(582at) Transgenic Expression of Thermomyces Lanuginosus and Candida Antarctica Lipases in Plants for the Enzymatic Production of Biodiesel

Enzymatic transesterification with lipase as the catalyst eliminates soap formation.  Unlike alkali-based reactions, the products can easily be collected and separated. Moreover, enzymes require much less alcohol to perform the reaction, and can be reused despite some loss in activity at the end of each cycle. We have genetically engineered plants to constitutively express a lipase for biodiesel production from spent oils. We have cloned the gene of a lipase with known transesterification activity from Thermomyces lanuginosus, and Candida antarctica. Cloning of TL enzyme involved isolation of total RNA, reverse transcription of the mRNA into cDNA and PCR amplification of the lipase gene using specific primers. The gene was first inserted into a cloning vector (pCR8/GW/TOPO) and sequenced to confirm its identity. The gene has been inserted into a plant destination vector (pGWB408 and pMDC83) via LR clonase reaction. Nicotiana tabacum (tobacco) leaf was transformed with the lipase gene using Agrobacterium tumefaciens (strain GV3101) and transferred onto selection media plates.  We have also grown Arabidopsis thaliana from seeds that were transformed using the floral dip transformation method. The recombinant enzyme was collected from the genetically engineered plants, purified, and tested for both hydrolytic and synthetic activity. The activity results will be compared with enzyme catalysts from commercial Thermomyces lanuginosus and Candida antarctica, and the effect of solvent addition will also be presented.