Stepnov Anton A, Forsberg Zarah, Sørlie Morten, Nguyen Giang-Son, Wentzel Alexander, Røhr Åsmund K, Eijsink Vincent G H
Faculty of Chemistry, Biotechnology and Food Science, NMBU-Norwegian University of Life Sciences, Ås, Norway.
Department of Biotechnology and Nanomedicine, SINTEF Industry, Trondheim, Norway.
Biotechnol Biofuels. 2021 Jan 21;14(1):28. doi: 10.1186/s13068-021-01879-0.
Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative depolymerization of industrially relevant crystalline polysaccharides, such as cellulose, in a reaction that depends on an electron donor and O or HO. While it is well known that LPMOs can utilize a wide variety of electron donors, the variation in reported efficiencies of various LPMO-reductant combinations remains largely unexplained.
In this study, we describe a novel two-domain cellulose-active family AA10 LPMO from a marine actinomycete, which we have used to look more closely at the effects of the reductant and copper ions on the LPMO reaction. Our results show that ascorbate-driven LPMO reactions are extremely sensitive to very low amounts (micromolar concentrations) of free copper because reduction of free Cu(II) ions by ascorbic acid leads to formation of HO, which speeds up the LPMO reaction. In contrast, the use of gallic acid yields steady reactions that are almost insensitive to the presence of free copper ions. Various experiments, including dose-response studies with the enzyme, showed that under typically used reaction conditions, the rate of the reaction is limited by LPMO-independent formation of HO resulting from oxidation of the reductant.
The strong impact of low amounts of free copper on LPMO reactions with ascorbic acid and O, i.e. the most commonly used conditions when assessing LPMO activity, likely explains reported variations in LPMO rates. The observed differences between ascorbic acid and gallic acid show a way of making LPMO reactions less copper-dependent and illustrate that reductant effects on LPMO action need to be interpreted with great caution. In clean reactions, with minimized generation of HO, the (O-driven) LPMO reaction is exceedingly slow, compared to the much faster peroxygenase reaction that occurs when adding HO.
裂解多糖单加氧酶（LPMOs）是一种单铜酶，可催化工业相关结晶多糖（如纤维素）的氧化解聚反应，该反应依赖于电子供体和O₂或H₂O₂。虽然众所周知LPMOs可以利用多种电子供体，但各种LPMO-还原剂组合的报道效率差异在很大程度上仍未得到解释。
在本研究中，我们描述了一种来自海洋放线菌的新型双结构域纤维素活性家族AA10 LPMO，我们用它来更仔细地研究还原剂和铜离子对LPMO反应的影响。我们的结果表明，抗坏血酸驱动的LPMO反应对极低量（微摩尔浓度）的游离铜极为敏感，因为抗坏血酸还原游离Cu（II）离子会导致H₂O₂的形成，从而加速LPMO反应。相比之下，使用没食子酸会产生稳定的反应，几乎不受游离铜离子存在的影响。各种实验，包括对该酶的剂量反应研究，表明在通常使用的反应条件下，反应速率受还原剂氧化产生的与LPMO无关的H₂O₂形成的限制。
少量游离铜对LPMO与抗坏血酸和O₂反应的强烈影响，即在评估LPMO活性时最常用的条件，可能解释了报道的LPMO速率差异。抗坏血酸和没食子酸之间观察到的差异表明了一种使LPMO反应减少对铜依赖的方法，并说明对LPMO作用的还原剂影响需要非常谨慎地解释。在H₂O₂生成最小化的清洁反应中，与添加H₂O₂时发生的更快的过氧合酶反应相比，（O₂驱动的）LPMO反应极其缓慢。
