Key Laboratory of Renewable Energy, Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, People's Republic of China; University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China.
Key Laboratory of Renewable Energy, Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, People's Republic of China.
Enzyme Microb Technol. 2022 Oct;160:110072. doi: 10.1016/j.enzmictec.2022.110072. Epub 2022 Jun 2.
The construction of methanol-resistant lipases with high catalytic activity is world-shattering for biodiesel production. A semi-rational method has been constructed to enhance the properties of Rhizomucor miehei lipase with propeptide (ProRML) by introducing N-glycosylation sites in the Loop structure. The enzyme activities of the mutants N288 (1448.89 ± 68.64 U/mg) and N142 (1073.68 ± 33.87 U/mg) increased to 56.09 and 41.56 times relative to that of wild type ProRML (WT, 25.83 ± 0.73 U/mg), respectively. After incubation in 50 % methanol for 2.5 h, the residual activities of N314 and N174-1 were 95 % and 85%, which were higher than the WT (27 %). Additionally, the biodiesel yield of all mutants was increased after a one-time addition of methanol for 24 h. Among them, N288 increased the quantity of biodiesel from colza oil from 9.49 % to 88 %, and N314 increased the amount of biodiesel from waste soybean oil from 8.44% to 70%. This study provides an effective method to enhance the properties of lipase and improve its application potential in biodiesel production.
构建具有高催化活性的甲醇抗性脂肪酶对生物柴油生产具有重要意义。本研究采用半理性方法，通过在 Rhizomucor miehei 脂肪酶前肽（ProRML）的Loop 结构中引入 N-糖基化位点，增强了该酶的性质。突变体 N288（1448.89±68.64 U/mg）和 N142（1073.68±33.87 U/mg）的酶活分别提高到野生型 ProRML（WT，25.83±0.73 U/mg）的 56.09 倍和 41.56 倍。在 50%甲醇中孵育 2.5 h 后，N314 和 N174-1 的剩余活性分别为 95%和 85%，高于 WT（27%）。此外，所有突变体在甲醇一次性添加 24 h 后，生物柴油的产率均有所提高。其中，N288 将菜籽油的生物柴油产量从 9.49%提高到 88%，N314 将废弃大豆油的生物柴油产量从 8.44%提高到 70%。该研究为增强脂肪酶的性质，提高其在生物柴油生产中的应用潜力提供了一种有效方法。
