Brander Søren, Lausten Stine, Ipsen Johan Ø, Falkenberg Kristoffer B, Bertelsen Andreas B, Nørholm Morten H H, Østergaard Lars H, Johansen Katja S
Department of Geosciences and Natural Resource Management, University of Copenhagen, 1958, Copenhagen, Denmark.
Department of Plant and Environmental Sciences, University of Copenhagen, 1871, Copenhagen, Denmark.
Biotechnol Biofuels. 2021 Feb 27;14(1):51. doi: 10.1186/s13068-021-01902-4.
Lytic polysaccharide monooxygenases (LPMOs) are important industrial enzymes known for their catalytic degradation of recalcitrant polymers such as cellulose or chitin. Their activity can be measured by lengthy HPLC methods, while high-throughput methods are less specific. A fast and specific LPMO assay would simplify screening for new or engineered LPMOs and accelerate biochemical characterization.
A novel LPMO activity assay was developed based on the production of the dye phenolphthalein (PHP) from its reduced counterpart (rPHP). The colour response of rPHP oxidisation catalysed by the cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A), was found to increase tenfold by adding dehydroascorbate (DHA) as a co-substrate. The assay using a combination of rPHP and DHA was tested on 12 different metallo-enzymes, but only the LPMOs catalysed this reaction. The assay was optimized for characterization of TaAA9A and showed a sensitivity of 15 nM after 30 min incubation. It followed apparent Michaelis-Menten kinetics with k = 0.09 s and K = 244 µM, and the assay was used to confirm stoichiometric copper-enzyme binding and enzyme unfolding at a temperature of approximately 60 °C. DHA, glutathione and fructose were found to enhance LPMO oxidation of rPHP and in the optimized assay conditions these co-substrates also enabled cellulose degradation.
This novel and specific LPMO assay can be carried out in a convenient microtiter plate format ready for high-throughput screening and enzyme characterization. DHA was the best co-substrate tested for oxidation of rPHP and this preference appears to be LPMO-specific. The identified co-substrates DHA and fructose are not normally considered as LPMO co-substrates but here they are shown to facilitate both oxidation of rPHP and degradation of cellulose. This is a rare example of a finding from a high-throughput assay that directly translate into enzyme activity on an insoluble substrate. The rPHP-based assay thus expands our understanding of LPMO catalysed reactions and has the potential to characterize LPMO activity in industrial settings, where usual co-substrates such as ascorbate and oxygen are depleted.
裂解多糖单加氧酶（LPMOs）是重要的工业酶，以其催化降解纤维素或几丁质等难降解聚合物而闻名。它们的活性可以通过冗长的高效液相色谱法（HPLC）来测定，而高通量方法的特异性较低。一种快速且特异的LPMO检测方法将简化新型或工程化LPMO的筛选，并加速生化特性分析。
基于由还原型酚酞（rPHP）生成染料酚酞（PHP），开发了一种新型LPMO活性检测方法。发现通过添加脱氢抗坏血酸（DHA）作为共底物，嗜热栖热菌（TaAA9A）的纤维素特异性LPMO催化的rPHP氧化反应的颜色响应增加了10倍。使用rPHP和DHA组合的检测方法在12种不同的金属酶上进行了测试，但只有LPMOs催化了该反应。该检测方法针对TaAA9A的特性进行了优化，孵育30分钟后显示出15 nM的灵敏度。它遵循表观米氏动力学，k = 0.09 s，K = 244 μM，该检测方法用于确认在约60°C温度下化学计量的铜 - 酶结合和酶解折叠。发现DHA、谷胱甘肽和果糖可增强rPHP的LPMO氧化，并且在优化的检测条件下，这些共底物也能促进纤维素降解。
这种新型且特异的LPMO检测方法可以在方便的微孔板形式中进行，适用于高通量筛选和酶特性分析。DHA是测试的用于rPHP氧化的最佳共底物，这种偏好似乎是LPMO特异性的。所鉴定的共底物DHA和果糖通常不被视为LPMO共底物，但在此显示它们既促进rPHP的氧化又促进纤维素的降解。这是一个罕见的例子，即高通量检测的发现直接转化为对不溶性底物的酶活性。基于rPHP的检测方法因此扩展了我们对LPMO催化反应的理解，并有可能在工业环境中表征LPMO活性，在这种环境中通常的共底物如抗坏血酸和氧气会耗尽。
