No description available.

Thesis (doctoral)--Freie Universität Berlin, 1980.

The polyphenolic pigments of black tea include the theaflavins (TF), the flavonol glycosides (FG) and a group of uncharacterised brown pigments referred to as thearubigins (TR). The TR are shown to be a complex, heterogeneous group of compounds, the majority of which are unresolvable by reverse phase HPLC. Fractionation procedures have been modified, developed, combined and applied to black tea liquor, to improve the separation of black tea components and to allow fractions of less complexity be obtained. A degree of resolution has been achieved for TR fractions, unresolvable with reverse phase HPLC, using size exclusion HPLC. Calibration of the column suggested there may be little material above 2,000 daltons associated with the TR. Two heterogeneous TR fractions have been subjected to Porter's autoxidative assay for proanthocyanidins. The resulting solutions were analysed by reverse phase HPLC and shown to contain proanthocyanidins (9.4 and 16.1 %) and galloyl ester (7.1 and 7.4 %), some 73 and 84 % remains uncharacterised. A novel group of yellow pigments has been isolated for which the name theacitrins (TC) has been proposed. Data are presented that suggest the TC are possibly derived from the TF by oxidative opening of the dihydroxybenzene ring associated with the benztropolone structure. The contribution of the phenolic pigments to tea cream was investigated using reversed phase HPLC and found to be ? 86 % TR, 12 % TF and 2 % FG. Evidence is also presented for a synergistic interaction between TF and TR during cream formation. A method has been developed, using caffeine precipitation, for the isolation of TR material that is free from TF and FG Tea pigments have been stored in aqueous solutions to evaluate their potential for use as commercial colourants. The TF and TC were found to possess poor stability. The FG exhibited greater stability but possess low tinctoral power and poor water solubility. Many of the stability trials exhibited a darkening of colour associated with an increase in unresolvable TR material. Storage of an aqueous solution of a TF enriched fraction at 37 °C resulted in the production of unresolvable (TR-like) material. This material was found to possess molecular weights in excess of 2,000, indicating polymerisation. Evidence is presented to suggest the involvement of galloyl groups in the formation of non-hydrolysable bonds within this polymeric material.

Univ., Diss.--Münster, 1997.

Citrus fruits are some of the most widely consumed fruits in the world and contain significant levels of flavonoids, a category of plant secondary metabolites which control taste, color, plant defense, and overall marketability. In citrus and other plants, flavonoids are found in their glucosylated form. Glucosyltransferases (GT’s) are enzymes that add glucose to secondary metabolites like flavonoids. They make up a diverse class of enzymes ubiquitous throughout the plant and animal kingdoms. While many GT’s have been identified, they vary greatly in their structural identity, and their chemical properties make it such that only a small percentage of existing GT’s have been functionally characterized. Research on GT structure function relationships strengthens the reliability of genomic databases and makes significant contributions to the field of enzyme biotechnology. Bioenergy research and custom enzyme synthesis rely on GT structural data, making this research critical to the success of many promising current and future projects. A GT was isolated from grapefruit and was shown to glucosylate the flavonol class of flavonoids at the 3-OH position, called CP3GT. Subsequent analysis showed there are specific arrangements of amino-acids inside the catalytic cleft of CP3GT that likely account for its specificity with flavonols. These interactions are not fully understood and make CP3GT an excellent model for elucidating unique structure function relationships of a GT enzyme. X-ray crystallography is one of the best methods for structure determination that allows a 3D image of the protein in question to be resolved at the molecular level. This method has vast potential for advancing plant enzymology, yet to date only 6 plant glucosyltransferases have had their crystal structures solved. The structural similarities and complementary specificities that CP3GT shares with these crystallized GT’s make CP3GT an excellent candidate for crystallization. This research hypothesizes that there are unique structural features that give CP3GT its specificity, and that these features can be elucidated using x-ray crystallography. Wild type CP3GT and 3 recently characterized mutants are being prepared for crystallization. The crystallization of 3 CP3GT mutants in addition to wild type will compliment structure/function analysis by providing insight into how structural modifications can alter enzyme function. It is recommended that protein be in its native form for crystallization, thus a thrombin-cleavage site was inserted into WT CP3GT and 3 mutants to remove tags following purification. Some studies have suggested that the presence of tags alters enzyme activity, thus this presented the opportunity to test the effect of tags by assaying both native and tagged enzyme. Initial results showed that WT CP3GT treated with thrombin retained 70 percent activity after a 2-hour treatment at 4o C. Additional assays will be conducted to fully determine tag effects and will run concurrently with crystallization experiments

