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Enrichment of flavonoids in food is often limited by their off-tastes, which might be counteracted by the use of food proteins as carriers of flavonoids. Various milk proteins, egg proteins, and gelatin hydrolysates were compared for their binding characteristics to two flavan-3-ols. Among the proteins tested for their affinities toward epigallocatechin gallate (EGCG), β-casein and gelatin hydrolysates, in particular fish gelatin, were found to be the most promising carriers with an affinity on the order of 10(4) M(-1). A flexible open structure of proteins, as present in random coil proteins, was found to be important. The saturation of binding observed at high flavonoid/protein ratios was used to estimate the maximal binding capacity of each protein. To reach a daily intake of EGCG that has been associated with positive health effects, only 519 mg of gelatin B and 787 mg of β-casein were required to complex EGCG on the basis of their maximal binding capacity. When the absence of turbidity is taken into account, β-casein prevails as carrier. Three selected proteins were further investigated for their binding potential of representative flavonoids differing in their C-ring structure. An increase in hydrophobicity of flavonoids was related to a higher affinity for proteins, and the presence of a gallic acid ester on the C-ring showed an overall higher affinity.

Triglyceride analogues were synthesized in which one of the primary acyl ester functions has been replaced by an alkyl group and the secondary acyl ester bond has been replaced by an acyl amino bond.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.

No abstract

Fatty acid hydroperoxide lyase (HPO-lyase) was puri®ed 300-fold from tomatoes. The enzymatic activity appeared to be very unstable, but addition of Triton X 100 and b-mercaptoethanol to the buer yielded an active enzyme that could be stored for several months at À808C. The enzyme was inhibited by desferoxamine mesylate (desferal), 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone), nordihydroguaiaretic acid (NDGA), n-propyl gallate and butylated hydroxyanisole, suggesting the involvement of free radicals in the reaction mechanism and the existence of a prosthetic group in the active center. However, no heme group could be demonstrated with the methods commonly used to identify heme groups in proteins. Only 13-hydroperoxides from linoleic acid (13-HPOD) and a-linolenic acid (a-13-HPOT) were cleaved by the tomato enzyme, with a clear preference for the latter substrate. The pH-optimum was 6.5, and for concentrations lower than 300 mM a typical Michaelis±Menten curve was found with a K m of 77 mM. At higher a-13-HPOT concentrations inhibition of the enzyme was observed, which could (at least in part) be attributed to 2E-hexenal. A curve of the substrate conversion as a function of the enzyme concentration revealed that 1 nkat of enzyme activity converts 0.7 mmol a-13-HPOT before inactivation. Headspace analysis showed that tomato HPO-lyase formed hexanal from 13-HPOD and 3Z-hexenal from a-13-HPOT. A trace of the latter compound was isomerized to 2E-hexenal. In addition to the aldehydes, 12-oxo-9Z-dodecenoic acid was found by GC/MS analysis. To a small extent, isomerization to 12-oxo-10E-dodecenoic acid occurred. #