Nguyen Lan Huong

Biochemical Characterization of Recombinant UDP-Glucose:Sterol 3-O-Glycosyltransferase from Micromonospora rhodorangea ATCC 31603 and Enzymatic Biosynthesis of Sterol-3-O-β-Glucosides.

A uridine diphosphate-glucose:sterol glycosyltransferase-encoding gene was isolated and cloned from the established fosmid library of Micromonospora rhodorangea ATCC 27932 that usually produces the aminoglycoside antibiotic geneticin. The gene consists of 1,185 base pairs and encodes a 41.4 kDa protein, which was heterologously expressed in Escherichia coli BL21(DE3). In silico analyses of the deduced gene product suggested that it is a member of the family 1 glycosyltransferases. The recombinant protein MrSGT was able to catalyze the transfer of a glucosyl moiety onto the C-3 hydroxy function in sterols (β-sitosterol, campesterol, and cholesterol), resulting in the corresponding steryl glucosides (β-sitosterol-3-O-β-D-glucoside, campesterol-3-O-β-D-glucoside, and cholesterol-3-O-β-D-glucoside). This enzyme prefers phytosterols to cholesterol, and also shows substrate flexibility to some extent, in that it could recognize a number of acceptor substrates.

Structural characterization of cyclosporin A, C and microbial bio-transformed cyclosporin A analog AM6 using HPLC-ESI-ion trap-mass spectrometry.

Cyclosporin A (CyA), a cyclic undecapeptide produced by a number of fungi, contains 11 unusual amino acids, and has been one of the most commonly prescribed immunosuppressive drugs. To date, there are over sixty different analogs reported as congeners and analogs resulting from precursor-directed biosynthesis, human CYP-mediated metabolites, or microbial bio-transformed analogs. However, there is still a need for more structurally diverse CyA analogs in order to discover new biological potentials and/or improve the physicochemical properties of the existing cyclosporins. As a result of the complexity of the resulting mass spectrometric (MS) data caused by its unusual amino acid composition and its cyclic nature, structural characterization of these cyclic peptides based on fragmentation patterns using multiple tandem MS analyses is challenging task. Here, we describe, an efficient HPLC-ESI-ion trap MS(n) (up to MS(8)) was developed for the identification of CyA and CyC, a (Thr(2))CyA congener in which L-aminobutyric acid (Abu) is replaced by L-threonine (Thr). In addition, we examined the fragmentation patterns of a CyA analog obtained from the cultivation of a recombinant Streptomyces venezuelae strain fed with CyA, assigning this analog as (γ-hydroxy-MeLeu(6))CyA (otherwise, known as an human CYP metabolite AM6). This is the first report on both the MS(n)-aided identification of CyC and the structural characterization of a CyA analog by employing HPLC-ESI-ion trap MS(n) analysis.

Ultra-High Performance Liquid Chromatography with Electrospray Ionization Tandem Mass Spectrometry for the Determination of Caffeine in Energy Drinks

A simple and highly sensitive method for the determination of caffeine in energy beverages was developed and validated. Sample preparation utilizing solid-phase extraction (SPE) was simple and reliable. Separation by isocratic ultra-high performance liquid chromatography (UPLC) with a reversed-phase C18 column was performed within 6 min. The use of SPE with UPLC coupled with electrospray ionization-multiple tandem mass spectrometry detection (ESI-MSn) was accurate, reproducible, and validated for the determination of caffeine in energy drink matrices. The limit of quantification for caffeine was approximately 2.1 ng mL−1. The intra-day and inter-day precisions were less than 4%, and the accuracy of the measurements was between 85.1% and 93.2%. Results for caffeine concentrations in eighteen beverages were compared to the values on the labels. This paper describes the first use of the UPLC–ESI-ion trap MSn technique for quality-control purposes of caffeine present in energy drinks.

