RESUMEN
Apple pomace was studied as a raw material for the production of xylitol and 2G ethanol, since this agroindustrial residue has a high concentration of carbohydrate macromolecules, but is still poorly studied for the production of fermentation bioproducts, such as polyols. The dry biomass was subjected to dilute-acid hydrolysis with H2SO4 to obtain the hemicellulosic hydrolysate, which was concentrated, detoxified and fermented. The hydrolyzate after characterization was submitted to submerged fermentations, which were carried out in Erlenmeyer flasks using, separately, the yeasts Candida guilliermondii and Kluyveromyces marxianus. High cellulose (32.62%) and hemicellulose (23.60%) contents were found in this biomass, and the chemical hydrolysis yielded appreciable quantities of fermentable sugars, especially xylose. Both yeasts were able to metabolize xylose, but Candida guilliermondii produced only xylitol (9.35 g L-1 in 96 h), while K. marxianus produced ethanol as the main product (10.47 g L-1 in 24 h) and xylitol as byproduct (9.10 g L-1 xylitol in 96 h). Maximum activities of xylose reductase and xylitol dehydrogenase were verified after 24 h of fermentation with C. guilliermondii (0.23 and 0.53 U/mgprot, respectively) and with K. marxianus (0.08 e 0.08 U/mgprot, respectively). Apple pomace has shown potential as a raw material for the fermentation process, and the development of a biotechnological platform for the integrated use of both the hemicellulosic and cellulosic fraction could add value to this residue and the apple production chain.
Asunto(s)
Biotecnología/métodos , Etanol/química , Malus/metabolismo , Xilitol/química , Aldehído Reductasa/química , Biomasa , Reactores Biológicos , Candida , Celulosa/metabolismo , D-Xilulosa Reductasa/química , Fermentación , Glucosa/metabolismo , Hidrólisis , Kluyveromyces , Polímeros/química , Polisacáridos/química , Saccharomycetales , Factores de Tiempo , Xilosa/metabolismoRESUMEN
This chapter describes the use of lenses obtained from rats as a model of cataractogenesis. At the molecular level, this is visualized as reduced activity of oxidative reductive enzymes such as aldose reductase and increased proteolysis of lens structural proteins including vimentin. In this chapter, protocols for assessment of these two pathways are presented. Specifically, this analysis shows a comparison of aldose reductase activity and vimentin cleavage in male and female rat lenses. This is because female rats are more susceptible to cataract formation compared to males.
Asunto(s)
Aldehído Reductasa/química , Catarata/fisiopatología , Cristalinas/aislamiento & purificación , Biología Molecular/métodos , Aldehído Reductasa/genética , Animales , Catarata/etiología , Catarata/genética , Cristalinas/química , Femenino , Humanos , Cristalino/química , Masculino , Estrés Oxidativo/genética , Ratas , Vimentina/química , Vimentina/genéticaRESUMEN
Plant aldo-keto reductases of the AKR4C subfamily play key roles during stress and are attractive targets for developing stress-tolerant crops. However, these AKR4Cs show little to no activity with previously-envisioned sugar substrates. We hypothesized a structural basis for the distinctive cofactor binding and substrate specificity of these plant enzymes. To test this, we solved the crystal structure of a novel AKR4C subfamily member, the AKR4C7 from maize, in the apo form and in complex with NADP(+). The binary complex revealed an intermediate state of cofactor binding that preceded closure of Loop B, and also indicated that conformational changes upon substrate binding are required to induce a catalytically-favorable conformation of the active-site pocket. Comparative structural analyses of homologues (AKR1B1, AKR4C8 and AKR4C9) showed that evolutionary redesign of plant AKR4Cs weakened interactions that stabilize the closed conformation of Loop B. This in turn decreased cofactor affinity and altered configuration of the substrate-binding site. We propose that these structural modifications contribute to impairment of sugar reductase activity in favor of other substrates in the plant AKR4C subgroup, and that catalysis involves a three-step process relevant to other AKRs.
Asunto(s)
Aldehído Reductasa/química , Aldehído Reductasa/ultraestructura , NADP/química , NADP/ultraestructura , Proteínas de Plantas/química , Proteínas de Plantas/ultraestructura , Aldo-Ceto Reductasas , Sitios de Unión , Coenzimas/química , Coenzimas/ultraestructura , Activación Enzimática , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica , Especificidad por SustratoRESUMEN
Cells of Candida guilliermondii (ATCC 201935) were permeabilised with surfactant treatment (CTAB or Triton X-100) or a freezing-thawing procedure. Treatments were monitored by in situ activities of the key enzymes involved in xylose metabolism, that is, glucose-6-phosphate dehydrogenase (G6PD), xylose reductase (XR) and xylitol dehydrogenase (XD). The permeabilising ability of the surfactants was dependent on its concentration and incubation time. The optimum operation conditions for the permeabilisation of C. guilliermondii with surfactants were 0.41 mM (CTAB) or 2.78 mM (Triton X-100), 30°C, and pH 7 at 200 rpm for 50 min. The maximum permeabilisation measured in terms of the in situ G6PD activity observed was, in order, as follows: CTAB (122.4±15.7U/g(cells)) > freezing-thawing (54.3 ± 1.9U/g(cells))>Triton X-100 (23.5 ± 0.0U/g(cells)). These results suggest that CTAB surfactant is more effective in the permeabilisation of C. guilliermondii cells in comparison to the freezing-thawing and Triton X-100 treatments. Nevertheless, freezing-thawing was the only treatment that allowed measurable in situ XR activity. Therefore, freezing-thawing permeabilised yeast cells could be used as a source of xylose reductase for analytical purposes or for use in biotransformation process such as xylitol preparation from xylose. The level of in situ xylose reductase was found to be 13.2 ± 0.1 U/g(cells).
