This research is focused on the study of the effect of mutating proline 145 to threonine on the substrate and regiospecificity of flavonol specific 3-O-glucosyltransferase (Cp3GT). While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to retaining activity with flavonols. HPLC was used for product identification and showed mutant P145T glucosylated naringenin at the 7-OH position forming naringenin-7-O-glucoside and flavonols at the 3-OH position. Homology modeling and docking was done to predict the acceptor substrate recognition pattern and models were validated by experimental results. In other related work, a thrombin cleavage site was inserted into wild type Cp3GT and recombinant P145T enzyme between the enzyme and the C-myc tags in order to be able to cleave off tags. This provides the tool needed for future efforts to crystallize these proteins for structural determination.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xvii, 122 p. : ill. Includes abstract. Includes bibliographical references (p. 46-53).

Flavonoid and limonoid glycosides influence taste properties as well as marketability of Citrus fruit and products, particularly grapefruit. In this work, nine grapefruit putative natural product glucosyltransferases (PGTs) were resolved by either using degenerate primers against the semiconserved PSPG box motif, SMART-RACE RT-PCR, and primer walking to full-length coding regions; screening a directionally cloned young grapefruit leaf EST library; designing primers against sequences from other Citrus species; or identifying PGTs from Citrus contigs in the harvEST database. The PGT proteins associated with the identified full-length coding regions were recombinantly expressed in Escherichia coli and/or Pichia pastoris and then tested for activity with a suite of substrates including flavonoid, simple phenolic, coumarin, and/or limonoid compounds. A number of these compounds were eliminated from the predicted and/or potential substrate pool for the identified PGTs. Enzyme activity was detected in some instances with quercetin and catechol glucosyltransferase activities having been identified.

Flavonoids are plant secondary metabolites that play diverse roles in plants and human health. These compounds in most part exist in the glucosylated form. Grapefruit accumulates high levels of glucosylated flavonoids. Plant secondary product glucosyltransferases (GTs) catalyze the glucosylation reaction, but due to low homology at both the nucleotide and amino acid sequence level of different GTs, it is not possible to ascribe function based on sequence only. The hypotheses that PGT clones 9 and 11 are plant secondary product GTs and are biochemically regulated were tested. PGT 9 has been cloned into Pichia pastoris using the pPICZA and pPICZAα vectors, expressed, enriched, and screened for GT activity with a variety of phenolic substrates. Initial screens show catechol, gentisic acid, vanillin, and p-hydroxylphenylacetic acid as potential substrates for the PGT 9 protein. PGT 11 has been successfully cloned into pPICZA for transformation into yeast, expression, and subsequent characterization.

The grapefruit plant, Citrus paradisi, tends to accumulate high levels of flavonoid glycosides such as flavanones and flavones. Flavonoids have a vast array of important functions in plants and also in humans. Glucosyltransferases (GTs) are enzymes responsible for glucosylation reactions. In our pursuit to study the structure and function of flavonoid GTs, we have used molecular approaches to identify, clone, express, and functionally characterize the enzymes. This research was designed to test the hypothesis that PGT3 is a flavonoid glucosyltransferase and is subject to biochemical regulation. PGT3 has been tested for GT activity with compounds representing subclasses of flavonoids as well as some simple phenolics. Results indicate GT activity with 6 substrates, p-hydroxybenzoic acid, vanillin, vanillic acid, p-hydroxyphenylpyruvate, gentisic acid, and catechol. A second project designed to clone putative PGT8 into the Pichia expression system has been completed.

Flavonoids are a broad class of low molecular weight, secondary plant phenolics characterized by the fl avan nucleus. Widely distributed in plants, food and traditional herbal medicines, more than 6000 fl avonoids have been identifi ed up to date. They are present mainly as glycosides whose phenolic hydrogen or hydrogens are substituted to sugar moiety. An increasing number of fl avonoids have attracted much attention in relation to their biological activities, including anti-viral, anti-infl ammatory, anti-bacterial, and vasodilatory activities. Present work is to understand the structure and function of a fl avonol specifi c glucosyltransferase from Citrus paradisi. The study is one of the many steps towards custom designing of the protein. We employed homology modeling, site-directed mutagenesis and yeast expression system to generate mutants of glucosyltransferase and study their substrate specifi city, regiospecifi city and kinetic properties.