Kinetic studies on recombinant UDP-glucose: sterol 3- O -β-glycosyltransferase from Micromonospora rhodorangea and its bioconversion potential

Kinetics of a recombinant uridine diphosphate-glucose: sterol glycosyltransferase from Micromonospora rhodorangea ATCC 27932 (MrSGT) were studied using a number of sterols (including phytosterols) as glycosyl acceptors. The lowest Km value and the highest catalytical efficiency (kcat/Km) were found when β-sitosterol was the glycosyl acceptor in the enzymatic reaction. In contrast to the enzyme’s flexibility toward the glycosyl acceptor substrate, this recombinant enzyme was highly specific to uridine diphosphate (UDP)-glucose as the donor substrate. Besides, the UDP-glucose-dependent MrSGT was able to attach one glucose moiety specifically onto the C-3 hydroxyl group of other phytosterols such as fucosterol and gramisterol, yielding stereo-specific fucosterol-3-O-β-d-glucoside and gramisterol-3-O-β-d-glucoside, respectively. Based on kinetic data obtained from the enzyme’s reactions using five different sterol substrates, the significance of the alkene (or ethylidene) side chains on the C-24 position in the sterol scaffolds was described and the possible relationship between the substrate structure and enzyme activity was discussed. This is the first report on the enzymatic bioconversion of the above two phytosteryl 3-O-β-glucosides, as well as on the discovery of a stereospecific bacterial SGT which can attach a glucose moiety in β-conformation at the C-3 hydroxyl group of diverse sterols, thus highlighting the catalytic potential of this promiscuous glycosyltransferase to expand the structural diversity of steryl glucosides.

Ultra-Performance Liquid Chromatography with Electrospray Ionization Mass Spectrometry for the Determination of Coenzyme Q10 as an Anti-Aging Ingredient in Edible Cosmetics

A simple and rapid method for the determination of coenzyme Q10 in edible cosmetics was developed and validated. Cleanup utilizing a solid-phase extraction technique provided a simple and reliable method for extracting both coenzyme Q10 and its internal standard coenzyme Q9 from the lecithin-rich samples with efficiencies of 92 ± 4% and 89 ± 3%, respectively. The separation of analytes through a reversed-phase C18 column was carried out within 10 min. By employing authentic coenzyme Q10 and the internal standard spiked into blank matrices, the combined use of solid phase extraction cleanup and chromatographic separation coupled with electrospray ionization-tandem mass spectrometry detection was shown to be sufficiently accurate to detect coenzyme Q10 in edible cosmetic matrices. The limit of quantification of coenzyme Q10 determined by the validated method was 54 ng/g in the matrices. In addition, the intra- and inter-day precision was <4% and the accuracy ranged from 87.1 to 92.3%. Quantitative results for coenzyme Q10 levels in two different edible cosmetic samples obtained by the established analytical protocol were reproducible. This is the first report of the determination of coenzyme Q10 in lipophilic edible cosmetic matrices by liquid chromatography–electrospray ionization tandem mass spectrometry.

Regio-selectively reduced streptogramin A analogue, 5,6-dihydrovirginiamycin M1 exhibits improved potency against MRSA

A newly reduced macrocyclic lactone antibiotic streptogramin A, 5,6‐dihydrovirginiamycin M1 was created by feeding virginiamycin M1 into a culture of recombinant Streptomyces venezuelae. Its chemical structure was spectroscopically elucidated, and this streptogramin A analogue showed twofold higher antibacterial activities against methicillin‐resistant Staphylococcus aureus (MRSA) compared with its parent molecule virginiamycin M1. Docking studies using the model of streptogramin A acetyltransferase (VatA) suggested that the newly generated analogue binds tighter with overall lower free energy compared with the parent molecule virginiamycin M1. This hypothesis was validated experimentally through the improvement of efficacy of the new analogue against MRSA strains. The biotransformation approach presented herein could have a broad application in the production of reduced macrocyclic molecules.

Ultra-Performance Liquid Chromatography with Electrospray Ionization Tandem Mass Spectrometry for the Determination of Ketoconazole in Anti-Dandruff Shampoo

A fast and highly sensitive method for the quantification of ketoconazole in antidandruff emulsion formulas was developed and validated. Sample preparation utilizing solid-phase extraction was a simple and reliable method for extracting both ketoconazole and its internal standard miconazole from the samples: 97 ± 3% for ketoconazole and 93 ± 4% for the internal standard. The separation by isocratic ultra-high performance liquid chromatography and multiple-stage mass spectrometry with a reversed-phase C18 column was performed within 10 min. The method, which operated in a selective reaction monitoring mode specific to ketoconazole, was validated for quantitative use. The intra- and inter-day precision values were <5% and their accuracies ranged from 89.6 to 96.2%. The ketoconazole concentrations in two samples obtained by the established protocol were comparable to the concentrations indicated on the labels of the formulations. Thus, this paper describes the first use of ultra-high performance liquid chromatography and multiple-stage mass spectrometry for the determination of ketoconazole.