Lipase activity of Lecitase® Ultra: characterization and applications in enantioselective reactions

Abstract

The general properties of Lecitase® Ultra, a phospholipase manufactured and marketed by Novozymes, Denmark, have been studied after purification by ultrafiltration. The enzyme has a molecular mass of 35 KD, pH-optimum of 8.5, and appears to possess a single active site which exhibits both the lipase and phospholipase activities that increase in the presence of Ca²⁺ and Mg²⁺ ions. The enzyme is inhibited by heavy metal ions and surfactants, and does not accept p-nitrophenyl acetate and glycerol triacetate. Substrates, such as glycerol tributyrate and p-nitrophenyl palmitate, esters of N-acetyl-α-amino acids and α-hydroxy acids are readily accepted. Amino acids with aliphatic residues, such as alanine, isoleucine, and methionine, are hydrolyzed with high enantioselectivity for the l-enantiomer (E >100), but amino acids with aromatic residues such as phenylalanine and phenylglycine, and esters of α-hydroxy acids are hydrolyzed with low enantioselectivity (E = 1–5). Immobilization of the enzyme in a gelatin matrix (gelozyme) leads to a marginal improvement in the enantioselectivity for these substrates. However, a dramatic improvement in enantioselectivity is observed for ethyl 2-hydroxy-4-oxo-4-phenylbutyrate (E value increases from 4.5 to 19.5 with S-selectivity). Similarly, glycidate esters, such as ethyl trans-(±)-3-phenyl glycidate and methyl trans-(±)-3-(4-methoxyphenyl) glycidate, are selectively hydrolyzed with a remarkable selectivity towards the (2S,3R)-enantiomer providing unreacted (2R,3S)-glycidate esters (ee >99%, conversion 52–55%) by the immobilized enzyme.