Signal peptide-deleted lipase, Lipd25A, with transesterification activity from Pseudomonas aeruginosa B10 was overexpressed in Escherichia coli (E. coli) as a fusion with Jatropha curcas oleosin 2 (Jco2). The fusion protein Jco2-Lipd25A which coexpressed with molecular chaperone LipB was named Jco2-Lipd25AB. J. curcas seed oil bodies and Jco2-Lipd25AB were used to prepare reconstructed oil bodies (ROBs) for biodiesel production as immobilized lipase (ROBs-Jco2-Lipd25AB). Results indicated that Jco2-Lipd25AB could be immobilized on ROBs efficiently. The lytic cycle of ROBs was 30 d, meaning that it could be reused three times in series, as evidenced by the stability and reusability analysis. Interestingly, methanol toxicity test suggested that ROBs-Jco2-Lipd25AB presented transesterification activity in a high methanol concentration (300 mmol/L) environment without significant change of transesterification rate. The orthogonal L-25(5(6)) test design showed that the amount of ROBs-Jco2-Lipd25AB and solvent reaction system are the two most important factors in the transesterification reaction. The highest transesterification rate (75.19%) reached under the following optimal conditions: 2 mL ROBs-Jco2-Lipd25AB with a 3/1 ratio of methanol to oil (n/n), water content (%) 1 at 33 degrees C, with shaking at 180 rpm in a petroleum ether reaction system. Due to low cost, easy operation and friendly to environmental, natural oil body mediated immobilized lipase presents great prospects for biodiesel production. (C) 2014 Published by Elsevier Ltd.