Citrus paradisi 3-O-glucosyltransferase (Cp3GT, Genbank Protein ID: ACS15351) and Citrus sinensis 3-O-glucosyltransferase (Cs3GT, Genbank Protein ID: AAS00612.2) share 95% amino acid sequence identity. Cp3GT was previously established as a flavonol-specific 3-O-glucosyltransferase by direct enzymatic analysis. Cs3GT is annotated as a flavonoid-3-O-glucosyltransferase and predicted to use anthocyanidins as substrates based on gene expression analysis correlated with the accumulation of anthocyanins in C. sinensis cv. Tarocco, a blood orange variety. Mutant enzymes in which amino acids found in Cs3GT were substituted for position equivalent residues in Cp3GT were generated, heterologously expressed in yeast, and characterized for substrate specificity. Structure–function relationships were investigated for wild type and mutant glucosyltransferases by homology modelling using a crystallized Vitis viniferaanthocyanidin/flavonol 3-O-GT (PDB: 2C9Z) as template and subsequent substrate docking. All enzymes showed similar patterns for optimal temperature, pH, and UDP/metal ion inhibition with differences observed in kinetic parameters. Although changes in the activity of the mutant proteins as compared to wild type were observed, cyanidin was never efficiently accepted as a substrate.

[ES] La tendencia alimentaria actual indica que el consumo diario de frutas y verduras es parte fundamental de una dieta saludable. El formato de fruta deshidratada en polvo aumenta la vida u'til del producto, adema's de ofrecer otras ventajas relacionadas con el menor espacio necesario para su transporte y almacenamiento. En la medida en que el proceso utilizado para la obtencio'n del polvo mantenga estable la calidad de la fruta, incluido su valor funcional, e'sta podri'a ser otra forma de ofrecer al consumidor. En este trabajo se ha estudiado el impacto de la incorporacio'n de distintos biopoli'meros en algunas propiedades de un polvo de pomelo obtenido por atomizacio'n, con el fin de seleccionar el mejor de ellos, asi' como su estabilidad y viabilidad como nutrace'utico. Para ello, se ha trabajado con diferentes mezclas de goma ara'biga (GA), carboximetilcelulosa (CMC), maltodextrina (MD), un asilado de protei'na de suelo de leche (WPI) y almido'n modificado con anhi'drido octenil succi'nico (OSA). A todos los productos atomizados obtenidos se les ha estudiado, adema's del rendimiento del proceso, la humedad, porosidad y color del polvo obtenido. Adema's, en algunos casos, se ha analizado tambie'n la actividad antioxidante, el contenido de vitamina C y en fenoles totales. Los resultados obtenidos de esta parte del estudio no recomiendan el uso de CMC, ni sola ni combinada con GA pues, si bien mejora las propiedades del producto obtenido, disminuye el rendimiento del proceso. Por otra parte, la utilizacio'n de OSA en la formulacio'n del pomelo en polvo, aunque parece interesante porque permite obtener polvos ma's porosos, ma's secos y con mayor contenido en vitamina C, disminuye el rendimiento y el contenido en compuestos feno'licos con respecto a las formulaciones preparadas con los otros biopoli'meros. Los resultados de esta parte del estudio permiten proponer, como condiciones para la obtencio'n del mejor producto, preparar el licuado de pomelo an¿adiendo, sin dilucio'n previa, 9.4 g GA + 1.25 g MD + 1.4 g WPI/100 g licuado y atomizar a 148 ºC. Con el producto en polvo obtenido se programo' un estudio de almacenamiento para comprobar su estabilidad. Se estudio' la evolucio'n con el tiempo de la vitamina C, fenoles y licopeno, actividad antioxidante, color y propiedades meca'nicas del polvo, expuesto o no a la luz y a diferentes humedades relativas. Los resultados obtenidos ponen de manifiesto la gran inestabilidad de los carotenoides y recomiendan, para asegurar la fluidez del polvo y la estabilidad del color, de la vitamina