Citrus fruits are widely consumed and can offer various health benefits. One enzyme found in grapefruits, Citrus paradisi flavonol specific 3-O-glucosyltransferase (CP3GT), catalyzes the addition of glucose to one specific flavonoid class and at only one site. These flavonoids are plant secondary metabolites that can be used in a variety of plant functions including signaling and protection. The only class of flavonoids that CP3GT glucosylates is flavonols, and this specificity is of interest to study for potential benefits in biotechnology and enzyme modeling. In order to study this enzyme and its structure, a variety of mutants were created using site-directed mutagenesis. One mutant, P297F, exhibited a loss of function. This mutant was previously studied by inserting a thrombin cleavage site, extracting the plasmid expressing the mutation and sequencing it. The gene sequence was previously verified to be in frame and contain the needed thrombin cleavage site to remove tags used for protein purification and identification. The plasmid was then linearized, and transformed into yeast. After this, conditions for protein expression were tested over a 72-hour period. The protein was found to have optimal expression at 50 hours with a constant temperature of 28 °C and methanol concentration of 0.5 %. However, numerous protein expression experiments indicated very low protein expression. For this reason, the P297F gene was amplified through colony PCR, extracted and sent for sequencing to verify the transformation of the gene into yeast and identify possible reasons for low protein production. Analysis of this sequencing data showed a single nucleotide addition early in the tag sequence causing a frameshift after this location. Reanalysis of the previous plasmid sequencing data showed this same mutation, indicating improper conclusions were drawn. Efforts should be made to identify a plasmid without the mutation or correct the frameshift mutation so that the tag sequence produces the correct amino acids.

No description available.

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.

This research was designed to determine the conditions necessary to remove c-myc and 6x-His tags from a flavonol specific glucosyltransferase found in grapefruit (CP3GT) using thrombin in preparation for crystallization. X-ray crystallography of CP3GT crystals may elucidate structural features that account for flavonol specificity in some glucosyltransferase enzymes. A thrombin cleavage site was inserted into WT CP3GT and one mutant. Recombinant CP3GT was expressed in yeast and purified. Optimal conditions for thrombin digestion were explored. Digestion with 100U of thrombin for 2 hours at 4o C was optimal for removing tags from CP3GT. Storage at 4o C for 2 hours resulted in approximately 70% retention of activity. The effect of thrombin treatment on CP3GT activity was tested. Purified CP3GT protein with and without tags was tested for activity with the flavonol quercetin. Data showed no significant difference in overall activity between tagged and native protein.

Three 2-oxoglutarate-dependent dioxygenases required for flavonoid biosynthesis were characterized in Arabidopsis thaliana. Genes encoding flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), and leucoanthocyanidin dioxygenase (LDOX) were cloned and sequenced. The predicted proteins encoded by each of these Arabidopsis genes shared high homology with all F3H, FLS, or LDOX sequences available in Genbank. Low-stringency DNA blot analysis indicated that F3H and LDOX are encoded by a single gene in Arabidopsis, while FLS may be encoded by two or three genes. RNA blot analysis was performed to determine the expression patterns of these three genes relative to previously-cloned genes encoding flavonoid biosynthetic enzymes. Light-induction experiments and analysis of regulatory mutants showed that the CHS, CHI, F3H, and FLS1 are coordinately regulated in Arabidopsis seedlings, encode enzymes acting near the beginning of the pathway, and are therefore referred to as "early" genes. The same experiments showed that DFR and LDOX are regulated distinctly from "early" genes, share similar expression patterns in response to light, and are not expressed in the ttg mutant. DFR and LDOX are therefore referred to as "late" genes due to the timing of expression in response to light and the fact that they encode enzymes acting late in flavonoid biosynthesis. To determine whether any of the previously-identified transparent testa mutants were defective in F3H, FLS, or LDOX, the chromosomal locations of these genes in the Arabidopsis genome were determined. The positions of these genes suggested that no previously-identified tt mutant was defective in the cloned FLS or LDOX structural genes, while tt6 was potentially the F3H locus. The coding region of F3H was amplified by PCR from tt6 genomic DNA and sequenced, and several point mutations were found in the coding region of this allele, three of which are predicted to result in amino acid substitutions. Polyclonal antibodies were also developed using four different purified, recombinant flavonoid enzymes as antigens. These antibodies were used to determine the pattern of accumulation of flavonoid enzymes in developing seedlings. Immunoblot analysis was also performed to determine whether mutations in genes encoding specific flavonoid enzymes or an enzyme in pathways that compete for or provide substrate for flavonoid biosynthesis (mutants defective in tryptophan or ferulic acid biosynthesis) affect the levels of flavonoid enzymes. These analyses showed that mutant seedlings which lacked specific flavonoid or tryptophan biosynthetic enzymes accumulated higher steady-state levels of other enzymes in the pathway. These results suggest that the accumulation of specific flavonoid intermediates or indole can lead directly or indirectly to higher levels of flavonoid enzymes. / Ph. D.