Asunto(s)
Aldehído Reductasa/aislamiento & purificación , Candida/enzimología , Fraccionamiento Celular/métodos , Compuestos de Cetrimonio/química , D-Xilulosa Reductasa/aislamiento & purificación , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Octoxinol/química , Aldehído Reductasa/química , Candida/aislamiento & purificación , Cetrimonio , D-Xilulosa Reductasa/química , Congelación , Glucosafosfato Deshidrogenasa/química , PermeabilidadRESUMEN
Inhibitory activities of flavonoid derivatives against aldose reductase (AR) enzyme were modelled by using CoMFA, CoMSIA and GALAHAD methods. CoMFA and CoMSIA methods were used for deriving quantitative structure-activity relationship (QSAR) models. All QSAR models were trained with 55 compounds, after which they were evaluated for predictive ability with additional 14 compounds. The best CoMFA model included both steric and electrostatic fields, meanwhile, the best CoMSIA model included steric, hydrophobic and H-bond acceptor fields. These models had a good predictive quality according to both internal and external validation criteria. On the other hand, GALAHAD was used for deriving a 3D pharmacophore model. Twelve active compounds were used for deriving this model. The obtained model included hydrophobe, hydrogen bond acceptor and hydrogen bond donor features; it was able to identify the active AR inhibitors from the remaining compounds. These in silico tools might be useful in the rational design of new AR inhibitors.
Asunto(s)
Aldehído Reductasa/antagonistas & inhibidores , Aldehído Reductasa/química , Inhibidores Enzimáticos/química , Flavonoides/química , Modelos Moleculares , Relación Estructura-Actividad Cuantitativa , Enlace de Hidrógeno , Estructura MolecularRESUMEN
Xylitol enzymatic production can be an alternative to chemical and microbial processes, because of advantages like higher conversion efficiency. However, for an adequate conversion, it is necessary to investigate the effect of many parameters, such as buffer initial concentration, pH, temperature, agitation, etc. In this context, the objective of this work was to evaluate xylitol enzymatic production under different Tris buffer initial concentrations in order to determine the best condition for this parameter to begin the reaction. The best results were obtained when Tris buffer initial concentration was 0.22 M, reaching 0.31 g L(-1) h(-1) xylitol volumetric productivity with 99% xylose-xylitol conversion efficiency. Although the increase in buffer concentration allowed better pH maintenance, it hindered the catalysis. The results demonstrate that this bioreaction is greatly influenced by involved ions concentrations.
Asunto(s)
Aldehído Reductasa/química , Proteínas Fúngicas/química , Trometamina/análisis , Xilitol/química , Tampones (Química) , Candida/enzimología , Concentración de Iones de Hidrógeno , Cinética , Xilosa/químicaRESUMEN
Maize aldose reductase (AR) is a member of the aldo-keto reductase superfamily. In contrast to human AR, maize AR seems to prefer the conversion of sorbitol into glucose. The apoenzyme was crystallized in space group P2(1)2(1)2(1), with unit-cell parameters a = 47.2, b = 54.5, c = 100.6 A and one molecule in the asymmetric unit. Synchrotron X-ray diffraction data were collected and a final resolution limit of 2.0 A was obtained after data reduction. Phasing was carried out by an automated molecular-replacement procedure and structural refinement is currently in progress. The refined structure is expected to shed light on the functional/enzymatic mechanism and the unusual activities of maize AR.
Asunto(s)
Aldehído Reductasa/química , Zea mays/enzimología , Cristalización , Cristalografía por Rayos XRESUMEN
The effect of glucose on xylose-xylitol metabolism in fermentation medium consisting of sugarcane bagasse hydrolysate was evaluated by employing an inoculum of Candida guilliermondii grown in synthetic media containing, as carbon sources, glucose (30 g/L), xylose (30 g/L), or a mixture of glucose (2 g/L) and xylose (30 g/L). The inoculum medium containing glucose promoted a 2.5-fold increase in xylose reductase activity (0.582 IU/mgprot) and a 2-fold increase in xylitol dehydrogenase activity (0.203 IU/mgprot) when compared with an inoculum-grown medium containing only xylose. The improvement in enzyme activities resulted in higher values of xylitol yield (0.56 g/g) and productivity (0.46 g/[L.h]) after 48 h of fermentation.
Asunto(s)
Aldehído Reductasa/biosíntesis , Candida/crecimiento & desarrollo , Candida/metabolismo , Medios de Cultivo/metabolismo , Glucosa/metabolismo , Deshidrogenasas del Alcohol de Azúcar/biosíntesis , Xilitol/metabolismo , Xilosa/metabolismo , Aldehído Reductasa/análisis , Aldehído Reductasa/química , Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Medios de Cultivo/análisis , Medios de Cultivo/química , D-Xilulosa Reductasa , Activación Enzimática , Tasa de Depuración Metabólica , Deshidrogenasas del Alcohol de Azúcar/análisis , Deshidrogenasas del Alcohol de Azúcar/químicaRESUMEN
Inhibitory activity against aldose reductase enzyme of flavonoid derivatives were modelled using 11 kinds of molecular descriptors from Dragon software. Model with four Galvez Charge Indices described 67% of data variance and overtaken other models using the same number of variables. Galvez indices showed to contain important information on the relationship between the inhibitor structures and its activity by describing the molecular topology and charge transfer through the molecule. In addition, artificial neural networks were trained using charge indices from the linear models but the obtaining networks overfitted the data having low predictive power.