(316b) Effects of Solvent and Enzyme Source for Transesterification of Waste Oils

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. Several strains of lipases have been found to have transesterification activity, namely Candida antarctica, Pseudomonas cepacia, and Thermomyces lanuginosus. The two primary obstacles in enzyme-catalyzed reactions are i), the immiscibility of the two substrates, namely hydrophilic methanol and hydrophobic triglyceride, resulting in the formation of an interface leading to mass transfer resistance; and ii), the strong polarity of methanol, which tends to strip the active water from the enzyme’s active site leading to enzyme deactivation. The addition of an organic solvent as the medium to the reaction system simultaneously overcomes the two limitations by enhancing the solubility of oil and methanol in the solvent, and by limiting the concentration of methanol surrounding the enzyme.

We are genetically engineering 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, a thermophylic fungus. Cloning 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 a commercial Thermomyces lanuginosus, and the effect of solvent will also be presented.