米曲霉裂解性多糖单加氧酶的异源表达与性质分析

Heterologous expression and characterization of Aspergillus oryzae lytic polysaccharide monooxygenases

摘要:

[目的] 裂解性多糖单加氧酶（lytic polysaccharide monooxygenases，LPMOs）是一类以氧化方式断裂多聚糖糖苷键的新型木质纤维素降解酶，本文旨在挖掘新型LPMOs并研究其性质。[方法] 从米曲霉中克隆LPMO基因，利用毕赤酵母表达系统进行异源表达，研究其酶学性质和还原剂对其活性的影响，进一步探讨LPMO与糖苷水解酶协同作用时的底物结合现象。[结果] AoLPMO2和AoLPMO5序列分析显示，两种蛋白都为辅助酶类9家族的LPMOs；电击转化至真核毕赤酵母GS115中，获得双拷贝转化子GS/AO5-4，经1%甲醇诱导4 d后，上清液蛋白表达量为0.19±0.01 g/L。重组蛋白分子量约34 kDa，高于理论分子量，推测可能存在翻译后修饰。酶学性质分析表明，AoLPMO5对刺槐豆胶的最适反应温度和pH分别为60℃和5.0，K_m和V_max分别为8.72±1.99 mg/mL和109.4±12.8μmol/（s·mg）。0.1 mmol/L Cu²⁺促进酶活性提高（7.10±1.32）%（P<0.05），0.5、2.0和2.5 mmol/L H₂O₂分别促进酶活性提高（21.11±6.17）%（P<0.01）、（20.22±1.13）%（P<0.01）和（18.40±2.86）%（P<0.01），而没食子酸和维生素C对活性无明显作用。在反应前期，AoLPMO5与刺槐豆胶底物结合从而影响甘露聚糖酶BsMAN3的降解作用。而在反应后期，AoLPMO5与BsMAN3则表现出协同增效作用。[结论] AoLPMO5是一种全新的生物质降解酶，阐明其酶学性质和底物作用方式，将为天然木质纤维素类底物的高效转化与生物炼制，如第二代生物乙醇、功能性低聚寡糖等生产建立基础。

Abstract:

[Objective] Lytic polysaccharide monooxygenases (LPMOs) are new lignocellulose-degrading enzymes that break glycosidic bonds of polysaccharides via oxidation. This study aims to explore and characterize novel LPMOs. [Methods] LPMO genes were cloned from Aspergillus oryzae and expressed in Pichia pastoris. [Results] Sequence analysis indicated that both AoLPMO2 and AoLPMO5 belong to auxiliary activity 9 family. AoLPMO5 was transformed into P. pastoris GS115 to obtain a 2-copy insertion yeast GS/AO5-4. After induction by 1% methanol for 4 d, protein yield in culture supernatant reached 0.19±0.01 g/L. The molecular mass of recombinant protein was approximate 34 kDa, which was higher that the theorical calculation, indicating probably post-translation modification proceeded in yeast host. The optimal temperature and pH of AoLPMO5 to catalyze locust bean gum were 60℃ and 5.0. Its K_m and V_max were 8.72±1.99 mg/mL and 109.4±12.8 μmol/(s·mg), respectively. Addition of 0.1 mmol/L Cu²⁺, 0.5, 2.0 and 2.5 mmol/L H₂O₂ enhanced its catalytic activity by (7.10±1.32)% (P<0.05), (21.11±6.17)% (P<0.01), (20.22±1.13)% (P<0.01) and (18.40±2.86)% (P<0.01), respectively, while gallic acid or vitamin C did not show promotion. The AoLPMO5 was found to bind with locust bean gum substrate at the early stage of reaction, resulting in temporary inhibition of mannanase (BsMAN3) activity. However, these two enzymes showed synergic effects on substrate deconstruction at the later period. [Conclusion] AoLPMO5 is a novel biomass-degrading enzyme. Insights into its enzymatic properties and substrate-degrading patterns will contribute to natural lignocellulosic biomass conversion and biorefinery, especially second-generation bioethanol and functional oligosaccharides production.