Filandr Frantisek, Man Petr, Halada Petr, Chang Hucheng, Ludwig Roland, Kracher Daniel
1BioCeV-Institute of Microbiology, The Czech Academy of Sciences, Prumyslova 595, 252 50 Vestec, Czech Republic.
2Faculty of Science, Charles University, Hlavova 2030/8, 128 43 Prague 2, Czech Republic.
Biotechnol Biofuels. 2020 Mar 5;13:37. doi: 10.1186/s13068-020-01673-4. eCollection 2020.
Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent redox enzymes that cleave recalcitrant biopolymers such as cellulose, chitin, starch and hemicelluloses. Although LPMOs receive ample interest in industry and academia, their reaction mechanism is not yet fully understood. Recent studies showed that HO is a more efficient cosubstrate for the enzyme than O, which could greatly affect the utilization of LPMOs in industrial settings.
We probe the reactivity of LPMO9C from the cellulose-degrading fungus with a turbidimetric assay using phosphoric acid-swollen cellulose (PASC) as substrate and HO as a cosubstrate. The measurements were also followed by continuous electrochemical HO detection and LPMO reaction products were analysed by mass spectrometry. Different systems for the in situ generation of HO and for the reduction of LPMO's active-site copper were employed, including glucose oxidase, cellobiose dehydrogenase, and the routinely used reductant ascorbate.
We found for all systems that the supply of HO limited LPMO's cellulose depolymerization activity, which supports the function of HO as the relevant cosubstrate. The turbidimetric assay allowed rapid determination of LPMO activity on a cellulosic substrate without the need for time-consuming and instrumentally elaborate analysis methods.
裂解多糖单加氧酶（LPMOs）是一类依赖铜的氧化还原酶，可裂解诸如纤维素、几丁质、淀粉和半纤维素等难降解生物聚合物。尽管LPMOs在工业界和学术界备受关注，但其反应机制尚未完全明晰。近期研究表明，与氧气相比，过氧化氢是该酶更有效的共底物，这可能会极大地影响LPMOs在工业环境中的应用。
我们使用磷酸膨胀纤维素（PASC）作为底物，过氧化氢作为共底物，通过比浊法探究了纤维素降解真菌中LPMO9C的反应活性。测量过程中还进行了连续的电化学过氧化氢检测，并通过质谱分析了LPMO反应产物。采用了不同的原位生成过氧化氢和还原LPMO活性位点铜的系统，包括葡萄糖氧化酶、纤维二糖脱氢酶以及常用的还原剂抗坏血酸。
我们发现，对于所有系统而言，过氧化氢的供应限制了LPMO的纤维素解聚活性，这支持了过氧化氢作为相关共底物的功能。比浊法能够快速测定LPMO在纤维素底物上的活性，无需耗时且仪器复杂的分析方法。
