et al., 1993) with a view to assess their potential as biocatalyst for the enrichment of
definite fatty acids that are of considerable interest due to their biomedical (Innis,
1991) and technical properties (Vulfson, 1994; Gandhi, 1997).
Specificity of lipases towards individual fatty acids can be determined from the
overall reaction rates in hydrolysis, esterification and interesterification. Using mix-
tures of triacylglycerols or fatty acids as substrates, preference for or discrimination
against definite acyl moieties/fatty acids can be determined by comparing their re-
lative rates of conversion.
Kinetic analysis under competitive conditions can be carried out according to
Rangheard et al. (1989). For example, kinetic analysis of lipase-catalyzed esterifica-
tion of fatty acids withn-butanol is carried out by reacting 250 mM of each of the
fatty acids, individually, together with 250 mM of myristic or oleic acid, the refer-
ence standard, and 500 mMn-butanol in 1 mL hexane in the presence of 5–10 % (w/
w) of substrate of the lipase preparation (Mukherjee et al., 1993). The reaction pro-
ducts consisting of butyl esters and unesterified fatty acids, are treated with diazo-
methane to convert the unreacted fatty acids to their methyl esters. Subsequently, the
mixtures of butyl esters and methyl esters are analyzed by gas chromatography.
Overall reaction rates are calculated from the composition of the reaction products
aslmol butyl esters formed per g lipase per min.
Specificity constants are calculated according to Rangheard et al. (1989) as fol-
lows. For two substrates competing for the lipase, the ratio of the reaction rates of
each substrate (v1 andv2) is derived from
v1/v 2 ¼a(Ac1X)/(Ac2X)24 2 Fractionation of Fatty Acids and Other Lipids Using Lipases
Figure 1. Scheme of kinetic resolution for the enrichment of fatty acids/acyl moieties via lipase-ca-
talyzed hydrolysis of triacylglycerols or esterification of fatty acids withn-butanol.