考文献 Reference |
|---|---|---|
| PcLPMO9D、NcLPMO9C、MtLPMO9E | 毕赤酵母(pPICZαA) P. pastoris(pPICZαA) | [ |
| SmLPMO10A、BlLPMO10A、JdLPMO10A、 ScLPMO10C、MaLPMO10B、CjLPMO10A | 大肠埃希菌(基于XylS/Pm系统的新型LPMO表达克隆载体) E. coli(novel LPMO expression cloning vector based on the XylS/Pm system) | [ |
| CjLPMO10A | 大肠埃希菌(pET22b) E. coli(pET22b) | [ |
| 米曲霉AA11家族LPMO A. oryzae AA11 family LPMO | 大肠埃希菌(pET32a)和毕赤酵母(pPIC9K) E. coli(pET32a) and P. pastoris(pPIC9K) | [ |
| TfLPMO10A、ScLPMO10B、 BatLPMO10、LsAA9A | 大肠埃希菌(pLyGo-Ec-1、pLyGo-Ec-2、pLyGo-Ec-3等多种载体) E. coli(various vectors including pLyGo-Ec-1, pLyGo-Ec-2, pLyGo-Ec-3) | [ |
表1 LPMO的基因工程表达系统
Table 1 Expression system of LPMO genetic engineering
| LPMO名称 LPMO name | 表达系统 Expression system | 参考文献 Reference |
|---|---|---|
| PcLPMO9D、NcLPMO9C、MtLPMO9E | 毕赤酵母(pPICZαA) P. pastoris(pPICZαA) | [ |
| SmLPMO10A、BlLPMO10A、JdLPMO10A、 ScLPMO10C、MaLPMO10B、CjLPMO10A | 大肠埃希菌(基于XylS/Pm系统的新型LPMO表达克隆载体) E. coli(novel LPMO expression cloning vector based on the XylS/Pm system) | [ |
| CjLPMO10A | 大肠埃希菌(pET22b) E. coli(pET22b) | [ |
| 米曲霉AA11家族LPMO A. oryzae AA11 family LPMO | 大肠埃希菌(pET32a)和毕赤酵母(pPIC9K) E. coli(pET32a) and P. pastoris(pPIC9K) | [ |
| TfLPMO10A、ScLPMO10B、 BatLPMO10、LsAA9A | 大肠埃希菌(pLyGo-Ec-1、pLyGo-Ec-2、pLyGo-Ec-3等多种载体) E. coli(various vectors including pLyGo-Ec-1, pLyGo-Ec-2, pLyGo-Ec-3) | [ |
表2 LPMO的基因工程改造结果对比
Table 2 Comparison of LPMO genetic engineering results
| 改造内容 Modification content | 具体结果 Specific result | 参考文献 Reference |
|---|---|---|
| 优化密码子和替换同义密码子, 对LPMO基因进行优化,通过优 化培养条件提高酶表达量,增强 酶活性 Codon optimization and synonymous codon replacement for LPMO gene, enhanced enzyme expression and activity through optimized culture conditions | 优化培养时间、诱导温度与甲醇添加量后,重组毕赤酵母菌株产LPMO能力较优化前增加了12.51%,重组菌株的酶活性最高达到(25.64±0.21) U·L-1;以1%微晶纤维素为底物时,添加LPMO可以使酶解6 h后水解率提升26.46%;以汽爆玉米秸秆为底物时,添加LPMO和一定浓度抗坏血酸可以缩短纤维素酶的降解启动时间,并在酶解9 h后水解效率提升13.07% After optimizing culture time, induction temperature, and methanol addition, the LPMO production of recombinant P. pastoris increased by 12.51% compared with that before optimization. The maximum enzyme activity reached (25.64±0.21) U·L-1. When 1% microcrystalline cellulose was used as the substrate, the hydrolysis rate increased by 26.46% after 6 h of enzymatic hydrolysis with LPMO. When steam-exploded corn stover was used as the substrate, the addition of LPMO and a certain concentration of ascorbic acid shortened the degradation initiation time of cellulase and increased the hydrolysis efficiency by 13.07% after 9 h of enzymatic hydrolysis | [ |
| 构建含有不同信号肽的表达载体, 提高酶表达量 Construction of expression vectors with different signal peptides to enhance enzyme expression | 使用结合了XylS启动子/激活子系统和不同LPMO信号序列的载体,该新型表达系统能可靠产生成熟的LPMO,并且使用SmLPMO10A的信号肽时受试LPMO的表达水平显著提高,SmLPMO10A、BlLPMO10A、JdLPMO10A的产量分别达到22、7、10 mg·L-1 Using a carrier combining the XylS promoter/activator system and different LPMO signal sequences, this novel expression system reliably produces mature LPMO. The expression levels of the tested LPMOs were significantly increased when using the signal peptide of SmLPMO10A, with yields of SmLPMO10A, BlLPMO10A, and JdLPMO10A reaching 22, 7, and 10 mg·L-1, respectively | [ |
