Key Laboratory of Geriatric Nutrition and Health, Beijing Technology and Business University, Ministry of Education, Beijing 100048, China; Key Laboratory of Brewing Microbiome and Enzymatic Molecular Engineering, China General Chamber of Commerce, Beijing 100048, China; School of Food and Health, Beijing Technology and Business University (BTBU), Beijing 100048, China.
Key Laboratory of Geriatric Nutrition and Health, Beijing Technology and Business University, Ministry of Education, Beijing 100048, China; Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU), Beijing 100048, China; Beijing Association for Science and Technology-Food Nutrition and Safety Professional Think Tank Base, Beijing 100048, China; School of Food and Health, Beijing Technology and Business University (BTBU), Beijing 100048, China.
Int J Biol Macromol. 2024 Nov;279(Pt 3):135399. doi: 10.1016/j.ijbiomac.2024.135399. Epub 2024 Sep 6.
Acidic xylanase PjxA from Penicillium janthinellum MA21601, with good eosinophilic and enzymatic activity, is an excellent candidate for xylan degradation to achieve effective utilization of biomass materials. However, the low thermal stability of PjxA has become a major bottleneck in its application. In this study, the flexible sites of PjxA were identified and rigidified through computational simulations of structure and sequence analysis combined with folding free energy calculations. Finally, a combined mutase PjxA-DS was constructed by rational integration of the two single mutants S82N and D45N. Compared to PjxA, PjxA-DS showed a 115.11-fold longer half-life at 50 °C and a 2.02-fold higher specific enzyme activity. Computer simulation analysis showed that S82N and D45N acted synergistically to improve the thermostability of PjxA. The stabilization of the N-terminus and the active center of PjxA, the increase in surface positive charge and hydrophilicity are the main reasons for the improved thermostability and catalytic activity of PjxA. Rigidification of the flexible site is an effective method for improving the thermostability of enzymes, S82N and D45N can be used as effective targets for the thermostability engineering modification of GH11 acidic xylanase.
从青霉（Penicillium janthinellum）MA21601 中提取的酸性木聚糖酶 PjxA 具有良好的嗜酸性和酶活性，是降解木聚糖以实现生物质材料有效利用的优秀候选酶。然而，PjxA 的低热稳定性已成为其应用的主要瓶颈。在这项研究中，通过结构和序列分析结合折叠自由能计算的计算模拟，确定了 PjxA 的柔性位点，并使其刚性化。最后，通过合理整合两个单点突变 S82N 和 D45N，构建了组合突变酶 PjxA-DS。与 PjxA 相比，PjxA-DS 在 50°C 下的半衰期延长了 115.11 倍，比酶活提高了 2.02 倍。计算机模拟分析表明，S82N 和 D45N 协同作用提高了 PjxA 的热稳定性。PjxA 的 N 端和活性中心的稳定化、表面正电荷和亲水性的增加是提高 PjxA 热稳定性和催化活性的主要原因。柔性位点的刚性化是提高酶热稳定性的有效方法，S82N 和 D45N 可以作为 GH11 酸性木聚糖酶热稳定性工程改造的有效靶点。
