Xiao Dunchi, Li Xun, Zhang Yu, Wang Fei
Co-Innovation Center for Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, China.
Jiangsu Provincial Key Lab for the Chemistry and Utilization of Agro-Forest Biomass, Nanjing Forestry University, Nanjing, 210037, China.
Appl Biochem Biotechnol. 2025 Apr;197(4):2163-2179. doi: 10.1007/s12010-024-05115-x. Epub 2024 Dec 18.
A dual lipase system has been developed to convert soybean oil into biodiesel through synergistic catalysis of Thermomyces lanuginosus lipase (TLL) and Yarrowia lipolytica lipase 2 (YLL) in this study. Pichia pastoris recombinant strains expressing lipases were successfully constructed, and the activities of TLL and YLL in the fermentation supernatant reached 23,142.71 ± 280.54 U/mL and 895.44 ± 27.31 U/mL, respectively. Immediately thereafter, free lipase was used to catalyze the preparation of biodiesel from soybean oil. After optimizing reaction conditions, 80 U/g oil TLL and 20 U/g oil YLL were used to catalyze the production of biodiesel, and a 95.56% biodiesel yield was obtained at 40 °C, 40% moisture content (water/oil, w/w), and stepwise addition of five molar equivalents of methanol. Double lipase plasmids (tll gene and yll gene in different proportions) were constructed in vitro and introduced into P. pastoris to construct a recombinant strain with optimal activity. Under the reaction conditions of 40% moisture content, 8% whole-cell biocatalyst dosage, and stepwise addition of five molar equivalents of methanol, the biodiesel yield reached 95.35% after 24 h at 40 °C. These results show that synergistic catalysis is an effective strategy for biodiesel synthesis and can not only improve the biodiesel yield but also shorten the reaction time. This study provides a scientific basis for biodiesel production by multi-enzyme compounding, with potential industrial applications.
本研究开发了一种双脂肪酶系统，通过嗜热栖热菌脂肪酶（TLL）和解脂耶氏酵母脂肪酶2（YLL）的协同催化将大豆油转化为生物柴油。成功构建了表达脂肪酶的毕赤酵母重组菌株，发酵上清液中TLL和YLL的活性分别达到23142.71±280.54 U/mL和895.44±27.31 U/mL。此后立即使用游离脂肪酶催化由大豆油制备生物柴油。优化反应条件后，使用80 U/g油的TLL和20 U/g油的YLL催化生物柴油的生产，在40℃、40%水分含量（水/油，w/w）以及逐步添加五摩尔当量甲醇的条件下，生物柴油产率达到95.56%。体外构建了不同比例的双脂肪酶质粒（tll基因和yll基因），并将其导入毕赤酵母中构建具有最佳活性的重组菌株。在40%水分含量、8%全细胞生物催化剂用量以及逐步添加五摩尔当量甲醇的反应条件下，40℃反应24 h后生物柴油产率达到95.35%。这些结果表明，协同催化是生物柴油合成的有效策略，不仅可以提高生物柴油产率，还可以缩短反应时间。本研究为多酶复合生产生物柴油提供了科学依据，具有潜在的工业应用价值。
