Halogenation of estrogens catalysed by a fungal chloroperoxidase.

Chloroperoxidase (CPO) is a haeme-thiolate enzyme able to catalyse the halogenation and oxidation of a wide range of organic substrates. In this work, the CPO-catalysed chlorination and bromination reaction of natural estrogens was characterised. Estradiol, estrone and equiline were efficiently converted to halogenated compounds in the presence of chloride or bromide and hydrogen peroxide. The catalytic efficiency of CPO in this reaction is similar to that measured for other aromatic substrates; as expected the bromination reaction proceeds more efficiently than the chlorination reaction. Three major products were detected for chlorination of estradiol; two of them were monohalogenated compounds while a third product was a dihalogenated compound at positions 2 and 4 of the aromatic ring A. Chlorinated compounds are not substrates for tyrosinase, suggesting that the halogenated form of estrogens is less susceptible to form o-quinones.

Determination of clenbuterol at trace levels in raw gelatin powder and jellies using ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry.

Clenbuterol is present in animal tissues and organs and, therefore, potentially present in gelatin derived from animal sources. The objective of this study was to develop a method for identify an quantify traces of clenbuterol in gelatin and jellies. The clenbuterol calibration curve showed linearity in the range of 20-1000 pg mL-1. The detection and quantification limits were 5 pg g-1 and 10 pg g-1, respectively. The recovery of the analyte ranged from 93.4 to 98.7% with an intra-day RSD% (n = 4) of 1.25%-3.25%, and an inter-day RSD% (n = 12) of 0.5%-2.25%, with good linearity (R2 = 0.99). The method developed and validated was successfully applied in 54 gelatin samples, 57.4% of which showed clenbuterol. This UHPLC-MS/MS method combines high sensitivity with good selectivity and short chromatographic run time.

Lignans from Bursera fagaroides: Chemistry, Pharmacological Effects and Molecular Mechanism. A Current Review.

Bursera fagaroides is a medicinal tree endemic to México, it belongs to the Burseraceae family and has proven antitumor activity. Modern research, performed principally with the bark extracts, have indicated that lignans are the main active constituents of B. fagaroides, with a high content of aryltetralin, aryldihydronaphtalene, dibenzylbutirolactone, and dibenzylbutane-type lignans as the constituents of the active extracts. In general, lignans from B. fagaroides exhibited potent anti-cancer activity, although antitumor, anti-bacterial, anti-protozoal, anti-inflammatory, and anti-viral properties have also been described. This review covers literature-reported lignans from B. fagaroides, chemical structures, nomenclature, chromatographic techniques of isolation, characterization strategies, and highlights the anti-cancer molecular mechanisms of lignans. Evaluation of the anticancer function of lignans has been extensively investigated since the cytotoxic in vitro results and in vivo assays in mice and zebrafish models to the tubulin molecular recognition by NMR. Also, we discuss the future direction for studying this important plant species and its lignan metabolites.

Deep Eutectic Solvents as New Reaction Media to Produce Alkyl-Glycosides Using Alpha-Amylase from Thermotoga maritima.

Deep Eutectic Solvents (DES) were investigated as new reaction media for the synthesis of alkyl glycosides catalyzed by the thermostable α-amylase from Thermotoga maritima Amy A. The enzyme was almost completely deactivated when assayed in a series of pure DES, but as cosolvents, DES containing alcohols, sugars, and amides as hydrogen-bond donors (HBD) performed best. A choline chloride:urea based DES was further characterized for the alcoholysis reaction using methanol as a nucleophile. As a cosolvent, this DES increased the hydrolytic and alcoholytic activity of the enzyme at low methanol concentrations, even when both activities drastically dropped when methanol concentration was increased. To explain this phenomenon, variable-temperature, circular dichroism characterization of the protein was conducted, finding that above 60 °C, Amy A underwent large conformational changes not observed in aqueous medium. Thus, 60 °C was set as the temperature limit to carry out alcoholysis reactions. Higher DES contents at this temperature had a detrimental but differential effect on hydrolysis and alcoholysis reactions, thus increasing the alcoholyisis/hydrolysis ratio. To the best of our knowledge, this is the first report on the effect of DES and temperature on an enzyme in which structural studies made it possible to establish the temperature limit for a thermostable enzyme in DES.