C y de la capacidad antioxidante durante al menos 6 meses, almacenar el producto a una humedad relativa ¿ 23.1% si la temperatura es de 20 ºC. Finalmente, para estudiar la viabilidad del producto en polvo obtenido como nutraceu'tico, se realizo' un estudio de biodisponibilidad de los compuestos bioactivos a trave's de un modelo intestinal 3D con la combinacio'n de li'neas celulares Caco-2 y HT29-MTX. Los compuestos bioactivos asimilados teo'ricamente por el sistema digestivo fueron identificados por cromatografi'a li'quida, electro-espray, ionizacio'n de espectrometri'a de masas (LC-ESI-MS). La delfinidina-3-gluco'sido y la hesperitina-7-O-gluco'sido presentaron una permeacio'n superior al 50%, seguida de la hesperidina que se aproximo' al 30%. Este trabajo permitio' establecer que la formulacio'n del polvo de pomelo propuesta tiene un gran potencial como nutraceu'tico. / [CAT] La tende¿ncia alimenta¿ria actual indica que el consum diari de fruites i verdures e's part fonamental d'una dieta saludable. El format de fruita deshidratada en pols augmenta la vida u'til del producte, a me's d'oferir altres avantatges relacionades amb el menor espai necessari per al seu transport i emmagatzemament. En la mesura que el proce's utilitzat per a l'obtencio' de la pols mantinga estable la qualitat de la fruita, inclo¿s el seu valor funcional, esta podria ser una altra forma d'oferir fruita al consumidor. En este treball s'ha estudiat l'impacte de la incorporacio' de distints biopoli'mers en algunes propietats d'una pols de pomelo obtingut per atomizacio', a fi de seleccionar el millor d'ells, aixi' com l'estabilitat i la viabilitat del producte com nutraceu'tic. Per ac¿o¿, s'ha treballat amb diferents mescles de goma ara¿biga (GA), carboximetilcel¿lulosa (CMC), maltodextrina (MD), un ai¿llat de protei'na de se¿rum de llet (WPI) i almido' modificat amb anhi'drid octenil succi'nic (OSA). Amb tots els productes atomitzats obtinguts s'ha estudiat, a me's del rendiment del proce's, la humitat, porositat i color de la pols obtinguda. A me's a me's, en alguns casos, s'ha analitzat tambe' l'activitat antioxidant, el contingut en vitamina C i en fenols totals. Els resultats obtinguts d'aquesta part de l'estudi no recomanen l'u's de CMC, ni sola ni combinada amb GA doncs, si be' millora les propietats del producte obtingut, disminueix el rendiment del proce's. D'altra banda, la utilizacio'n d'OSA en la formulacio' del pomelo en pols, encara que sembla interessant perque¿ permet obtenir pols me's porosos, me's secs i amb un major contingut en vitamina C, disminueix el rendiment i el contingut en compostos feno¿lics pel que fa a les formulacions preparades amb els altres biopoli'mers. Els resultats d'aquesta part de l'estudi permeten proposar, com a condicions per a la obtencio' del millor producte, preparar el liquat de pomelo afegint, sense dilucio' pre¿via, 9.4 g GA + 1.25 g MD + 1.4 g WPI/100 g liquat i atomitzar a 148 ºC. Amb el producte en pols obtingut es programa¿ un estudi d'emmagatzematge per a comprovar la seua estabilitat. S'estudia¿ la evolucio', amb el temps, de la vitamina C, fenols, licopen, activitat antioxidant, color i propietats meca¿niques de la pols, exposat o no a la llum i a diferents humitats relatives. Els resultats obtinguts posen de manifest la gran inestabilitat del licopen i recomanen, per a assegurar la flotabilitat de la pols i l'estabilitat del color, de la vitamina C i de la capacitat antioxidant durant almenys 6 mesos, emmagatzemar el producte a una humitat relativa ¿ 23.1% si la temperatura e's de 20 ºC. Finalment, per a estudiar la viabilitat del producte en pols com nutraceu'tic, s'analitza¿ la biodisponibilidad dels compostos bioactivos a trave's d'un model intestinal 3D amb la combinacio' de li'nies cel¿lulars Caco-2 i HT29-MTX. Els compostos bioactivos assimilats teo¿ricament pel