Substrate selectivity of Novozym 435 in the esterification of glycerol with an equimolar mixture of linoleic, conjugated linoleic, and pinolenic acids

Abstract

The selectivity of Novozym 435, an immobilized Candida antarctica lipase B toward linoleic (LA), conjugated linoleic (CLA), and pinolenic acids (PLA) was investigated in the esterification of glycerol with an equimolar (27.3 mol% each) mixture of the fatty acids (FAs) in a solvent-free system to prepare triacylglycerols (TAGs) with anti-obesity effects. The maximal TAG content of 98.9 wt% was achieved under the following conditions: temperature, 70 degrees C; reaction time, 24 h; glycerol-to-FA mixture molar ratio, 1: 3; enzyme loading, 10 wt% of total substrates; vacuum level, 0.4 kPa. During the initial phase of the reaction, Novozym 435 showed pronounced selectivity in the order of PLA > CLA > LA; however, no significant difference in the selectivity of Novozym 435 toward the three FAs was observed after the reaction plateaued. Novozym 435 showed the significant selectivity toward PLA over CLA and LA at the sn-2 position of TAGs throughout the reaction. The resulting TAGs had the same total content (similar to 27 mol% each) of the three FAs. However, the LA, CLA, and PLA contents at the sn-2 position were 27.7, 24.7, and 30.3 mol%, respectively, indicating that unlike LA, CLA, and PLA were unevenly distributed on the glycerol backbone of the TAGs. Practical applications: CLA and PLA have received much attention because of their anti-obesity effects. Structured TAGs containing both CLA and PLA not only maintain the health benefits of each but also may provide several advantages (e.g., enhanced oxidative stability, reduced incidence of off-flavor) to both for food applications compared to the free FA forms. The selectivity of a lipase toward FAs used as substrates for the lipase-catalyzed synthesis of structured TAGs affects the composition and positional distribution of FAs in the resulting structured TAGs. The present study reports a new finding about the selectivity of Novozym 435 toward LA, CLA, and PLA which are all C18-unsaturated FAs, in the esterification of glycerol with the FAs to prepare structured TAGs containing both CLA and PLA.