Hydroxytyrosol acyl esters: biosynthesis and activities.

We previously reported the production of high yields of hydroxytyrosol through the bioconversion of tyrosol. In the present work, hydroxytyrosol was subjected to the lipase catalyzed acylation aiming for the recovery of more lipophilic esters that might be easily incorporated in cosmetic and food preparations. Hydroxytyrosyl acetate and hydroxytyrosyl oleate were produced with respective molar esterification yields of 98% and 78%. DPPH free radical quenching potency demonstrated that the acylation of hydroxytyrosol did not alter its antioxidant activity. The acylated esters were shown to be more effective than the natural antioxidant: caffeic acid and two synthetic ones as BHA and BHT. Antiproliferative activity on human cervical cells (HeLa) resulted in IC(50) values of 0.46, 0.42 and 0.33 mM for hydroxytyrosol and its acetyl and oleyl esters, respectively. Additionally, when used at a non-cytotoxic concentration (100 µM), these compounds showed significant effectiveness in preventing iron-induced oxidative stress, resulting in a reduction of 30%, 36% and 38% in thiobarbituric acid-reactive substance production, respectively.

Solubilities inferred from the combination of experiment and simulation. Case study of quercetin in a variety of solvents.

A strategy to infer solubilities from the combination of experiment and all-atom simulations is presented. From a single experimental estimate, the solubility of a substrate can be predicted in various environments from the related free energies of solvation. In the case of quercetin, the methodology was shown to reproduce the experimental solubilities in chloroform, water, acetonitrile, acetone, and tert-amyl alcohol within 0.5 log unit. The reliability of the estimates is markedly correlated to the accuracy of the experimental measure and to both the accuracy and precision of the computed free energies of solvation.

Preliminary investigation of naringenin hydroxylation with recombinant E. coli expressing plant flavonoid hydroxylation gene.

Flavonoid hydroxylation is one way to increase the biological activities of these molecules and the number of hydroxyl groups needed for polymerization, esterification, alkylation, glycosylation and acylation reactions. These reactions have been suggested as a promising route to enhance flavonoid solubility and stability. In our preliminary study we hydroxylated naringenin (the first flavonoid core synthesized in plants) with recombinant E. coli harboring flavanone 3 hydroxylase (F3H). We demonstrated that recombinant E. coli harboring the F3H from Petroselinum crispum, can convert naringenin to dihydrokaempferol. The whole cell hydroxylase activity was often influenced by the stability of the plasmid harboring the cloned gene and the biomass yield. When the composition of the growth media became richer the amount of formed product decreased about twofold; the naringenin bioconversion yield in LB media was 70% and decreased to 33% in TB. However, the enrichment of culture media increased the biomass yield nearly threefold in LB media, only 0.5 g/L of bacteria was formed, but in TB there was 1.6 g/L. Thus, LB constitutes the best medium for naringenin bioconversion using the recombinant E. coli harboring the F3H; this allows for maximum bioconversion yield and plasmid stability when compared with the fourth tested culture medium. Consequently, E. coli harboring F3H from Petroselinum crispum can be used to produce flavonoids hydroxylated in position 3 that can serve in additional reactions like polymerization, glycosylation, and acylation,

Biotransformation of naringenin to eriodictyol by Saccharomyces cerevisiea functionally expressing flavonoid 3' hydroxylase.

To increase the biological activities of flavonoids and to enhance their stability and solubility by functionalization reactions (polymerization, esterification, alkylation, glycosylation and acylation), an increase in the number of hydroxyl groups in these molecules is needed. Hydroxylation reactions may be achieved using either chemical or enzymatic methods, the latter being more highly specific than the former. In our study, the flavonoid 3' hydroxylase (F3'H) from Gerbera hybrid, functionally expressed in Saccharomyces cerevisiae, was used to hydroxylate naringenin (the first flavonoid core synthesized in plants). Furthermore, we studied factors that may affect naringenin hydroxylation by recombinant cell-like yeast growth on selective or rich media and plasmid stability. The whole recombinant cells hydroxylated naringenin at position 3' to give eriodictyol. In a selective media, the yeast failed to grow to high cell densities (maximum 5 g/L), but the plasmid stability was nearly 90%, and naringenin hydroxylation reached 100%. In a rich complex media, the biomass reached 10 g/L, but the yield of naringenin hydroxylation reached only 71%, and the plasmid stability decreased. When yeast functionally expressing F3'H from Gerbera hybrid was used, in a selective media, 200 mg/L of eriodictyol from naringenin was produced.

Enzymatic acylation of flavonoids: effect of the nature of the substrate, origin of lipase, and operating conditions on conversion yield and regioselectivity.

