Lytic polysaccharide monooxygenases (LPMOs) are industrially important enzymes able to enhance the enzymatic lignocellulose saccharification in synergism with classical glycoside hydrolases. Fungal LPMOs have been classified as AA9, AA11, and AA13-16 families showing a diverse specificity for substrates such as soluble and insoluble beta-glucans, chitin, starch, and xylan, besides cellulose. These enzymes are still not fully characterized, and for example this is testify by their mechanism of oxidation regularly reviewed multiple times in the last decade. Noteworthy is that despite the extremely large abundance in the entire Tree of Life, our structural and functional knowledge is based on a restricted pool of LPMO, and probably one of the main reason reside in the challenging posed by their heterologous expression. Notably, the lack of a simple cloning protocol that could be universally applied to LPMO, hinders the conversion of the ever-increasing available genomic information to actual new enzymes. Here, we provide an easy and fast protocol for cloning, expression, and purification of active LPMOs in the following architecture: natural signal peptide, LPMO enzyme, TEV protease site, and His6-Tag. For this purpose, a commercial methanol inducible expression vector was initially modified to allow the LPMO expression containing the above characteristics. Gibson assembly, a one-step isothermal DNA assembly, was adopted for the direct assembly of intron-less or intron-containing genes and the modified expression vector. Moreover, His6-tagged LPMO constructs can be submitted to TEV proteolysis for removal of the questionable C-terminal His6-Tag, obtaining a close-to-native form of LPMO. We successfully applied this method to clone, express, and purify six LPMOs from AA9 family with different regioselectivities. The proposed protocol, provided as step-by-step, could be virtually applied in many laboratories thanks to the choice of popular and commons materials.

中文翻译：

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一种快速简便的裂解多糖单加氧酶可裂解 His6-Tag 克隆、表达和纯化策略

裂解多糖单加氧酶 (LPMO) 是工业上重要的酶，能够与经典的糖苷水解酶协同增强酶促木质纤维素糖化。真菌 LPMO 已被分类为 AA9、AA11 和 AA13-16 家族，除纤维素外，它们对可溶性和不溶性 β-葡聚糖、几丁质、淀粉和木聚糖等底物具有不同的特异性。这些酶仍然没有完全表征，例如，这可以通过它们在过去十年中多次定期审查的氧化机制来证明。值得注意的是，尽管整个生命之树的丰度极大，但我们的结构和功能知识是基于有限的 LPMO 池，主要原因之一可能在于它们的异源表达带来的挑战。尤其，缺乏可普遍应用于 LPMO 的简单克隆方案阻碍了将不断增加的可用基因组信息转化为实际的新酶。在这里，我们提供了一种简单快速的方案，用于在以下架构中克隆、表达和纯化活性 LPMO：天然信号肽、LPMO 酶、TEV 蛋白酶位点和 His6-Tag。为此，最初对商业甲醇诱导型表达载体进行了修饰，以允许包含上述特征的 LPMO 表达。Gibson 组装是一种一步等温 DNA 组装，用于直接组装无内含子或含内含子的基因和修饰的表达载体。此外，His6 标记的​​ LPMO 构建体可以提交给 TEV 蛋白水解以去除有问题的 C 端 His6-Tag，获得接近本机形式的 LPMO。我们成功地应用这种方法克隆、表达和纯化了来自 AA9 家族的六个具有不同区域选择性的 LPMO。由于选择了流行和通用材料，所提议的协议分步提供，实际上可以在许多实验室中应用。