sistema digestiu van ser identificats per cromatografia liquida electro- sprai, ionitzacio' d'espectro-materia de masses. El delphinidin-3-glucoside i el hesperetin- 7-O-glucoside mostren una permeabilitat superior al 50%, seguit de la hesperidina que s'aproxima¿ al 30%. Aquest treball va permetre establir que la formulacio' de la pols de pomelo proposada te' un gran potencial com a nutraceu'tic. / [EN] The current food trend indicates that the daily consumption of fruits and vegetables is a fundamental part of a healthy diet. The dried fruit powder format increases the product's shelf-life, in addition to offering other advantages related to the lower volume and easier handle required for its transport and storage. To the extent that the process used to obtain the powder retains the quality of the fruits, including its functional value, this could be another way of offer fruit to the consumer. In this study, the impact of the incorporation of different biopolymers on some properties of a powdered grapefruit obtained by spray- drying, in order to select the best of them, as well as the powder stability and viability as a nutraceutical, has been considered. To this end, different mixtures of gum Arabic (GA), carboxymethylcellulose (CMC), maltodextrin (MD), whey protein isolate (WPI) and octenyl succinic anhydride modified- starch (OSA) have been tested. To all the obtained spray dried products, besides the process yield, the water content, porosity and color were analyzed. In addition, in some cases, antioxidant activity, vitamin C and total phenols content have also been analyzed. The results obtained from this part of the study do not recommend the use of CMC, either alone or combined with GA, because, although it improves the properties of the obtained product, the process yield decreases. On the other hand, the use of OSA in the grapefruit powder formulation, although it seems interesting because it allows to obtain more porous and dried powders, with a higher vitamin C content, it decreases the yield and the content of phenolic compounds with respect to the formulations prepared with the other biopolymers. The results of this part of the study allow us to propose, in order to obtain the best product, to prepare the liquidized grapefruit by adding, without previous dilution, 9.4 g GA + 1.25 g MD + 1.4 g WPI/100 g liquidized and spray-drying at 148 ºC. Whit the obtained powdered product, a storage study was scheduled to check its stability. The evolution over time of vitamin C, total phenols, lycopene, antioxidant activity, color and mechanical properties of the powder, exposed or not to light and at different relative humidities, were studied. The obtained results showed the great instability of the lycopene and recommend, to ensure the powder flowability and the color, vitamin C and antioxidant capacity stability for at least six months, to store the product at a relative humidity ¿ 23.1 % if the temperature is 20 ºC. Finally, to study the viability of the powdered product as a nutraceutical, the bioavailability of the bioactive compounds was analyzed through a 3D intestinal model with the combination of cell lines Caco-2 and HT29-MTX. The bioactive compounds assimilated theoretically by the digestive system were identified by liquid chromatography- electrospray ionization-mass spectrometry (LC-ESI-MS). Delphinidin-3-glucoside and hesperetin-7-O-glucoside showed a permeation greater than 50%, followed by hesperidin that approached to 30%. This study allowed to establish that the proposed grapefruit powder formulation has a great potential as a nutraceutical. / González Zamora, FJ. (2019). OPTIMIZACIÓN DE LA FORMULACIÓN Y TEMPERATURA DE ATOMIZACIÓN DE UN LICUADO DE POMELO PARA LA OBTENCIÓN DE UN POLVO DE ALTA CALIDAD Y ESTABILIDAD [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/118797 / TESIS