| 引入二硫键,提高热稳定性、化学 稳定性和活性 The disulfide bond was introduced to improve the thermal stability, chemical stability and activity | 与野生型相比,突变体在60 ℃下的半衰期增加3倍,表观Tm值上升7 ℃,化学变性中点浓度(midpoint concentration of chemical denaturation, Cm值)提高0.3 mol·L-1,显示出野生型1.5倍的酶活性 Compared with the wild type, the mutant had a 3-fold increased half-life at 60 ℃, a 7 ℃ increase in apparent Tm value, a 0.3 mol·L-1 increase in midpoint concentration of chemical denaturation(Cm value), and 1.5 times the enzyme activity of the wild type | [ |
| 异源表达,提高酶表达量和催化 活性 Heterologous expression to enhance enzyme expression and catalytic activity | 在大肠埃希菌中获得了较高的表达量,重组LPMO的表达量达到了600 mg·L-1,在毕赤酵母中则实现了简单快速的分泌表达,利于工业化应用,异源表达后与几丁质酶共同作用于壳聚糖时比色法显示在40 h内产生的还原糖量显著高于野生型组 High expression was achieved in E. coli, with a recombinant LPMO yield of 600 mg·L-1. Simple and rapid secretory expression was realized in P. pastoris, facilitating industrial application. When co-acting with chitinase on chitosan after heterologous expression, the amount of reducing sugar produced within 40 h was significantly higher than that of the wild-type group, as shown by colorimetric method | [ |
| 优化密码子或添加N端标签, 构建含有不同信号肽的表达载体, 提高酶表达量和催化活性 Codon optimization or addition of N-terminal tags, construction of expression vectors with different signal peptides to enhance enzyme expression and catalytic activity | 所有LPMO均在大肠埃希菌周质成功表达,产量至少为40 mg·L-1,在胞质表达的产量在100~800 mg·L-1之间,BatLPMO10产量范围为0.5~2 g·L-1,使用天然信号肽产生的蛋白质量最多,而密码子优化的LsAA9A突变体催化活性高于天然序列,约为天然序列的2倍 All LPMOs were successfully expressed in the periplasm of E. coli, with yields of at least 40 mg·L-1. The yields in cytoplasmic expression ranged from 100 to 800 mg·L-1, and the yield range of BatLPMO10 was 0.5-2 g·L-1. The highest protein production was achieved using the native signal peptide, while the codon-optimized LsAA9A mutant had higher catalytic activity than the native sequence, approximately twice that of the native sequence | [ |
表2 LPMO的基因工程改造结果对比
Table 2 Comparison of LPMO genetic engineering results
| 改造内容 Modification content | 具体结果 Specific result | 参考文献 Reference |
|---|---|---|
| 优化密码子和替换同义密码子, 对LPMO基因进行优化,通过优 化培养条件提高酶表达量,增强 酶活性 Codon optimization and synonymous codon replacement for LPMO gene, enhanced enzyme expression and activity through optimized culture conditions | 优化培养时间、诱导温度与甲醇添加量后,重组毕赤酵母菌株产LPMO能力较优化前增加了12.51%,重组菌株的酶活性最高达到(25.64±0.21) U·L-1;以1%微晶纤维素为底物时,添加LPMO可以使酶解6 h后水解率提升26.46%;以汽爆玉米秸秆为底物时,添加LPMO和一定浓度抗坏血酸可以缩短纤维素酶的降解启动时间,并在酶解9 h后水解效率提升13.07% After optimizing culture time, induction temperature, and methanol addition, the LPMO production of recombinant P. pastoris increased by 12.51% compared with that before optimization. The maximum enzyme activity reached (25.64±0.21) U·L-1. When 1% microcrystalline cellulose was used as the substrate, the hydrolysis rate increased by 26.46% after 6 h of enzymatic hydrolysis with LPMO. When steam-exploded corn stover was used as the substrate, the addition of LPMO and a certain concentration of ascorbic acid shortened the degradation initiation time of cellulase and increased the hydrolysis efficiency by 13.07% after 9 h of enzymatic hydrolysis | [ |
