Liquid lipase-mediated alcoholysis for biodiesel production has drawn an increasing interest worldwide due to the faster reaction rate and lower cost compared to conventional reactions catalyzed by immobilized lipase.BTL2 from Bacillus thermocatenulatusis a thermo-stable lipase with high specific activity and is hopefully applied to the catalysis for biodiesel production.We explored the potential of constructing lipase BTL2 gene into Pichia pastoris strain X33 for high level extracellular expression and then discussed the possibility of using lipase BTL2 for biodiesel production.The engineered strain was constructed successfully and the hydrolytic activity of lipase BTL2 in the culture supernatant reached 2360 U/mL in a 5-L bioreactor.It was found that the crude lipase BTL2 from fermentation culture could be applied directly to catalyze the methanolysis reaction for biodiesel production.The initial water content,lipase activity,methanol adding strategy and temperature showed significant influence on liquid lipase BTL2-mediated reaction.A biodiesel yield of 87%was obtained under the conditions of 45 ℃,20%initial water content,10000 U BTL2 and stepwise addition of 30%,25%,20%,15%,10%methanol of the total volume at 0 h,1 h,2 h,3 h and4 h,respectively.