Lipase regioselectivity is the ability to distinguish between primary (i.e., sn-1,3) and secondary (sn-2) ester functionalities in a triacylglycerol molecule, which is of importance in the manufacture of structured lipids. Unlike existing methods of assessment, which utilize hydrolysis reactions, an alternative technique to assess the regioselectivity of lipases in triacylglycerol transesterification reactions has been developed. An acidolysis reaction is performed between triolein and decanoic, lauric, or stearic acids under conditions that minimize acyl migration, and products are analyzed by silver-ion complexation liquid chromatography, enabling detection of specific triacylglycerol positional isomers. From the rate of formation of these isomers the relative selectivity of the lipase for sn-2 and sn-1,3 ester bonds is determined. With lipases known to lack regioselectivity, the rate of reaction at sn-2 was similar to that at sn-1,3 from the start of the reaction. With sn-1,3 selective lipases, the formation of triacylglycerol isomers with decanoic acid in the secondary position was not detected at any point in the reaction. Regioselectivity as a function of reaction progress was monitored. Two lipases from the genus Pseudomonas exhibited activity toward all positions, but the rate at sn-2 was much reduced, and no incorporation of decanoic acid into this position was detectable until a high degree of conversion had been achieved. FIG. 3. Reaction composition (A) and first-order rate plots (B) for the acidolysis of triolein with decanoic acid catalyzed by nonregioselective lipase (Candida antarctica A). For abbreviations and key see Figures 1 and 2; OOO not shown. FIG. 4. Reaction composition (A) and first-order rate plots (B) for the acidolysis of triolein with decanoic acid catalyzed by partially regioselective lipase (Lipase P). For abbreviations and key see Figures 1 and 2; OOO, not shown.
Under certain reaction conditions, the acidolysis of tripalmitin with oleic acid using immobilized lipase from Rhizomucor miehei resulted in a higher level of monosubstituted oleoyldipalmitoyl (OPP) triglycerides than had been predicted according to kinetic modeling. The reaction products were subjected to chiral analysis by high-performance liquid chromatography (HPLC), which indicated that the enzyme was more active at the sn-1 position of the triglyceride than at the sn-3 position, resulting in synthesis of the chiral triglyceride 1-oleoyl-2,3-dipalmitoyl-sn-glycerol. A kinetic model was developed and was correlated with the HPLC method to provide a simple means to predict the stereoselectivity of lipase-catalyzed reactions. By using the model, the stereoselectivity of immobilized Rhizomucor miehei lipase was found to depend strongly on the initial water activity (a w ) of the reaction mixture, with greater selectivity occurring at lower a w . The sn-1 selectivity was essentially maintained using various solvents, or without solvent, when a w was kept constantly low. Variation in the fatty acid composition of the triglyceride indicated that shorter-chain fatty acids result in greater stereoselectivity, while variation of the chainlength of the free fatty acid indicated an enhancement by the longest chainlength. The stereoselectivity of this lipase was confirmed using a new 13 C nuclear magnetic resonance method. By using immobilized R. miehei lipase at low a w , approximately 80% of the chiral triglyceride found in the reaction mixture was the sn-1 enantiomer, at high reaction conversion. JAOCS 75, 1513-1518 (1998).
The generation of off-flavors in soybean homogenates such as n-hexanal via the lipoxygenase (LOX) pathway can be a problem in the processed food industry. Previous studies have examined the effect of using soybean varieties missing one or more of the 3 LOX isozymes on n-hexanal generation. A dynamic mathematical model of the soybean LOX pathway using ordinary differential equations was constructed using parameters estimated from existing data with the aim of predicting how n-hexanal generation could be reduced. Time-course simulations of LOX-null beans were run and compared with experimental results. Model L2, L3, and L12 beans were within the range relative to the wild type found experimentally, with L13 and L23 beans close to the experimental range. Model L1 beans produced much more n-hexanal relative to the wild type than those in experiments. Sensitivity analysis indicates that reducing the estimated Km parameter for LOX isozyme 3 (L-3) would improve the fit between model predictions and experimental results found in the literature. The model also predicts that increasing L-3 or reducing L-2 levels within beans may reduce n-hexanal generation.Practical Application: This work describes the use of mathematics to attempt to quantify the enzyme-catalyzed conversions of compounds in soybean homogenates into undesirable flavors, primarily from the compound n-hexanal. The effect of different soybean genotypes and enzyme kinetic constants was also studied, leading to recommendations on which combinations might minimize off-flavor levels and what further work might be carried out to substantiate these conclusions.
Elucidating the stereoselectivity of lipases in synthetic reactions of triacylglycerols has hitherto been carried out using traditional analytical techniques to determine the composition of the reaction products. These methods are laborious and are not always appropriate for analysis of certain triacylglycerol types. A direct method, utilizing a stereospecific deuteriumlabeled triacylglycerol substrate, has been developed where the stereoisomeric composition of the reaction product is determined by ultra-high resolution 13 C nuclear magnetic resonance (NMR) spectroscopy. Through lipase-catalyzed transesterification of deuterium-labeled trilauroylglycerol with oleic acid, chemical shifts were induced in the 13 C NMR spectrum by the deuterium atom and olefinic double bonds, enabling unambiguous stereospecific assignment of triacylglycerol species. By this method of analysis, we found an effect of the degree of reaction conversion on the extent of stereoisomerism in the triacylglycerol product. Stereoselectivity was greatest (for sn-1) with lipase from Rhizomucor miehei. Lipases from Rhizopus niveus, Candida rugosa, Carica papaya, and the cutinase from Fusarium sp. were also found to exhibit stereoselectivity, with preference for either sn-1 or sn-3 acyl exchange.Paper no. J9811 in JAOCS 78, 953-958 (September 2001).
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