Department of Microbial and Plant Biotechnology, Centro de Investigaciones Biológicas Margarita Salas, Spanish National Research Council (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), 1462 Ås, Norway.
Int J Mol Sci. 2021 Dec 19;22(24):13611. doi: 10.3390/ijms222413611.
The first lytic polysaccharide monooxygenase (LPMO) detected in the genome of the widespread ascomycete (TamAA9A) has been successfully expressed in and characterized. Molecular modeling of TamAA9A showed a structure similar to those from other AA9 LPMOs. Although fungal LPMOs belonging to the genera or have not been analyzed in terms of regioselectivity, phylogenetic analyses suggested C1/C4 oxidation which was confirmed by HPAEC. To ascertain the function of a C-terminal linker-like region present in the wild-type sequence of the LPMO, two variants of the wild-type enzyme, one without this sequence and one with an additional C-terminal carbohydrate binding domain (CBM), were designed. The three enzymes (native, without linker and chimeric variant with a CBM) were purified in two chromatographic steps and were thermostable and active in the presence of HO. The transition midpoint temperature of the wild-type LPMO (Tm = 67.7 °C) and its variant with only the catalytic domain (Tm = 67.6 °C) showed the highest thermostability, whereas the presence of a CBM reduced it (Tm = 57.8 °C) and indicates an adverse effect on the enzyme structure. Besides, the potential of the different LPMO variants for their application in the saccharification of cellulosic and lignocellulosic materials was corroborated.
第一个在广泛分布的子囊菌（TamAA9A）基因组中检测到的溶细胞多糖单加氧酶（LPMO）已成功在 中表达并进行了表征。TamAA9A 的分子建模显示出与其他 AA9 LPMO 相似的结构。虽然尚未从种系发生分析的角度分析属于 或 的真菌 LPMO 的区域选择性，但系统发育分析表明 C1/C4 氧化，这通过 HPAEC 得到了证实。为了确定 LPMO 野生型序列中存在的 C 末端连接子样区域的功能，设计了两种野生型酶的变体，一种没有该序列，另一种带有额外的 C 末端碳水化合物结合结构域（CBM）。三种酶（天然、无连接子和带有 CBM 的嵌合变体）通过两步色谱法进行纯化，并且在 HO 存在下热稳定且具有活性。野生型 LPMO（Tm = 67.7°C）及其仅具有催化结构域的变体（Tm = 67.6°C）的过渡中点温度显示出最高的热稳定性，而 CBM 的存在降低了其热稳定性（Tm = 57.8°C），这表明对酶结构有不利影响。此外，还证实了不同的 LPMO 变体在纤维素和木质纤维素材料的糖化中的应用潜力。
