Lytic polysaccharide monooxygenases (LPMOs) are redox enzymes of high biotechnological interest due to their capacity to degrade recalcitrant polysaccharides, such as cellulose, by an oxidative mechanism. The characterization of LPMOs is challenging since they generate a variety of catalytic products which include native oligosaccharides (non-oxidized) and oligosaccharides oxidized at the reducing end (C1 position), the non-reducing end (C4 position), or both ends, with different degrees of polymerization. Moreover, oxidized products exist in equilibrium with their hydrated forms, which further complicates their identification. The lack of commercial analytical standards for all these possible forms of oxidized oligosaccharides and the low concentration of these products make LPMO functional characterization dependent on advanced mass spectrometry techniques capable of identifying the profile of oxidized products. Here, a new approach for the characterization of cellulose-active LPMOs based on the combination of MALDI-TOF MS and hydrophilic interaction UHPLC-ESI-MS was proposed and optimized. The LPMO TrAA9A from Trichoderma reesei was used as a model enzyme to develop and test the MS methods. MALDI-TOF MS and UHPLC-ESI-MS methods were both capable of identifying putative C1, C4 and C1/C4 oxidized cello-oligosaccharides as well as their native counterparts generated after cellulose treatment with LPMO, which allows their utilization to characterize type I (C1-oxidizer), type II (C4-oxidizer) and type III (C1- and C4-oxidizer) LPMOs. Moreover, both methods were complementary since MALDI-TOF MS was capable of detecting oligosaccharides with higher degrees of polymerization (DP3–DP10), while UHPLC-ESI-MS allowed the evaluation of smaller oligosaccharides (DP1–DP5). The combined use of both methods offers a comprehensive description of LPMO catalytic products.

中文翻译：

---

结合使用MALDI-TOF MS和UHPLC-ESI-MS表征裂解多糖单加氧酶活性

溶菌多糖单加氧酶（LPMO）是具有高生物技术意义的氧化还原酶，因为它们能够通过氧化机制降解难降解的多糖，例如纤维素。LPMO的表征具有挑战性，因为它们会产生多种催化产物，包括天然的低聚糖（未氧化）和在还原端（C1位置），非还原端（C4位置）或两端被氧化的低聚糖。不同的聚合度。而且，氧化产物与它们的水合形式平衡存在，这进一步使它们的鉴定复杂化。缺少所有可能形式的氧化寡糖的商业分析标准，且这些产品的浓度低，使得LPMO的功能表征取决于能够识别氧化产物分布的先进质谱技术。在此，提出并优化了一种基于MALDI-TOF MS和亲水相互作用UHPLC-ESI-MS的表征纤维素活性LPMO的新方法。LPMO来自里氏木霉的Tr AA9A被用作模型酶来开发和测试MS方法。MALDI-TOF MS和UHPLC-ESI-MS方法均能够鉴定推定的C1，C4和C1 / C4氧化纤维寡糖，以及经过LPMO纤维素处理后生成的天然对应物，从而可以利用它们表征I型（ C1氧化剂），II型（C4氧化剂）和III型（C1和C4氧化剂）LPMO。此外，这两种方法是互补的，因为MALDI-TOF MS能够检测聚合度较高的寡糖（DP3-DP10），而UHPLC-ESI-MS可以评估较小的寡糖（DP1-DP5）。两种方法的组合使用提供了LPMO催化产物的全面描述。