| Commercial lipases, including Lipozyme IM 20 from Rhizomucor meihei and Novozym 435 from Candida antarctica , were investigated to determine their capacity as biocatalysts for the biosynthesis of phenolic lipids, using dihydroxyphenylacetic acid (DHPA) and fish liver oil (FLO) as substrates. Novozym 435 showed a higher bioconversion yield (63%) as compared to that (18%) for Lipozyme IM 20. As a result, Novozym 435 was subsequently used for further investigations. The phenolic lipids were recovered and characterized by thin-layer chromatography, high-performance liquid chromatography and gas-liquid chromatography. The structural analyses indicated the synthesis of five major phenolic lipids. In order to optimize the initial enzymatic activity and bioconversion yield, selected parameters, including lipase amount (20 to 60 mg solid enzyme or 200 to 600 U), organic solvent ratios (hexane:2-butanone; 65:35 to 85:15, v/v), substrate molar ratios (FLO/phenolic acid, 1:1 to 8:1), Silica gel (1.5 to 5.5 mg/mL) and molecular sieve (10 mg/mL, Type 4A; 8-12 mesh), were investigated. The experimental results showed that the use of 50 mg of solid Novozym 435 (500 U) resulted in a maximum bioconversion yield of 83% of total phenolic lipids, at a molar substrate ratio 4:1. The bioconversion yield of phenolic monoacylglycerols (MAGs) increased from 11 to 70%, when the ratio of the hexane:2-butanone reaction medium was changed from 85:15 to 75:25 (v/v), respectively, whereas that of phenolic diacylglycerols (DAGs) remained relatively unchanged (13 to 16%). However, the addition of molecular sieve and Silica gel to the reaction medium resulted in a decrease in the bioconversion yield by 34% and 62%, respectively. Under the optimized conditions, the phenolic lipids showed a significant relative increase in C20:5 n-3 and C22:6 n-3 content in the phenolic MAGs and phenolic DAGs as compared to that in the unmodified FLO. The phenolic lipids demonstrated radical scavenging properties of approximately half to that of alpha-tocopherol, but also less than that of DHPA. |