A novel two-step enzymatic synthesis of blastose, a ß-d-fructofuranosyl-(2↔6)-d-glucopyranose sucrose analogue.

Blastose, a natural disaccharide found in honey, is usually found as a byproduct of fructo-oligosaccharide synthesis from sucrose with fructosyltransferases. In this study, we describe a novel two-step biosynthetic route to obtain blastose, designed from a detailed observation of B. subtilis levansucrase (SacB) acceptor structural requirements for fructosylation. The strategy consisted first in the synthesis of the trisaccharide O-ß-d-Fruf-(2↔6)-O-α-d-Glcp-(1↔1)-α-d-Glcp, through a regioselective ß-d-transfructosylation of trehalose (Tre) which acts as acceptor in a reaction catalyzed by SacB using sucrose or levan as fructosyl donor. In this reaction, levansucrase (LS) transfers regioselectively a fructosyl residue to either C6-OH group of the glucose residues in Tre. The resulting trisaccharide obtained in 23% molar yield based on trehalose, was purified and fully characterized by extensive NMR studies. In the second step, the trisaccharide is specifically hydrolyzed by trehalase, to obtain blastose in 43.2% molar yield based on the trisaccharide. This is the first report describing the formation of blastose through a sequential transfuctosylation-hydrolysis reaction.

Acceptor-induced modification of regioselectivity in CGTase-catalyzed glycosylations of p-nitrophenyl-glucopyranosides.

Cyclodextrin glycosyltransferases (CGTase) are reported to selectively catalyze α(1 → 4)-glycosyl transfer reactions besides showing low hydrolytic activity. Here, the effect of the anomeric configuration of the glycosyl acceptor on the regioselectivity of CGTase catalyzed glycosylations was investigated. For this purpose, the α and ß anomers of p-nitrophenyl-D-glucopyranoside were used as glycosyl acceptors, Bacillus macerans and Thermoanaerobacter sp. CGTases were used as biocatalysts and ß-cyclodextrin as the glycosyl donor. As expected, p-nitrophenyl-α-D-glucopyranosyl-(1 → 4)-O-α-D-glucopyranoside was produced when p-nitrophenyl-α-D-glucopyranoside was used as acceptor with B. macerans CGTase. Surprisingly, when p-nitrophenyl-ß-D-glucopyranoside was used as glycosyl acceptor, besides the expected α(1 → 4)-glycosylation products both α(1 → 3)- and α(1 → 6)-transfer products were also obtained. This unexpected change in B. macerans CGTase regioselectivity leading to α(1 → 4)-, α(1 → 3)- and α(1 → 6)-glycosylation products was also observed for Thermoanaerobacter sp. CGTase with the ß anomer. It is shown, applying time course analyses, that all isomers can be synthesized efficiently by adequate selection of enzyme and reaction conditions. In fact, when using Thermoanaerobacter sp. CGTase the yield of p-nitrophenyl-ß-D-isomaltoside (the α(1 → 6)-transfer product) was the highest at long reaction time (19% yield). The previously unknown capacity of α(1 → 6)-glycosidic linkages formation by CGTases demonstrates an unexpected broader regioselectivity of CGTases in glycosyl-transfer reactions as well as an acceptor dependent transfer selectivity.

Screening and characterization of extracellular polysaccharides produced by Leuconostoc kimchii isolated from traditional fermented pulque beverage.

We report the screening and characterization of EPS produced by LAB identified as Leuconostoc kimchii isolated from pulque, a traditional Mexican fermented, non-distilled alcoholic beverage produced by the fermentation of the sap extracted from several (Agave) maguey species. EPS-producing LAB constitutes an abundant bacterial group relative to total LAB present in sap and during fermentation, however, only two EPS-producing colony phenotypes (EPSA and EPSB, respectively) were detected and isolated concluding that despite the high number of polymer-producing LAB their phenotypic diversity is low. Scanning electron microcopy analysis during EPS-producing conditions revealed that both types of EPS form a uniform porous structure surrounding the bacterial cells. The structural characterization of the soluble and cell-associated EPS fractions of each polymer by enzymatic and acid hydrolysis, as by 1D- and 2D-NMR, showed that polymers produced by the soluble and cell-associated fractions of EPSA strain are dextrans consisting of a linear backbone of linked α-(1→6) Glcp in the main chain with α-(1→2) and α-(1→3)-linked branches. The polymer produced by the soluble fraction of EPSB strain was identified as a class 1 dextran with a linear backbone containing consecutive α-(1→6)-linked D-glucopyranosyl units with few α-(1→3)-linked branches, whereas the cell-associated EPS is a polymer mixture consisting of a levan composed of linear chains of (2→6)-linked ß-D-fructofuranosyl residues with ß-(2→6) connections, and a class 1 dextran. According to our knowledge this is the first report of dextrans and a levan including their structural characterization produced by L. kimchii isolated from a traditional fermented source.