The general objective of this study was to analyse freeze-drying (FD) and spray- drying (SD) and to select the best process to obtain a grapefruit powder of high nutritional, functional and sensorial quality. With this objective in mind, the optimization of each dehydration process was carried out using the response surface methodology. As variables of the processes, in both cases, the concentration of gum arabic (GA) and bamboo fibre (BF), added as carriers, were considered, as well as the feed inlet moisture (Xw) in the case of FD or air inlet temperature (T) in SD. The properties of the obtained products analyzed were the water content, hygroscopicity, porosity, color, bioactive compounds (vitamin C, total carotenoids, total phenols) and antioxidant activity (AOA). In addition, the drying yield and product yield of SD were considered. The obtained results, duly validated, allow to propose for FD, the addition of 4.2g GA and 0.58g BF per 100g of grapefruit pulp and bringing the sample to 90g water/100g feed mixture. In the case of SD, the best product is obtained with T in the equipment is of 120 °C and the addition of 4g AG and 2g BF/100g liquidized grapefruit. The comparison of both products allows us to propose FD as being a better technique than spray-drying. In addition, the powder yield of FD is much higher compared to SD and far fewer by-products are generated. A specific study was carried out to confirm the encapsulating power of the two solutes used. The results obtained confirm the benefit of the joint addition of GA and BF, especially against the spray-drying temperature, offering a greater protection against the degradation of bioactive compounds and AOA, than when solutes are added separately. The characterization of the different bioactive compounds and their correlation with the AOA carried showed that the phenolic compounds contribute significantly to the free radical scavenging activity and the inhibition of the discoloration of ß -carotene, while ascorbic acid and ¿- tocopherol contribute to an increase in reducing power. In addition, the stability of the optimized powders during storage at 4 and 20 °C and different surrounding relative humidities has been studied. The modeling of the water sorption data together with the variation in the glass transition temperature (Tg) that takes place in line with the water content of the samples permits the determination of the critical values of water content and water activity that ensure the glassy state of the powdered product. In this sense, what is recommended when storing the powders is to maintain the products under refrigeration and with a surrounding relative humidity in the order of 10%. As soon as the grapefruit powder begins the Tg, the degradation of the bioactive compounds begins, with carotenoids being the most sensitive. In less restrictive storage conditions than those mentioned above, in just one month of storage there are already significant losses in the studied compounds, fewer in the FD product than in the SD. In order to detect any problems of caking in the free-flowing powders or changes in color, a larger quantity of the product must have changed to the rubbery state. In this sense, for the purposes of relating the loss in bioactive compounds, the change in the mechanical properties and the color change with the glass transition, the onset temperature, the midpoint or the end point of the Tg, respectively, should be taken into consideration. Finally, a sensory evaluation of the rehydrated powdered products was carried out in order to evaluate consumer acceptance of the juices obtained and their probability of purchase. In general, grapefruit juice, even the natural or commercial ones, did not enjoy a good level of consumer acceptance due to its astringency and high acidity. The penalty analysis showed that if the sweetness of the samples is improved and their astringency is reduced, their level of consumer acceptance will rise. / El objetivo general de este estudio fue seleccionar el mejor proceso entre la liofilización y la atomización para la obtención de pomelo en polvo de alta calidad nutritiva, funcional y sensorial. Para ello se llevó a cabo la optimización de ambos procesos, usando la metodología de superficie de respuesta. Como variables independientes se consideraron, en ambos casos, la cantidad de goma arábiga (GA) y de fibra de bambú (FB) incorporadas, y la cantidad de agua de la muestra (Xw) en la liofilización o la temperatura de atomización (T). Las propiedades analizadas a los productos obtenidos fueron la humedad, higroscopicidad, porosidad, color, vitamina C, carotenoides totales, fenoles totales y la actividad antioxidante (AOA). Además se consideró el rendimiento en producto de la atomización. Los resultados obtenidos, debidamente validados, permiten proponer, para la liofilización, la adición de 4.2g de GA y 0.58g de FB por cada 100 g de pomelo triturado y una Xw de 90 gagua/100gmezcla. En el caso de la atomización, el mejor producto se obtiene cuando la T es de 120 °C y al pomelo licuado se añaden 4g GA y 2g de FB por cada 100g. Al comparar ambos productos se propone a la liofilización como una mejor tecnología de secado que la atomización. Además, se llevó a cabo un estudio para confirmar el poder encapsulante de los dos solutos utilizados. Los resultados confirmaron el beneficio de la adición conjunta de GA y FB especialmente frente a la temperatura de atomización, ofreciendo una mayor protección de la degradación de los compuestos bioactivos y de la AOA que cuando se añaden por separado. Por su parte, la caracterización de los diferentes compuestos bioactivos y su correlación con la AOA realizada en esta parte del estudio, mostró que los compuestos fenólicos contribuyen de manera significativa a la actividad captadora de radicales libres y a la inhibición de la decoloración de ß-caroteno, mientras que el ácido ascórbico y el ¿-tocoferol contribuyen a incrementan el poder reductor. Además se