“EFFECT OF PHENOLIC COMPOUNDS ON THE ARGININE DEIMINASE ENZYMES”

ARENA ME; ALBERTO MR; MANCA DE NADRA MC

Badajoz, España

Conferencia; BioMicroWorld-2005 - 1st International Conference on Environmental, Industrial and Applied Microbiology.; 2005

Arginine is quantitatively one of the most important amino acids found in fruits. Lactobacillus hilgardii X1B, a wine Lactic Acid Bacteria (LAB) is able to degrade L-arginine, with the formation of ornithine, ammonia, ATP and CO2 [1]. The catabolism of arginine via Arginine Deiminase system (ADI) could be an important energy source for bacterial growth. Another concern about arginine metabolism by wine LAB is the formation of citrulline, a precursor of ethyl carbamate (EC). EC is known as an animal carcinogen. Grapes phenolic that are the main compounds responsible for color, taste and astringency of wines, may affect the bacterial growth and metabolism [2]. Alberto et al [3] demonstrated the degradation of phenolic compounds by a L. hilgardii 5w. This work is aimed to study the effect of different wine phenolic compounds on the enzymes involved in the ADI pathway: arginine deiminase (ADI) and ornithine trasncarbamylase (OTC) in L. hilgardii X1B. The composition of the reaction mixture for the enzymes determinations were:  for ADI, L-arginine-HCl 0.1M adjusted to pH 6.5; sodium phosphate buffer 0.2 M pH 6.5 and cell free extract and for OTC: L-citulline-HCl 0.1M; sodium acetate buffer 0.5 M pH 5.8; sodium arsenate 0.1M and cell free extract. Phenolic compounds were added in the following concentrations: gallic, protocatechuic and vanillic acids, 50-200 mg/l and caffeic acid 10-20 mg/l; the flavonoids quercetin and rutin, 25-50 mg/l and catechin, 50-200 mg/l. The reaction mixtures without phenolic compound were used as controls. Specific activities of the enzymes were defined as the micrograms of substrate consumed per hour per minute per µg of protein. Arginine, citrulline and ornithine concentration were determined by HPLC. Independently of the phenolic concentration in presence of caffeic and vanillic acids and quercetin, the arginine deiminase activity was about 38% higher than in the control without phenolic compounds. In presence of catechin and rutin, the enzyme activity increased in direct relation with the phenolic concentrations, been 21 and 31% higher in presence of 100 and 200mg/l catechin and 28 and 37% higher in presence of 25 and 50 mg/l rutin. Different results were observed in presence of 50, 100 and 200 mg/l gallic or protocatechuic acids. The activity of ADI decreased 20, 33, 53% for gallic acid and 38, 61 and 100% for protocatechuic acid, respectively. At all concentrations, OTC activity was not modified by gallic acid, protocatechuic acid and catechin. The activity increase 11 and 10% in presence of 10 and 20 mg/l caffeic acid, respectively and 10 and 12% in presence of 50 and 100mg/l of vanillic acid, respectively. With the addition of 25 and 50 mg/l rutin the OTC activity increased 9 and 8%, respectively. At the same concentrations, quercetin increased the activity 11 and 9%, respectively. The activities of ADI pathway enzymes varied considerably in presence of the different phenolic compounds. Only rutin, quercetin and caffeic and vanillic acids stimulated both enzymes of the ADI system. The formation of citrulline, precursor of EC, by ADI enzyme was increased by the flavonoid compounds quercetin and rutin and the non flavonoids caffeic and vanillic acids. The inhibitory effect of gallic and protocatechuic, acids on the arginine deiminase activity could be considered as a beneficial property because the diminution of citrulline, a precursor of EC formation. In addition these phenolic acids did not inhibit the OTC enzyme, so the formed citrulline will be degraded. The results about the inhibitory effect of gallic and protocatechuic acids on de enzymes involved in the ADI system are important considering the formation of the toxic compounds, EC. Acknowledgments This work was supported by  Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), and Consejo de Investigaciones de la Universidad Nacional de Tucumán (CIUNT). References [1] M.E. Arena, F.M. Saguir, M.C. Manca de Nadra. Int.  J. Food Microbiol. .52 (1999), p. 155. [2] M.R. Alberto, M.E. Farías, M.C. Manca de Nadra. J. Agric. Food Chem. 49 (2001), p.4359. [3] M.R. Alberto, C. Gómez Cordovés, M. C. Manca de Nadra. J. Agric. Food Chem. 52 (2004), p 6465.