Regioselective glucosylation of inositols catalyzed by Thermoanaerobacter sp. CGTase.

Monoglucosylated products of L-chiro-, D-chiro-, muco-, and allo-inositol were synthesized by regioselective α-D-glucosylation with cyclodextrin glucosyl transferase from Thermoanaerobacter sp. after hydrolysis of by products with Aspergillus niger glucoamylase. While the reactions carried out with D-chiro-, muco-, and allo-inositol resulted in the regioselective formation of monoglucosylated products, two products were obtained in the reaction with L-chiro-inositol. Through the structural characterization of the glucosylated inositols here we demonstrated that the selectivity observed in the glucosylation of several inositols by Thermoanaerobacter sp. CGTase, is analogous to the specificity observed for the glucosylation of ß-D-glucopyranose and equivalent glucosides.

FadD is required for utilization of endogenous fatty acids released from membrane lipids.

FadD is an acyl coenzyme A (CoA) synthetase responsible for the activation of exogenous long-chain fatty acids (LCFA) into acyl-CoAs. Mutation of fadD in the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti promotes swarming motility and leads to defects in nodulation of alfalfa plants. In this study, we found that S. meliloti fadD mutants accumulated a mixture of free fatty acids during the stationary phase of growth. The composition of the free fatty acid pool and the results obtained after specific labeling of esterified fatty acids with a Δ5-desaturase (Δ5-Des) were in agreement with membrane phospholipids being the origin of the released fatty acids. Escherichia coli fadD mutants also accumulated free fatty acids released from membrane lipids in the stationary phase. This phenomenon did not occur in a mutant of E. coli with a deficient FadL fatty acid transporter, suggesting that the accumulation of fatty acids in fadD mutants occurs inside the cell. Our results indicate that, besides the activation of exogenous LCFA, in bacteria FadD plays a major role in the activation of endogenous fatty acids released from membrane lipids. Furthermore, expression analysis performed with S. meliloti revealed that a functional FadD is required for the upregulation of genes involved in fatty acid degradation and suggested that in the wild-type strain, the fatty acids released from membrane lipids are degraded by ß-oxidation in the stationary phase of growth.

Phenylpropanoid glycoside analogues: enzymatic synthesis, antioxidant activity and theoretical study of their free radical scavenger mechanism.

Phenylpropanoid glycosides (PPGs) are natural compounds present in several medicinal plants that have high antioxidant power and diverse biological activities. Because of their low content in plants (less than 5% w/w), several chemical synthetic routes to produce PPGs have been developed, but their synthesis is a time consuming process and the achieved yields are often low. In this study, an alternative and efficient two-step biosynthetic route to obtain natural PPG analogues is reported for the first time. Two galactosides were initially synthesized from vanillyl alcohol and homovanillyl alcohol by a transgalactosylation reaction catalyzed by Kluyveromyces lactis ß-galactosidase in saturated lactose solutions with a 30%-35% yield. To synthesize PPGs, the galactoconjugates were esterified with saturated and unsaturated hydroxycinnamic acid derivatives using Candida antarctica Lipase B (CaL-B) as a biocatalyst with 40%-60% yields. The scavenging ability of the phenolic raw materials, intermediates and PPGs was evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) method. It was found that the biosynthesized PPGs had higher scavenging abilities when compared to ascorbic acid, the reference compound, while their antioxidant activities were found similar to that of natural PPGs. Moreover, density functional theory (DFT) calculations were used to determine that the PPGs antioxidant mechanism proceeds through a sequential proton loss single electron transfer (SPLET). The enzymatic process reported in this study is an efficient and versatile route to obtain PPGs from different phenylpropanoid acids, sugars and phenolic alcohols.