estudió la estabilidad de los polvos optimizados durante el almacenamiento, a 4 y a 20 °C y a diferentes humedades relativas (HR). La modelización conjunta de los datos de sorción de agua y de variación de la temperatura de transición vítrea (Tg) con la humedad de las muestras permite establecer los valores críticos de humedad y actividad del agua que aseguran el estado vítreo del producto en polvo. En este sentido, para su almacenamiento se recomienda la refrigeración y mantener la HR del entorno del orden del 10 %. En cuanto en el pomelo en polvo se inicia la Tg, comienza la degradación de los compuestos bioactivos, siendo los carotenoides los más sensibles. En condiciones de almacenamiento menos restrictivas a las comentadas, en tan sólo un mes de almacenamiento ya se observan pérdidas importantes de los compuestos estudiados, que son menores en el producto liofilizado que en el atomizado. Por otra parte, para que en el almacenamiento empiecen a detectarse problemas de apelmazamiento del polvo suelto o cambios de color, es necesario que una mayor cantidad de producto haya pasado a estado gomoso. En este sentido, para el estudio de la pérdida de compuestos bioactivos, del cambio en las propiedades mecánicas y del cambio de color se recomienda considerar la temperatura inicial, la del punto medio o la del punto final de la Tg, respectivamente. Finalmente se realizó un análisis sensorial de los productos en polvo rehidratados para evaluar el grado de aceptación de los zumos así obtenidos y su probabilidad de compra. En general el zumo de pomelo, no mostró una buena aceptación por parte del consumidor debido a su astringencia y elevada acidez. El análisis de penalización mostró que si se mejora el dulzor de las muestras y se disminuye su astringencia podrían mejorar los productos y cambiar su grado de aceptación. / L'objectiu general d'este estudi va ser seleccionar el millor procés entre la liofilització (LIO) i l'atomització (ATO) per a l'obtenció de pomelo en pols d'alta qualitat nutritiva, funcional i sensorial. Amb este objectiu es va dur a terme l'optimització de cada procés de deshidratació usant la metodologia de superfície de resposta. Com a variables dels processos es van considerar, en ambdós casos, la quantitat de goma aràbiga (GA) i de fibra de bambú (FB) incorporades com coadjuvants dels processos, així com la quantitat d'aigua de la mostra en el cas de la LIO o la temperatura d'ATO. Les propietats analitzades als productes obtinguts van ser la humitat, higroscopicidad, porositat, color, compostos bioactivos (vitamina C, carotenoides totals, fenols totals) i l'AOA. A més es va considerar el rendiment en producte de l'atomització. Els resultats obtinguts, degudament validats, permeten proposar, per a la LIO, la incorporació de 4.2g de GA i 0.58g de FB per cada 100g de pomelo triturat i portar a la mostra abans del seu processat fins una humitat de 90g agua/100g mezcla. En el cas de l'ATO, el millor producte s'obté quan la T en l'equip és de 120 °C i al liquat de pomelo s'afigen 4g de GA i 2g de FB per cada 100g. La comparació d'ambdós productes permet proposar a la LIO com una millor tecnologia d'assecat que l'ATO. A més el rendiment en producte de la LIO és molt major que el de l'ATO i els subproductes generats molt menors. D'altra banda, es va dur a terme un estudi específic per a confirmar el poder encapsulant dels dos soluts utilitzats. Els resultats permeten confirmar el benefici de l'addició conjunta de GA i FB especialment enfront de la temperatura d'ATO, que oferixen una major protecció de la degradació de compostos bioactivos i AOA que quan s'afigen per separat. La caracterització dels diferents compostos bioactivos i la seua correlació amb l'AOA realitzada en esta part de l'estudi, va mostrar que els compostos fenòlics contribuïxen de manera significativa a l'activitat captadora de radicals lliures i a la inhibició de la decoloració del ¿-caroteno, mentres que l'àcid ascòrbic i el ¿-tocoferol contribuïxen a incrementen el poder reductor. A més s'ha estudiat l'estabilitat de les pols optimitzats durant l'emmagatzemament, a 4 i 20 °C i a diferents humitats relatives (HR) de l'entorn. La modelització de les dades de sorción d'aigua i de variació de la temperatura de transició vítria (Tg) amb la humitat de les mostres permet establir els valors crítics d'humitat i activitat de l'aigua que asseguren l'estat vitri del producte en pols. En este sentit, per al seu emmagatzemament es recomana la refrigeració i mantindre la HR de l'entorn de l'orde del 10 %. En quant en el pomelo en pols s'inicia la Tg, comença la degradació dels compostos bioactivos. En condicions menys restrictives a les comentades, en tan sols un mes d'emmagatzemament ja s'observen pèrdues importants dels compostos estudiats, que són menors en el producte LIO que en l'ATO. D'altra banda, perquè en l'emmagatzemament comencen a detectar-se problemes d'atapeïment de la pols solta o canvis de color, és necessari que una major quantitat de producte haja passat a estat gomós. En este sentit, per a l'estudi de la pèrdua de compostos bioactivos, del canvi en les propietats mecàniques i del canvi de color es recomana considerar la temperatura inicial, la del punt mitjà o la del punt final de la Tg, respectivament. Finalment es va realitzar una anàlisi sensorial dels productes en pols rehidratats per a avaluar el grau d'acceptació dels sucs així obtinguts i la seua probabilitat de compra. En general el suc de pomelo, inclús el natural o el comercial, no va mostrar una bona acceptació per part del consumidor a causa de la seua astringència i elevada acidesa. L'anàlisi de penalització va mostrar que si es millora la dolçor de les mostres i es disminuïx la seua astringència podrien millorar els produc / Agudelo Sterling, CM. (2017). SELECCIÓN DEL MEJOR PROCESO PARA LA OBTENCIÓN DE POMELO EN POLVO (Citrus Paradisi) DE ALTA CALIDAD NUTRITIVA, FUNCIONAL Y SENSORIAL [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/80695 / TESIS

