A tyrosine-hydroxylating enzyme was partially puri®ed from betacyanin-producing callus cultures of Portulaca grandi¯ora Hook. by using hydroxyapatite chromatography and gel ®ltration. It was characterized as a tyrosinase (EC 1.14.18.1 and EC 1.10.3.1) by inhibition experiments with copper-chelating agents and detection of concomitant o-diphenol oxidase activity. The tyrosinase catalysed both the formation of L-(3,4-dihydroxyphenyl)-alanine (Dopa) and cyclo-Dopa which are the pivotal precursors in betalain biosynthesis. The hydroxylating activity with a pH optimum of 5.7 was speci®c for L-tyrosine and exhibited reaction velocities with L-tyrosine and D-tyrosine in a ratio of 1:0.2. Other monophenolic substrates tested were not accepted. The enzyme appeared to be a monomer with an apparent molecular mass of ca. 53 kDa as estimated by gel ®ltration and SDS-PAGE. Some other betalainproducing plants and cell cultures were screened for tyrosinase activity; however, activities could only be detected in red callus cultures and plants of P. grandiora as well as in plants, hairy roots and cell cultures of Beta vulgaris L. subsp. vulgaris (Garden Beet Group), showing a clear correlation between enzyme activity and betacyanin content in young B. vulgaris plants. We propose that this tyrosinase is speci®cally involved in the betalain biosynthesis of higher plants.Abbreviations: 4-COH 4-coumarate hydroxylating activity; Dopa L-(3,4-dihydroxyphenyl)-alanine; DO Dopa oxidase activity of the tyrosinase; IEF isoelectric focusing; KPi Kphosphate; MBTH = 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate; PPO polyphenol oxidase; TOH tyrosine hydroxylase activity of the tyrosinase; tyramineOH tyramine hydroxylating activity

The flavonoid composition of six varieties in either case of yellow and green French beans was investigated. Fresh beans were chopped under liquid nitrogen just after harvest and lyophilized subsequently. The flavonoids were exhaustively extracted, purified by selective adsorption to and stepwise elution from polyamide, and separated by RP-HPLC with UV detection. With one exception the qualitative chromatographic pattern of the 12 varieties was very similar. The main flavonoids were identified as 3-O-glucuronides (-3-O-glucur) and 3-O-rutinosides (-3-O-rut) of quercetin (Q) and kaempferol (K) by LC-TSP-MS comparing retention time, UV spectra, and mass spectra with those of pure flavonoids. The areas of HPLC peaks with identical aglycon were summed up and converted into the -3-O-rut of Q or K with the respective calibration curves. The total content of Q-3-O-glycosides ranged from 19.1 to 183.5 μg/g and that of K-3-O-glycosides from 5.6 to 14.8 μg/g of fresh bean, demonstrating a dependence on the variety without general difference between yellow and green representatives. Keywords: French bean (Phaseolus vulgaris L.); flavonoids; HPLC; LC-TSP-MS

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Abstract. Uridine 5'-diphosphoglucose-dependent glucosyltransferases (UDP-glucose:betanidin 5-0-and 6-0-glucosyltransferases; 5-GT and 6-GT; EC 2.4.1) catalyze the regiospecific transfer of glucose to the 5-and 6-hydroxy group of betanidin in the formation of betanin and gomphrenin I, respectively. Both GT activities were partially purified from cell suspension cultures of Dorotheanthus bellidiformis (Burro. f.) N.E. Br.. Isoelectric focusing of crude protein extracts indicated the presence of three 5-GT isoforms and a single 6-GT form. The 5-GT isoforms were partially separated from each other and completely from the 6-GT. Studies of the glucosyltransferase activities were focused on the major isoform of the 5-GTs and the 6-GT, which displayed the same pH optimum near 7.5 in K-phosphate buffer. A 3-and 2.5-fold enrichment and 11% and 10% recovery of the 5-GT and 6-GT, respectively, were routinely achieved; however, a 3300-fold enrichment of the major 5-GT isoform and a 6-fold enrichment of the 6-GT were also achieved. Both enzymes are monomers and displayed apparent native Mrs near 55 000. The maxima of the reaction temperature were at 50 ~ for the 5-GT and at 37 ~ for the 6-GT with respective apparent energies of activation of 51 and 53 kJ. tool-1. Kinetic studies indicated that the apparent Michaelis constants (apparent Km) of the GTs for one substrate were dependent on the concentration of the second substrate. However, the relationship between the apparent Km values and the dissociation constants (Ki) were different; Km> Ki applies for the 5-GT and Km< Ki for the 6-GT activity. Consequently, this results in a predominant formation of betanin at low substrate concentrations, but a predominant formation of gomphrenin I at high substrate concentrations, assuming that both enzymes may compete freely for their substrates. This might explain why we could not observe a correlation between extract-