State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211800, P. R. China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P. R. China.
J Agric Food Chem. 2020 Dec 23;68(51):15257-15266. doi: 10.1021/acs.jafc.0c05979. Epub 2020 Dec 8.
Lytic polysaccharide monooxygenases (LPMOs) play a crucial role in the enzymatic depolymerization of cellulose through oxidative cleavage of the glycosidic bond in the highly recalcitrant crystalline cellulose region. Improving the activity of LPMOs is of considerable importance for second-generation biorefinery. In this study, we identified a beneficial amino acid substitution (N526S) located in the cellulose binding module (CBM) of LPMO10 (LPMO of ) using directed evolution. The improved variant LPMO10 M1 (N526S) exhibits 2.1-fold higher activity for the HO production, 2.7-fold higher oxidation activity, and 1.9-fold higher binding capacity toward cellulose compared with those of the wild type (WT). Furthermore, M1 shows 2.1-fold higher activity for degradation of crystalline cellulose in synergy with cellulase, compared to the WT. Structural analysis through molecular modeling and molecular dynamics (MD) simulation revealed that the substitution N526S located in the CBM likely stabilizes the cellulose binding surface and enhances the binding capacity of LPMO10 to cellulose, thereby enhancing enzyme activity. These findings demonstrate the important role of the CBM in the catalytic function of LPMO.
溶细胞单加氧酶（Lytic polysaccharide monooxygenases，LPMOs）在通过氧化切割高度稳定的结晶纤维素区域中的糖苷键来酶解纤维素方面发挥着关键作用。提高 LPMO 的活性对于第二代生物炼制具有重要意义。在这项研究中，我们使用定向进化技术在 LPMO10（）的纤维素结合模块（Cellulose binding module，CBM）中鉴定出一个有益的氨基酸取代（N526S）。与野生型（WT）相比，改良变体 LPMO10 M1（N526S）在 HO 产生方面的活性提高了 2.1 倍，氧化活性提高了 2.7 倍，对纤维素的结合能力提高了 1.9 倍。此外，与 WT 相比，M1 与纤维素酶协同作用时对结晶纤维素的降解活性提高了 2.1 倍。通过分子建模和分子动力学（MD）模拟的结构分析表明，位于 CBM 中的取代 N526S 可能稳定了纤维素结合表面，并增强了 LPMO10 对纤维素的结合能力，从而提高了酶活性。这些发现表明 CBM 在 LPMO 的催化功能中起着重要作用。
