Hydrolysis of triglycerides by cutinase from Fusarium solani pisi causes in oil drop tensiometer experiments a decrease of the interfacial tension. A series of cutinase variants with amino acid substitutions at its molecular surface yielded different values of the steady state interfacial tension. This tension value poorly correlated with the specific activity as such nor with the total activity (defined as the specific activity multiplied by the amount of enzyme bound) of the cutinase variants. Moreover, it appeared that at activity levels above 15% of that of wild type cutinase the contribution of hydrolysis to the decrease of the tension is saturating. A clear positive correlation was found between the interfacial tension plateau value and the interfacial binding of cutinase, as determined with attenuated total reflection Fourier transformed infrared spectroscopy (ATR-FTIR). These results indicate that the interfacial steady state level is not determined by the rate of hydrolysis, but mainly by the interfacial binding of cutinase.
Changes of the oil-water interfacial tension resulting from binding of Fusarium solani pisi cutinase and subsequent lipid hydrolysis were investigated using the oil drop technique. An ELISA was developed to determine the amount of cutinase bound to the triolein-water interface after biotinylation of the enzyme. Cutinase irreversibly adsorbs to a maximum value of about 2 mg/m2. A minimal specific activity of 110 mumol/min/mg was calculated for cutinase acting on a single oil droplet, which is close to the activity found for triglyceride emulsions. At a maximum surface load cutinase could generate one monolayer of fatty acid products per second at the interface. It was found that oleic acid rapidly dissolves into the oil phase under the conditions used. The interfacial tension measured reflects the adsorption of cutinase to the oil droplet and also responds to the fate of the hydrolysis products. A model is presented that describes the catalytic events at the oil-water interface during lipid hydrolysis.
Title: Action of cutinase at the triolein-water interface. Characterisation of interfacial effects during lipid hydrolysis using the oil-drop tensiometer as a tool to study lipase kinetics Flipsen JA, van der Hijden HT, Egmond MR, Verheij HM Ref: Chemistry & Physic of Lipids, 84:105, 1996 : PubMed
Interfacial events during lipid hydrolysis by cutinase are described as measured with the oil-drop tensiometer. A linear relation between enzyme concentration and initial decrease of oil-water interface tension (gamma o/w) due to lipolytic activity was observed. The amount of hydrolysis products showed a non-linear relation with gamma o/w. Hydrolysis is linear with time, even when the area occupied by the fatty acid molecules exceeds the drop surface by a factor 7000. At pH 9.0, fatty acids were found to partition mainly in the oil phase. Formation of calcium soaps and ionization increase the impact of fatty acids on gamma o/w without affecting enzyme activity. The presence of fatty acids at the interface, added prior to cutinase, delayed hydrolysis effects on gamma o/w. Fatty acids in the water phase almost completely abolished adsorption effects on gamma o/w, when the concentration was over the critical micellar concentration (cmc).
Triglyceride analogues were synthesized in which one of the primary acyl ester functions has been replaced by an alkyl group and the secondary acyl ester bond has been replaced by an acyl amino bond. The chain length at either position was varied, and both (R)- and (S)-enantiomers of each compound were synthesized. These pseudo triglycerides contain only one hydrolyzable ester bond, and they are ideally suited to studying the influence of the chain length at the 1-, 2-, and 3-position on lipase activity and on stereopreference. These substrates were used to characterize cutinase from Fusarium solani pisi. Our results show that the activity of cutinase is very sensitive to the length and distribution of the acyl chains and that the highest activities are found when the chains at positions 1 and 3 contain three or four carbon atoms. The enzyme preferentially hydrolyzes the (R)-enantiomers, but this preference is strongly dependent on the acyl chain length distribution, with (R) over (S) activity ratios varying from about 30 to 1. This enantioselectivity was found in three different assay systems: a mixed micellar, a reverse micellar, and a monolayer study. Our data suggest that at least two alkyl chains of the pseudo triglycerides must be fixed during hydrolysis. Therefore, these substrates were used to characterize mutants of cutinase with mutations in putative lipid binding domains. Two mutants (A85F and A85W) have increased activities. The results obtained with these mutants suggest an interaction of the acyl chain of the scissile ester bond with a surface loop, comprising residues 80-90, in the enzyme-substrate complex.
1,2-Dioctylcarbamoylglycero-3-O-p-nitrophenyl alkylphosphonates, with alkyl being methyl or octyl, were synthesised and tested as irreversible inhibitors of cutinase from Fusarium solani pisi and Staphylococcus hyicus lipase. Rapid inactivation of these enzymes occurred with a concomitant release of one mole of p-nitrophenol per mole of enzyme. With both lipases a higher reactivity was observed when the alkyl substituent on the phosphonate is a methyl rather than an octyl chain. Both lipases are highly selective for the chirality of these compounds at glycerol and at phosphorus. Rapid inactivation at an inhibitor concentration of 0.1 mol% in 100 mM NaTDOC (t 1/2 < 60 min.) occurred when the glycerol moiety had the (R) configuration, while inhibitors of the (S) configuration react 4-10-fold more slowly. The isomer with the p-nitrophenyl octylphosphonate attached to the secondary hydroxyl group of glycerol hardly inhibited (t 1/2 > 1 day) the lipases. These results reflect the known positional- and stereopreference of these enzymes which preferentially release the fatty acid at sn-3 of natural triacylglycerols. The enzymes appeared to be even more selective for the chirality at phosphorus, the differences in reactivity of the faster and slower reacting isomers being as high as about 250-fold for the methylphosphonates and about 60-fold for the octylphosphonates. These phosphonates can be regarded as true active site-directed inhibitors. The inhibited enzymes can be considered as analogues of the tetrahedral intermediate in the acylation step that occurs during triacylglycerol hydrolysis.
