Wang_2024_Bioresour.Technol_413_131521

Lipases catalyze the synthesis of biodiesel, which is an important renewable alternative energy source. Cost-efficient conversion of waste acidified oil to biodiesel entails acid-tolerant lipases which have not been extensively studied. This study showed that the commonly used Thermomyces lanuginosus lipase TLL displayed a weak acid tolerance and an unsatisfactory performance in biodiesel production from acidified oil. Directed evolution of TLL identified one TLL-T3 variant with three residue substitutions (A69S/V150P/N222G). TLL-T3 displayed significantly enhanced acid tolerance, and its application in acidified oil treatment led to a biodiesel yield up to 90 % (w/w). A scaled-up production of TLL-T3 in Trichoderma reesei was further achieved and the highest extracellular lipase activity reached 16,123 U/mL after fermentation optimization. These results provide new insights into structural adaptation to acid tolerance by lipases and show that TLL-T3 holds great potential in commercial biodiesel production from waste acidified oil.