Background The availability of a sensitive and robust activity assay is a prerequisite for efficient enzyme production, purification, and characterization. Here we report on a spectrophotometric assay for lytic polysaccharide monooxygenase (LPMO), which is an advancement of the previously published 2,6-dimethoxyphenol (2,6-DMP)-based LPMO assay. The new assay is based on hydrocoerulignone as substrate and hydrogen peroxide as cosubstrate and aims toward a higher sensitivity at acidic pH and a more reliable detection of LPMO in complex matrices like culture media. Results An LPMO activity assay following the colorimetric oxidation of hydrocoerulignone to coerulignone was developed. This peroxidase activity of LPMO in the presence of hydrogen peroxide can be detected in various buffers between pH 4-8. By reducing the substrate and cosubstrate concentration, the assay has been optimized for minimal autoxidation and enzyme deactivation while maintaining sensitivity. Finally, the optimized and validated LPMO assay was used to follow the recombinant expression of an LPMO in Pichia pastoris and to screen for interfering substances in fermentation media suppressing the assayed reaction. Conclusions The biphenol hydrocoerulignone is a better substrate for LPMO than the monophenol 2,6-DMP, because of a ~ 30 times lower apparent K M value and a 160 mV lower oxidation potential. This greatly increases the measured LPMO activity when using hydrocoerulignone instead of 2,6-DMP under otherwise similar assay conditions. The improved activity allows the adaptation of the LPMO assay toward a higher sensitivity, different buffers and pH values, more stable assay conditions or to overcome low concentrations of inhibiting substances. The developed assay protocol and optimization guidelines increase the adaptability and applicability of the hydrocoerulignone assay for the production, purification, and characterization of LPMOs.

中文翻译：

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裂解多糖单加氧酶的改进分光光度法测定。

背景 灵敏且稳健的活性测定的可用性是高效酶生产、纯化和表征的先决条件。在这里，我们报告了裂解多糖单加氧酶 (LPMO) 的分光光度法测定，这是先前发表的基于 2,6-二甲氧基苯酚 (2,6-DMP) 的 LPMO 测定的进步。新的检测方法基于氢化coerulignone 作为底物和过氧化氢作为共底物，旨在提高酸性pH 下的灵敏度和更可靠地检测复杂基质（如培养基）中的LPMO。结果开发了一种在氢化coerulignone 比色氧化为coerulignone 后的LPMO 活性测定。LPMO 在过氧化氢存在下的这种过氧化物酶活性可以在 pH 4-8 之间的各种缓冲液中检测到。通过降低底物和共底物的浓度，该测定已针对最小的自氧化和酶失活进行优化，同时保持灵敏度。最后，优化和验证的 LPMO 测定用于跟踪 LPMO 在毕赤酵母中的重组表达，并筛选发酵培养基中抑制测定反应的干扰物质。结论 与单酚 2,6-DMP 相比，双酚氢钴木酮是 LPMO 更好的底物，因为其表观 KM 值低约 30 倍，氧化电位低 160 mV。当在其他相似的测定条件下使用氢化考鲁木酮而不是 2,6-DMP 时，这大大增加了测量的 LPMO 活性。改进的活性允许 LPMO 分析适应更高的灵敏度、不同的缓冲液和 pH 值，更稳定的测定条件或克服低浓度的抑制物质。已开发的检测方案和优化指南提高了氢化coerulignone 检测对LPMO 的生产、纯化和表征的适应性和适用性。