Teng Rong, Zhang Jin, Tu Zhui, He Qinghua, Li Yanping
State Key Laboratory of Food Science and Resources, Nanchang University, Nanchang 330047, China.
Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, China.
Foods. 2024 Dec 12;13(24):4023. doi: 10.3390/foods13244023.
Lipase, a green biocatalyst, finds extensive application in the food sector. Enhancing the thermal stability of lipase presents both challenges and opportunities within the food industry. This research employed multiple rounds of cross-screening using tools like FoldX and I-Mutant 3.0 to strategically design mutations for lipase (RML), resulting in eight unique single-point mutation designs. E230I, N120M, and N264M have been confirmed experimentally to be potential combination mutation candidates. The resulting triple mutant N120M/E230I/N264M showed a higher thermal stability, with an optimum temperature of 55 °C, 10 °C higher than that of the wild-type RML. The half-life was extended from 46 to 462 min at 50 °C. Furthermore, the catalytic activity of N120M/E230I/N264M on camphor tree seed oil increased by 140% to 600 U/mg, which aids in the production of novel structured lipids. Using molecular docking and molecular dynamics simulations, we analyzed the molecular mechanism of enhanced thermal stability. This study validated the efficacy and dependability of computer-aided design to generate heat-resistant RML mutants and indicated that RML N120M/E230I/N264M lipase can be used as an effective biocatalyst for fat processing in the food industry.
脂肪酶作为一种绿色生物催化剂，在食品领域有着广泛的应用。提高脂肪酶的热稳定性在食品工业中既带来了挑战，也带来了机遇。本研究利用FoldX和I-Mutant 3.0等工具进行多轮交叉筛选，对脂肪酶（RML）进行策略性突变设计，得到了8种独特的单点突变设计。实验证实E230I、N120M和N264M是潜在的组合突变候选者。所得的三重突变体N120M/E230I/N264M表现出更高的热稳定性，其最适温度为55℃，比野生型RML高10℃。在50℃下，半衰期从46分钟延长至462分钟。此外，N120M/E230I/N264M对樟树籽油的催化活性提高了140%，达到600 U/mg，这有助于新型结构脂质的生产。通过分子对接和分子动力学模拟，我们分析了热稳定性增强的分子机制。本研究验证了计算机辅助设计生成耐热RML突变体的有效性和可靠性，并表明RML N120M/E230I/N264M脂肪酶可作为食品工业中脂肪加工的有效生物催化剂。
