Immobilization of LipC12, a new lipase obtained by metagenomics, and its application in the synthesis of biodiesel esters

• We report the immobilization of a new metagenomic lipase, LipC12.
• LipC12 was immobilized directly from the crude extract onto Immobead 150.
• Immobilized LipC12 gave 98% conversion for ethyl-oleate synthesis in n-hexane.
• Conversions of over 90% were maintained during 10 cycles for the reaction in n-hexane.
• Immobilized LipC12 catalyzed the synthesis of ethyl-oleate in a solvent-free medium.

We immobilized a new metagenomic lipase, LipC12, and studied its application in the synthesis of ethyl-oleate, a model ester for biodiesel studies. Initially, we purified His-tagged LipC12 on a nickel column and immobilized it by adsorption on Accurel MP-1000 and by covalent bonding on Immobead 150. Preparations obtained with 10 mg of protein per gram of support were compared with respect to the synthesis of ethyl-oleate in n-hexane, using an ethanol to oleic acid molar ratio of 3:1. LipC12 gave a better conversion of oleic acid when immobilized on Immobead 150 (95% in 4 h) than when immobilized on Accurel MP-1000 (80% in 6 h). This led us to investigate the immobilization of His-tagged LipC12 directly from the crude extract on Immobead 150. At a protein loading of 200 mg g−1, the immobilized preparation (“Ibead-CELipC12”) gave a conversion of oleic acid of 99% in 60 min, for reactions performed in n-hexane, again using an ethanol to oleic acid molar ratio of 3:1. We obtained conversions of over 90% in ten successive 60-min reactions using the same batch of immobilized enzyme. When Ibead-CELipC12 was used to catalyze the esterification of oleic acid with ethanol in a solvent-free system at an ethanol to oleic acid molar ratio of 1:1, all activity was lost within 12 h, with a conversion of oleic acid of around 30%. However, when the reaction was repeated with the addition of ethanol in six equal aliquots during the course of the reaction, a conversion of oleic acid of 85% in 48 h was achieved. These results demonstrate that LipC12 has good potential to be used in the enzymatic synthesis of biodiesel. The results are particularly encouraging because it was possible to immobilize His-tagged LipC12 directly from the crude extract, thereby avoiding the need to purify the enzyme prior to immobilization.

Figure optionsDownload as PowerPoint slide