Purification and characterization of an extracellular proline iminopeptidase from Arthrobacter nicotianae 9458
Emanuele Smacchi
Istituto di Industrie Agrarie (Microbiologia), Facoltà di Agraria, Università degli Studi di Perugia, S. Costanzo, 06126 Perugia, Italy
Search for more papers by this authorCorresponding Author
Marco Gobbetti
Istituto di Produzioni e Preparazioni Alimentari, Facoltà di Agraria, Università degli Studi di Bari, Via Napoli 25, 71100 Foggia, Italy
*Corresponding author. Istituto di Industrie Agrarie (Microbiologia), Facoltà di Agraria, Università degli Studi di Perugia, S. Costanzo, 06126 Perugia, Italy. Tel.: +39 (75) 32387; Fax: +39 (75) 32387, E-mail address: [email protected]Search for more papers by this authorRosalba Lanciotti
Istituto di Produzioni e Preparazioni Alimentari, Facoltà di Agraria, Università degli Studi di Bari, Via Napoli 25, 71100 Foggia, