Characterization of a new sn-1,3-regioselective triacylglycerol lipase from Malbranchea cinnamomea.

The thermophilic ascomycetous fungus Malbranchea cinnamomea produces lipases (EC 3.1.1.3) that allow it to grow efficiently on medium containing triacylglycerol substrates such as plant oils or tributyrin as sole carbon source. In the transcriptome of M. cinnamomea grown on olive oil, we found one cDNA sequence encoding a putative extracellular lipase. This gene, termed as MclipA, was cloned and heterologously expressed in Pichia pastoris. The recombinant protein, rMclipA, catalyzed the hydrolysis of short-chain fatty acid ester such as p-nitrophenyl butyrate (C4) and long-chain fatty acid ester such as p-nitrophenyl myristate (C14). These results indicate that MclipA is a true triacylglycerol lipase. For rMclipA, the optimum lipase activity was obtained at 45 °C, and more than 93% of enzyme activity was retained after 24 H of incubation at temperatures up to 50 °C. rMclipA was active toward p-nitrophenyl esters of various carbon chain lengths with peak activity on long-chain fatty acid (C14). rMclipA displayed high sn-1,3-regioselectivity on hydrolyzing triolein. rMclipA can catalyze oleic acid methyl ester synthesis resulting in a 71% esterification degree after 24 H of reaction at 40 °C. These properties suggest that rMclipA has potential application in, for example, selective hydrolysis of oil, modification of triacylglycerol, and production of biodiesel.