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Applied and Environmental Microbiology, December 2002, p. 6193-6201, Vol. 68, No. 12
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.12.6193-6201.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Arginine Catabolism by Sourdough Lactic Acid Bacteria: Purification and Characterization of the Arginine Deiminase Pathway Enzymes from Lactobacillus sanfranciscensis CB1

Maria De Angelis,^1,2 Liberato Mariotti,¹ Jone Rossi,¹ Maurizio Servili,³ Patrick F. Fox,⁴ Graciela Rollán,⁵ and Marco Gobbetti^6*

Sezione di Microbiologia Agro-Alimentare,¹ Sezione di Industrie Agrarie, Dipartimento di Scienze degli Alimenti, Università degli Studi di Perugia, 06126 Perugia,³ Institute of Sciences of Food Production, CNR Bari,² Dipartimento di Protezione delle Piante e Microbiologia Applicata, Facoltà di Agraria di Bari, 70126 Bari, Italy,⁶ Department of Food Science and Technology, University College Cork, Cork, Ireland,⁴ Centro de Referenzia para Lactobacilos, CONNICET, 4000 Tucumán, Argentina⁵

Received 23 May 2002/ Accepted 10 September 2002

The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as Lactobacillus sanfranciscensis CB1; Lactobacillus brevis AM1, AM8, and 10A; Lactobacillus hilgardii 51B; and Lactobacillus fructivorans DD3 and DA106 showed all three enzyme activities. Lactobacillus plantarum B14 did not show CK activity. L. sanfranciscensis CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37°C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of Lactobacillus sakei. L. sanfranciscensis CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in L. sanfranciscensis CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by L. sanfranciscensis CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by L. sanfranciscensis CB1 enhanced cell growth, cell survival during storage at 7°C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds.

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* Corresponding author. Mailing address: Dipartimento di Protezione delle Piante e Microbiologia Applicata, Facoltà di Agraria di Bari, Via G. Amendola 165/a, 70126 Bari, Italy. Phone: 39 080 5442945. Fax: 39 080 5442911. E-mail: gobbetti{at}agr.uniba.it.

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Applied and Environmental Microbiology, December 2002, p. 6193-6201, Vol. 68, No. 12
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.12.6193-6201.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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