The conversion yield at equilibrium, the initial rate, and the regioselectivity of the enzymatic acetylation of aglycone flavonoids (quercetin, naringenin, hesperetin, and chrysin) were investigated and compared to those obtained with a glycosylated one (isoquercitrin). The effects of a wide range of operating conditions were quantified. Fourier transform infrared spectrometry (FT-IR), NMR, and high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) analyses showed that for glycosylated flavonoids, in the presence of Candida antarctica (CAL-B), the acetylation occurred on the 2''-OH, 3''-OH, and 6''-OH of the glucose part, while with Pseudomonas cepacea lipase (PSL-C) acetylation takes place on 6''-OH of the sugar and 4'-OH of the B-ring. For aglycone flavonoids, the acetylation occurred only with PSL-C on 4'-OH, 3'-OH, and 7-OH hydroxyls. The conversion yield and the number and the relative proportions of the synthesized products were found dependent on the nature of the enzyme, the molar ratio, and the flavonoid structure. The initial rate was affected only by the origin of the enzyme.

Dynamics of glutamate synthesis and excretion fluxes in batch and continuous cultures of temperature-triggered Corynebacterium glutamicum.

Corynebacterium glutamicum 2262 strain, when triggered for glutamate excretion, experiences a rapid decrease in growth rate and increase in glutamate efflux. In order to gain a better quantitative understanding of the factors controlling the metabolic transition, the fermentation dynamics was investigated for a temperature-sensitive strain cultivated in batch and glucose-limited continuous cultures. For non-excreting cells at 33 degrees C, increasing the growth rate resulted in strong increases in the central metabolic fluxes, but the intracellular glutamate level, the oxoglutarate dehydrogenase complex (ODHC) activity and the flux distribution at the oxoglutarate node remained essentially constant. When subjected to a temperature rise to 39 degrees C, at both high- and low-metabolic activities, the bacteria showed a rapid attenuation in ODHC activity and an increase from 28% to more than 90% of the isocitrate dehydrogenase flux split towards glutamate synthesis. Simultaneously to the reduction in growth rate, the cells activated a high capacity export system capable of expelling the surplus of synthesized glutamate.

Glutamate as an inhibitor of phosphoenolpyruvate carboxylase activity in Corynebacterium glutamicum.

The glutamate-producing bacterium, Corynebacterium glutamicum is known to possess two anaplerotic enzymes: pyruvate carboxylase (Pc) and phosphoenolpyruvate carboxylase (PEPc). In vitro, this latter enzyme appeared to be inhibited by different glutamic acid salts, whereas ammonium-glutamate had no influence on Pc activity. To investigate the in vivo relevance of PEPc activity inhibition, the intracellular concentration of glutamate was determined throughout the glutamate-producing process. The intracellular concentration was then shown to be sufficient to induce a dramatic inhibition of PEPc activity during the process. As a consequence, intracellular accumulation of glutamate could be at least partially responsible for the weak participation of PEPc within the anaplerosis activity in amino-acid-producing strains of C. glutamicum.

Instability of glutamate production by Corynebacterium glutamicum 2262 in continuous culture using the temperature-triggered process.

Kinetics and physiology of Corynebacterium glutamicum 2262 cultured for extended periods in continuous mode were investigated at 33, 39 and 41 degrees C. At 33 degrees C no glutamate production occurred whatever the dilution rates tested (ranging between 0.05 and 0.5 h(-1)). When the continuous culture was performed at 39 degrees C and D=0.05 h(-1), the glutamate was actively produced, while the activities of 2-oxoglutarate dehydrogenase complex (ODHC) and pyruvate dehydrogenase (PDH) were, respectively completely inhibited and 35% decreased. Simultaneously, the intracellular glutamate was 62% reduced compared to the level found at 33 degrees C and the co-metabolites lactate and trehalose were excreted. The decrease in PDH activity during the glutamate production was suggested to be responsible for the accumulation of by-products and for limiting the carbon flux required for glutamate synthesis. When the culture was prolonged for more than 100 h, a cell selection occurred, in favor of growth and to the detriment of glutamate production. In fact, these selected cells presented high levels of ODHC and PDH activities even at 39 degrees C, resulting in a complete inhibition of the glutamate production after 150 h of culture. A further temperature increase till 41 degrees C restored the glutamate production and abolished the ODHC activity of these selected cells.

Influence of glucose solubility and dissolution rate on the kinetics of lipase catalyzed synthesis of glucose laurate in 2-methyl 2-butanol.

The lipase catalyzed acylation of glucose by dodecanoic acid in 2-methyl 2-butanol was studied. The initial reaction rate was strongly dependent on the dissolved glucose concentration in the medium. Several methods were shown to increase dissolved glucose concentrations and initial reaction rates, namely, the use of solid beta-glucose, amorphous solid glucose, and supersaturated glucose solution. Supersaturated glucose solutions in 2-methyl 2-butanol showed a high stability even in the presence of solid crystalline glucose. During the reaction, the dissolved glucose concentration falls as the reaction proceeds, before recovering later as more of the excess solid dissolves. However, the ester synthesis rate continues to fall even after glucose concentration reaches its minimum, so glucose dissolution rate limitation is not responsible for the synthesis rate decline. Experiments with added molecular sieves show that the main reason is the accumulation of product water. In the presence of molecular sieves, 70% of glucose was converted to ester, independent of the initial soluble glucose in the medium.