| 构建含有不同信号肽的表达载体, 提高酶表达量 Construction of expression vectors with different signal peptides to enhance enzyme expression | 使用结合了XylS启动子/激活子系统和不同LPMO信号序列的载体,该新型表达系统能可靠产生成熟的LPMO,并且使用SmLPMO10A的信号肽时受试LPMO的表达水平显著提高,SmLPMO10A、BlLPMO10A、JdLPMO10A的产量分别达到22、7、10 mg·L-1 Using a carrier combining the XylS promoter/activator system and different LPMO signal sequences, this novel expression system reliably produces mature LPMO. The expression levels of the tested LPMOs were significantly increased when using the signal peptide of SmLPMO10A, with yields of SmLPMO10A, BlLPMO10A, and JdLPMO10A reaching 22, 7, and 10 mg·L-1, respectively | [ |
| 引入二硫键,提高热稳定性、化学 稳定性和活性 The disulfide bond was introduced to improve the thermal stability, chemical stability and activity | 与野生型相比,突变体在60 ℃下的半衰期增加3倍,表观Tm值上升7 ℃,化学变性中点浓度(midpoint concentration of chemical denaturation, Cm值)提高0.3 mol·L-1,显示出野生型1.5倍的酶活性 Compared with the wild type, the mutant had a 3-fold increased half-life at 60 ℃, a 7 ℃ increase in apparent Tm value, a 0.3 mol·L-1 increase in midpoint concentration of chemical denaturation(Cm value), and 1.5 times the enzyme activity of the wild type | [ |
| 异源表达,提高酶表达量和催化 活性 Heterologous expression to enhance enzyme expression and catalytic activity | 在大肠埃希菌中获得了较高的表达量,重组LPMO的表达量达到了600 mg·L-1,在毕赤酵母中则实现了简单快速的分泌表达,利于工业化应用,异源表达后与几丁质酶共同作用于壳聚糖时比色法显示在40 h内产生的还原糖量显著高于野生型组 High expression was achieved in E. coli, with a recombinant LPMO yield of 600 mg·L-1. Simple and rapid secretory expression was realized in P. pastoris, facilitating industrial application. When co-acting with chitinase on chitosan after heterologous expression, the amount of reducing sugar produced within 40 h was significantly higher than that of the wild-type group, as shown by colorimetric method | [ |
| 优化密码子或添加N端标签, 构建含有不同信号肽的表达载体, 提高酶表达量和催化活性 Codon optimization or addition of N-terminal tags, construction of expression vectors with different signal peptides to enhance enzyme expression and catalytic activity | 所有LPMO均在大肠埃希菌周质成功表达,产量至少为40 mg·L-1,在胞质表达的产量在100~800 mg·L-1之间,BatLPMO10产量范围为0.5~2 g·L-1,使用天然信号肽产生的蛋白质量最多,而密码子优化的LsAA9A突变体催化活性高于天然序列,约为天然序列的2倍 All LPMOs were successfully expressed in the periplasm of E. coli, with yields of at least 40 mg·L-1. The yields in cytoplasmic expression ranged from 100 to 800 mg·L-1, and the yield range of BatLPMO10 was 0.5-2 g·L-1. The highest protein production was achieved using the native signal peptide, while the codon-optimized LsAA9A mutant had higher catalytic activity than the native sequence, approximately twice that of the native sequence | [ |
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