In this work, the synthesis of acylglycerides with high nutritional value was carried out by enzymatic esterification at sn-2 position of 1,3-dicaprin with palmitic acid. A comparative study of the performance of several biocatalysts according to the obtained products was carried out. The results obtained with several of the biocatalysts evaluated are very interesting, and it would be possible to use them to obtain a mixture of acylglycerides to act as a fat substitute. The final product was composed of about 90% of nutritionally attractive glycerides. These glycerides were medium-chain length triglycerides, medium-long chain triglycerides (mainly triglycerides with medium chain fatty acids at sn-1 and sn-3 positions and long chain fatty acid at sn-2 position), and 1,3-diglycerides. Pseudomonas fluorescens lipase and Burkholderia cepacia lipase immobilized on chitosan demonstrated unusual high activity in the sn-2 esterification of 1,3-dicaprin with palmitic acid at 45 °C and 12 h with 33% yield to 1,3-dicaproyl-2-palmitoyl glycerol. Burkholderia cepacia lipase has the advantage of being immobilized; however, BCL/chitosan has the advantages of being immobilized and therefore its easy recovery from the reaction media.