Enzymatic Synthesis Of 1,2-diacylglycerols

1,2-Diacylglycerols(1,2-DAGs),an intracellular second messenger,is involved in regulating stimulating-response mechanisms to control the cell activities.1,2-DAGs is the precursor of phospholipids and triglycerides in vivo,and may improve myocardial dysfunction through the synthesis of myocardial protein.The synthesis of asymmetric triacylglycerols(TAGs)with 1,2-DAGs makes up the disadvantages of chemical methods--time consuming,high temperature and toxic reagents.Therefore,it is an efficient and green approach for the production of asymmetric TAGs,which is beneficial for studying the structure and function of TAGs.An enzymatic method for the synthesis of 1,2-DAGs with the advantages of lipase was studied based on such merits as strong specificity,high catalytic activity,low by-products and mild reaction conditions.A solvent crystallization method was reported to purify 1,2-DAGs,as well.The main contents were as follows.Firstly,a method for the synthesis of 1,2-DAGs by enzymatic ethanolysis of high oleic sunflower oil(HOSO)and alcohol was reported.The effects of lipase type,alcohol type,substrate molar ratio,lipase loading,reaction temperature and reaction time on the yield of1,2-DAGs in the ethanolysis reaction mixture were investigated.The optimal conditions were as follows: the reaction was conducted at 50°C for 1 h by reacting HOSO with ethanol(1:50of substrate molar ratio)using 6% Lipozyme 435 as catalyst(based on the total mass of substrate).Under the optimal conditions,88% yield of 1,2-DAGs were produced.Secondly,a two-step crystallization method was described to purify the 1,2-DAGs in the crude product.Solvent type,ratio of crude product to solvent(in the first step)and ratio of semi-purified product to solvent(in the second step),crystallization temperature and crystallization time were investigated.In the first step,one nonpolar solvent was adopted to remove the nonpolar impurities such as TAGs and fatty acid esters(FAEs).The optimal conditions identified by single factor experiments were as follows: the crude product was crystallized at-40?C for 18 h with hexane(the crude product was dissolved in hexane in a ratio of 1:10,m/v).Under the optimal conditions,the purity of 1,2-DAGs reached 85.5% in a90.3% yield.In the second step,one polar solvent was adopted to remove the polar impurities such as monoacylglycerols(MAGs)and glycerol.The optimal conditions identified by single factor experiments were as follows: the semi-purified product was crystallized at-20?C for 6h with methanol(the semi-purified product was dissolved in methanol in a ratio of 1:12,m/v).Under the optimal conditions,the purity of 1,2-DAGs reached 99.3% in a 85.6% yield.After two-step purification,the purity of 1,2-DAGs reached almost 100% in a 77.3% overall yield.The purification method of 1,2-DAGs by solvent crystallization is suitable for batch operation and consumes low energy cost,therefore providing a new approach for the purification of partial glycerides such as diacylglycerols(DAGs)and MAGs.