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

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.

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(R)-Ethyl 2-hydroxy-4-oxo-4-phenylbutanoateC12H14O4[α]D25=-6.0 (c 1, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (R)

Ethyl (2R,3S)-3-phenyloxirane-2-carboxylateC11H12O3Ee >99%[α]D25=-156 (c 1, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (2R, 3S)

Methyl (2R,3S)-3-(4-methoxyphenyl)oxirane-2-carboxylateC11H12O4Ee >99%[α]D25=-196 (c 1, ethanol)Source of chirality: enzymatic resolutionAbsolute configuration: (2R, 3S)

(2S,3S)-3-Hydroxy-2-(4-methoxyphenyl)-2,3-dihydrobenzo[b]-[1,4]thiazepin-4(5H)-oneC16H15NO3SEe >99%[α]D25=+115 (c 0.5, DMF)Source of chirality: enzymatic resolutionAbsolute configuration: (2S, 3S)

N-Acetyl-d-alanine methyl esterC6H11NO3Ee >99%[α]D25=+90.1 (c 1, water)Source of chirality: enzymatic resolutionAbsolute configuration: (R)

N-Acetyl-d-methionine methyl esterC8H15NO3SEe >99%[α]D25=+21 (c 1, ethanol)Source of chirality: enzymatic resolutionAbsolute configuration: (R)

N-Acetyl-d-isoleucine methyl esterC9H17NO3Ee >99%[α]D25=+6.2 (c 1, ethanol)Source of chirality: enzymatic resolutionAbsolute configuration: (R)