Application of lipases to develop novel acyltrnsferase substrates

1,2-Diacylglycerols (DG) are the native substrates for the diacylglycerol acyltransferase (DGAT). While DG containing saturated or unsaturated fatty acids are relatively easy to make chemically, it is difficult to chemically synthesize DG containing hydroxy fatty acids in specific positions on the glycerol backbone. An alternate approach is to start from acylglycerols containing hydroxy fatty acids, and selectively remove fatty acid chains to obtain the desired acylglycerol. Our goal was to produce both natural and "unnatural" substrates to evaluate the substrate specificity of the DGAT. This study identified optimal parameters for lipasecatalyzed methanolysis of triricinolein to produce 1,2(2,3 )-diricinolein, the substrate for biosynthesis of triricinolein in castor (Ricinus communis). We tested four different immobilized lipases, using n-hexane and diisopropyl ether (DIPE) as reaction media, and three different water activities. We followed the consumption of triricinolein and the formation of diricinolein, methyl ricinoleate and ricinoleic acid during the course of the reaction. Pénicillium roquefortii lipase gave the highest yield of l,2(2,3)-diricinolein and this lipase showed the highest specificity for the studied reaction, i.e. high selectivity for reaction with triricinolein, but low for diricinolein. The diacylglycerol produced can be acylated by the diacylglycerol acyltransferase from Arabidopsis thaliana. Therefore, the product of the lipase reaction is a suitable substrate for acyltransferase reactions. In order to produce an "unnatural" substrate, we evaluated conditions for synthesizing tri-hydroxy-stearin, using esterification of glycerol and 12-hydroxystearic acids in a solvent free system. We identified optimal parameters for lipase-catalyzed synthesis of tri-12- hydroxystearin (THS) from glycerol and 12-hydroxystearic acid (12-HSA), using Lipozyme RMIM 60 from Rhizomucor miehei (RML), a 1,3-specific lipase, as the biocatalyst. in this study. We obtained yields of THS up to 75% under conditions in which water was removed during the course of the reaction. Our future work with this product will be to produce 1,2 -di 12 hydroxy stearoyl glycerol.