Petunia hybrida and Citrus paradisi have significantly different flavonoid accumulation patterns. Petunia sp. tend to accumulate flavonol glycosides and anthocyanins while Citrus paradisi is known for its accumulation of flavanone diglycosides. One possible point of regulation of flavanone metabolism is flavanone 3-hydroxylase (F3H) expression. To test whether this is a key factor in the different flavanone usage by Petunia hybrida and Citrus paradisi, F3H mRNA expression in seedlings of different developmental stages was measured using semi-quantitative RT-PCR. Primers were designed to conserved regions of F3H and used to amplify an approximately 350 bp segment for quantitation by PhosphorImaging. Primary leaves of 32 day old grapefruit seedlings and a grapefruit flower bud had the highest levels of F3H mRNA expression. Petunia seedlings had much lower levels of F3H mRNA expression relative to grapefruit. The highest expression in petunia was in primary leaves and roots of 65 day old seedlings. These results indicate that preferential use of naringenin for production of high levels of flavanone glycosides in young grapefruit leaves cannot be attributed to decreased F3H mRNA expression.

Flavonoids are secondary metabolites that have significant roles in plant defense and human nutrition. Glucosyltransferases (GTs) catalyze the transfer of sugars from high energy sugar donors to other substrates. Several different secondary product GTs exist in the tissues of grapefruit making it a model plant for studying their structure and function. The goal of this investigation was to determine the expression patterns of seven putative secondary product GTs during grapefruit growth and development by quantifying mRNA expression levels in the roots, stems, leaves, flowers, and mature fruit to establish whether the genes are expressed constitutively or if one or more could be expressed in a tissue specific manner and/or developmentally regulated. Six growth stages were defined from which RNA was extracted, and expression levels were quantified by standardized densitometry of gene-specific RT-PCR products. Results show that there were variable degrees of PGT expression in different tissues and at different developmental stages. These results add to the growing knowledge base of dynamics of expression and potential regulation of secondary metabolism in Citrus paradisi.

Glucosylation is a predominant flavonoid modification reaction affecting the solubility, stability, and subsequent bioavailability of these metabolites. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. In this work, a Citrus paradisi glucosyltransferase gene was identified, cloned, and introduced into the pET recombinant protein expression system utilizing primers designed against a predicted flavonoid glucosyltransferase gene (AY519364) from Citrus sinensis. The encoded C. paradisi protein is 51.2 kDa with a predicted pI of 6.27 and is 96% identical to the C. sinensis homologue. A number of compounds from various flavonoid subclasses were tested, and the enzyme glucosylated only the flavonol aglycones quercetin (Kmapp = 67 μ M; Vmax = 20.45 pKat/μg), kaempferol (Kmapp = 12 μ M; Vmax = 11.63 pKat/μg), and myricetin (Kmapp = 33 μ M; Vmax = 12.21 pKat/μg) but did not glucosylate the anthocyanidin, cyanidin. Glucosylation occurred at the 3 hydroxyl position as confirmed by HPLC and TLC analyses with certified reference compounds. The optimum pH was 7.5 with a pronounced buffer effect noted for reactions performed in Tris-HCl buffer. The enzyme was inhibited by Cu2+, Fe2+, and Zn2+ as well as UDP (Kiapp = 69.5 μ M), which is a product of the reaction. Treatment of the enzyme with a variety of amino acid modifying compounds suggests that cysteine, histidine, arginine, tryptophan, and tyrosine residues are important for activity. The thorough characterization of this C. paradisi flavonol 3-O-glucosyltransferase adds to the growing base of glucosyltransferase knowledge, and will be used to further investigate structure-function relationships.