Utilization of the recombinant human β-carotene-15,15′-monooxygenase gene in <Emphasis Type="Italic">Escherichia coli</Emphasis> and mammalian cells

In animals, β-carotene 15,15′-monooxygenase (BCMO) is the key enzyme involved in the metabolism of plant β-carotene to retinal. In the present study, we utilized β-carotene-producing Escherichia coli to screen for mutants with higher BCMO activity which was monitored by color changes derived from β-carotene cleavage. Recombinant wild-type and T381L mutant BCMO proteins were purified to near homogeneity in E. coli, and their enzymatic activities were determined by HPLC analysis. The catalytic efficiency for β-carotene and retinal production of the mutant were 1.5-fold and 1.7-fold higher than those of wild-type, respectively. Further BCMO function in mammalian cells was analyzed by a retinoic acid receptor reporter assay, which responds to the metabolic conversion of β-carotene to retinoic acid in vivo. Overall, these tools can be used to screen more active BCMO for the industrial and pharmacological purpose of retinal production from β-carotene.     

Efficacy validation of synthesized retinol derivatives In vitro: stability,toxicity, and activity

Retinol (vitamin A) is used as an antiwrinkle agent in the cosmetics industry. However, its photo-instability makes it unsuitable for use in general cosmetic formulations. To improve the photo-stability of retinol, three derivatives (3, 4, and 5) were synthesized and their biological activities were analyzed. 1H NMR and HPLC analysis indicated that derivatives 3 and 5 were much more stable than retinol under our sunlight exposure conditions. When human adult fibroblasts were treated, the IC(50) of derivative 3 was 96 microM, which is similar to that of retinol, as determined by the MTT assay. Derivatives 4 and 5 were 2.5 and 8 times more toxic than retinol, respectively. At 1 microM treatment, like retinol, derivatives 3 and 4 were specifically active for RARalpha out of six retinoid receptors (RAR/RXRalpha, beta, gamma). Dose-dependent analysis confirmed that derivative 4 was as active as retinol and the other two derivatives were less active for RARalpha. The effect of our derivatives on the expression of collagenase, an indicator of wrinkle formation, was measured using the transient co-expression of c-Jun and RT-PCR in HaCaT cells. Collagenase promoter activity, which is increased by c-Jun expression, was reduced 42% by retinol treatment. The other derivatives inhibited collagenase promoter activity similarly. These results were further confirmed by RT-PCR analysis of the collagenase gene. Taken together, our results suggest that retinol derivative 3 is a promising antiwrinkle agent based on its higher photo-stability, lower RARalpha activity (possibly indicating reduced side effects), and similar effect on collagenase expression.     

Synthesis of new glycyrrhetinic acid (GA) derivatives and their effects on tyrosinase activity

To synthesize glycyrrhetinic acid (GA) derivatives (3, 4, 5, 10, 13, 14, 15, and 16), we first removed the ketonic group in the C-11 position, and the carboxylic function at the C-30 position was kept intact, reduced to an alcohol, or transformed to an aldehyde corresponding derivatives 10 and 13. Glycyrrhetinic acid (GA) derivatives (3, 4, 5, 15, and 16) were coupled with 4-amino piperpyridine derivatives (12 and 14) and 4-fluorobenzyl bromide at C-30 carboxylic acid position of glycyrrhetinic acid. In subsequent tyrosinase assays, we found that GA derivatives 4, 5, and 16 were not active at early time points, but strongly inhibited tyrosinase activity at late time points. Of the GA derivatives examined, derivative 5 was most active, with an IC₅₀ value of 50 μM after 2 h reaction. IC₅₀ values of derivatives 4 and 16 were 120 and 170 μM, respectively. Further kinetic data indicated that these derivatives are slow-binding inhibitors of tyrosinase. The time-dependent inhibition was reversed when vitamin C or kojic acid was used, that is, both compounds showed active inhibition at early time points. These results suggest that GA derivatives are much more stable than vitamin C or kojic acid, although their intrinsic inhibitory potentials are relatively low. Higher stability and activity suggest that GA derivative 5 might be a useful candidate for skin whitening.     

Production of conjugated linoleic acid by isolated Bifidobacterium strains

Two isolates from Korean faecal samples converted linoleic acid (LA) into conjugated linoleic acid (CLA), and were identified as Bifidobacterium breve and Bifidobacterium pseudocatenulatum by analysis of 16S rRNA sequences. In both cases, the major CLA was the cis-9, trans-11 isomer and CLA production paralleled the increase in cell biomass with both bacteria and was maximal at 30 h. The quantities of supernatant CLA produced by B. breve and B. pseudocatenulatum were 160 and 135 mg l^–1, from 500 mg LA l^–1, respectively. In the presence of 0.05% LA, the growth of B. breve was weakly inhibited but that of B. pseudocatenulatum was not affected over 6 days fermentation. During fermentation, the majority of CLA isomers were in the culture supernatant, but with washed cells obtained at the early stationary phase, 36 h, about 40% was detected in the cellular lipid. The optimal culture age with equal concentrations of washed cells for CLA production by the two bifidobacteria was determined to be 36 h. At this culture age, total CLA conversion of supernatant and cell pellets was 78% in B. breve and 69% in B. pseudocatenulatum from 0.01% LA.