Biodiesel Production from Soybean Oil Using a Free-Enzyme and Whole-Cell Dual Lipase System as a Biocatalyst.

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.