Flavonoids are a group of plant secondary metabolites that are vital to the cell systems of plants. The intake of these chemicals is advantageous to animals for their antioxidant properties that affect the function of immune and inflammatory cells. The bitter taste of grapefruit (Citrus paradise) and other citrus species is caused by the accumulation of glycosylated flavonoids. Glucosyltransferases (GTs) are enzymes that add glucose moieties to a carbon or hydroxyl group of natural products. The function of a putative secondary product GT clone was tested. In previous research, putative GT 4 was cloned into a pCD1 modified pET expression system, heterologously expressed in E.coli, and screened for activity with only a few substrates, and little GT activity was found. Issues of protein localized to inclusion bodies in bacteria are being addressed. PGT 4 is being heterologously expressed in yeast (Pichia pastoris) to allow for protein production and analysis. PGT 4 will be screened for GT activity with different flavonoid subclass representatives and simple phenolics. PGT 4’s significant impact on the biochemical regulation of Citrus paradise will be elucidated with its characterization and determination of PGT 4’s structure and function.

Flavonoids are an important class of secondary metabolites widely distributed in plants. The majority of naturally occurring flavonoids are found in glucosylated form. Glucosyltransferases are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor flavonoid substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi (Cp3OGT) has strict substrate and regiospecificity. In this study, amino acid residues that could potentially alter the rigidity observed in this enzyme were mutated to position equivalent residues of a putative anthocyanin specific glucosyltransferase from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling followed by site directed mutagenesis to identify candidate regions, three double mutations were made. To test the basis of substrate specificity, biochemical analysis of the three recombinant mutant proteins was carried out. Recombinant protein with mutation S20G+T21S revealed that the enzyme retained activity similar to the wildtype (Cp3OGT) (WT- Km app-104.8 µM; Vmax = 24.6 pmol/min/µg, Mutant- Km app-136.42 µM; Vmax -25pmol/min/µg) but the mutant was more thermostable compared to the WT. The (S290C+S319A) mutant protein retained 40% activity relative to wildtype and has an optimum pH shifted towards the acidic side (pH 6) (Km app-8.27 µM; Vmax-90.9 pmol/min/µg). Mutation of Glutamine87 and Histine154 (H154Y+Q87I) have rendered this recombinant protein inactive with every class of flavonoid tested. Interestingly, the single point mutations H154Y and Q871I had significant activity, slightly greater than that of wildtype enzyme. The two active recombinant proteins will further be analyzed to determine whether the mutations have altered regiospecificity of the original enzyme. Product identification is being conducted using HPLC.

Glucosylation is a common alteration reaction in plant metabolism and is regularly associated with the production of secondary metabolites. Glucosylation serves a number of roles within metabolism including: stabilizing structures, affecting solubility, transport, and regulating the bioavailability of the compounds for other metabolic processes. The enzymes that lead to glucoside formation are known as glucosyltransferases (GTs), and characteristically accomplish this task by transferring a UDP-activated glucose to a corresponding acceptor molecule. GTs involved in secondary metabolism share a conserved 44 amino acid residue motif (60–80% identity) known as the plant secondary product glucosyltransferase (PSPG) box, which has been demonstrated to include the UDP-sugar binding moiety. Among the secondary metabolites, flavonoid glycosides and limonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. The research focus of our lab is to establish the function of putative secondary product glucosyltransferase clones identified from Citrus paradisi. In the present study, we report on the activity and biochemical characterization of two clones, PGT 7 (Flavonol-3-O-GT) and PGT8 (Limonoid GT) which were expressed in Pichia pastoris.

Glucosyltransferases (GTs) are enzymes that expedite the incorporation of UDP-activated glucose to a corresponding acceptor molecule. This enzymatic reaction stabilizes structures and affects solubility, transport, and bioavailability of flavonoids for other metabolic processes. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant flavonol-3-O-glucosyltransferase (F-3-O-GT) proteins using site-directed mutagenesis and testing the effect of each mutation on substrate specificity, regiospecificity and kinetic properties of the enzyme. Mutations were selected on the basis of sequence similarity between grapefruit F-3-O-GT, an uncharacterized GT gene in blood orange (98%), and grape F3GT (82%). Grapefruit F-3-O-GT prefers flavonol as a substrate whereas the blood orange sequence is annotated to be a flavonoid 3GT and the grape GTs could glucosylate both flavonols and anthocyanidins. Mutants of F-3-O-GT were generated by substituting L41M, N242K, E296K and N242K+E296K and proteins were expressed in Pichia pastoris using the pPICZA vector. Analysis of these mF-3-O-GTs showed that all of them preferred flavonols over flavanone, flavone, isoflavones, or anthocyanidin substrates and showed decrease in enzyme activity of 16 to 51% relative to the wild type F-3-O-GT.

