College of Chemical and Biological Engineering, Shandong University of Science and Technology, Qingdao 266590, PR China; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300450, PR China.
Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300450, PR China.
Enzyme Microb Technol. 2023 Aug;168:110255. doi: 10.1016/j.enzmictec.2023.110255. Epub 2023 May 10.
Lytic polysaccharide monooxygenases (LPMOs) can oxidatively cleave the glycosidic bonds of crystalline polysaccharides, providing more accessible sites for polysaccharide hydrolases and promoting efficient conversion of biomass. In order to promote industrial applications of LPMOs, the stability of an LPMO of Myceliophthora thermophila C1 (MtC1LPMO) was improved by adding disulfide bonds in this study. Firstly, the structural changes of wild-type (WT) MtC1LPMO at different temperatures were explored using molecular dynamics simulations, and eight mutants were selected by combining the predicted results from Disulfide by Design (DBD), Multi agent stability prediction upon point mutations (Maestro) and Bridge disulfide (BridgeD) websites. Then, the enzymatic properties of the different mutants were determined after their expression and purification, and the mutant S174C/A93C with the highest thermal stability was obtained. The specific activities of unheated S174C/A93C and WT were 160.6 ± 1.7 U/g and 174.8 ± 7.5 U/g, respectively, while those of S174C/A93C and WT treated at 70 °C for 4 h were 77.7 ± 3.4 U/g and 46.1 ± 0.4 U/g, respectively. The transition midpoint temperature of S174C/A93C was 2.7 °C higher than that of WT. The conversion efficiency of S174C/A93C for both microcrystalline cellulose and corn straw was about 1.5 times higher than that of WT. Finally, molecular dynamics simulations revealed that the introduction of disulfide bonds increased the β-sheet content of the H1-E34 region, thus improving the rigidity of the protein. Therefore, the overall structural stability of S174C/A93C was improved, which in turn improved its thermal stability.
溶细胞多糖单加氧酶（LPMOs）能够氧化切割结晶多糖的糖苷键，为多糖水解酶提供更多可及的作用位点，并促进生物质的有效转化。为了促进 LPMO 的工业应用，本研究通过在嗜热真菌（Myceliophthora thermophila C1）C1LPMO 中添加二硫键来提高其稳定性。首先，通过分子动力学模拟研究了野生型（WT）MtC1LPMO 在不同温度下的结构变化，并结合 Disulfide by Design（DBD）、Multi agent stability prediction upon point mutations（Maestro）和 Bridge disulfide（BridgeD）网站的预测结果，选择了 8 个突变体。然后，对不同突变体的表达和纯化后的酶学性质进行了测定，得到了热稳定性最高的突变体 S174C/A93C。未经热处理的 S174C/A93C 和 WT 的比活性分别为 160.6±1.7 U/g 和 174.8±7.5 U/g，而在 70°C 处理 4 h 后，S174C/A93C 和 WT 的比活性分别为 77.7±3.4 U/g 和 46.1±0.4 U/g。S174C/A93C 的转变中点温度比 WT 高 2.7°C。S174C/A93C 对微晶纤维素和玉米秸秆的转化率约为 WT 的 1.5 倍。最后，分子动力学模拟表明，二硫键的引入增加了 H1-E34 区域的β-折叠含量，从而提高了蛋白质的刚性。因此，S174C/A93C 的整体结构稳定性得到提高，进而提高了其热稳定性。