Flavonoids are a class of secondary metabolites, the majority of which are present in glucosylated form. Glucosyltransferases catalyze glucosylation by transferring glucose from UDP-activated sugar donor to the acceptor substrates. This research is focused on the study of the effect of a single point mutation on enzyme activity, characterization of a flavonol specific 3-O-glucosyltransferase (Cp-3-O-GT) mutant- P145T, and further modification of the clone to cleave off tags from recombinant wild type and P145T mutant proteins in order to crystallize the proteins. Multiple sequence alignment and homology modeling was done to identify candidate residues for mutation. Cp-3-O-GT was modeled with a flavonoid 3-O-GT from Vitis vinifera (VvGT) that can glucosylate both flavonols and anthocyanidins. We identified a proline residue at position 145 of Cp-3-O-GT that corresponded to a threonine residue in VvGT and designed a Cp-3-O-GTP145T mutant to test the hypothesis that that mutation of proline by threonine in Cp-3-O-GT could alter substrate or regiospecificity of Cp-3-O-GT. While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to flavonols. This is significant because flavanones and flavones do not contain a 3-OH group. HPLC was performed to identify the reaction products. Early results indicated that the mutant protein glucosylates naringenin at the 7-OH position forming prunin. Results are being used to revisit and refine the structure model. In other related work, a thrombin cleavage site was inserted into wild type and recombinant P145Tenzyme and we are currently working on transformation into yeast for recombinant protein expression. Cleaving off tags is a pre-requisite to future efforts to crystallize the proteins. Solving the crustal structures will make a significant contribution to the structural and functional study of plant flavonoid GTs in general and Cp-3- O-GT in particular.

Glucosyltransferases (GTs) are enzymes that expedite the incorporation of UDP-activated glucose to a corresponding acceptor molecule. This enzymatic reaction stabilizes structures and affects solubility, transport, and bioavailability of flavonoids for other metabolic processes. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant flavonol-3-O-glucosyltransferase (F-3-O-GT) proteins using site directed mutagenesis and testing the effect of each mutation on substrate specificity, regiospecificity and kinetic properties of the enzyme. Mutations were selected on the basis of sequence similarity between grapefruit F-3- O-GT, an uncharacterized GT gene in blood orange (98%), and grape F3GT (82%). Grapefruit F-3-O-GT prefers flavonol as a substrate whereas the blood orange sequence is annotated to be a flavonoid 3GT and the grape GTs could glucosylate both flavonols and anthocyanidins. Mutants of F-3-O-GT were generated by substituting N242K, E296K and N242K+E296K and proteins were expressed in Pichia pastoris using the pPICZA vector. Analysis of these mF-3-O-GTs showed that all of them preferred flavonols over flavanone, flavone, isoflavones, or anthocyanidin substrates and showed decrease in enzyme activity of 16 to 51% relative to the wild type F-3- O-GT.

No description available.

Flavonoids are a group of plant secondary metabolites that are vital to the cell systems of plants. The intake of these chemicals is advantageous to animals for their antioxidant properties that affect the function of immune and inflammatory cells. The bitter taste of grapefruit (Citrus paradisi) and other citrus species is caused by the accumulation of glycosylated flavonoids. Glucosyltransferases (GTs) are enzymes that add glucose moieties to a carbon or hydroxyl group of natural products. The function of a putative secondary product GT clone was tested. In previous research, putative GT 4 was cloned into a pCD1 modified pET expression system, heterologously expressed in E.coli, and screened for activity with a few substrates; little GT activity was found. Issues of protein localized to inclusion bodies in bacteria were addressed. PGT 4 is being heterologously expressed in yeast (Pichia pastoris) to allow for protein production and analysis. PGT 4 was screened for GT activity with different flavonoid subclass representatives and simple phenolics.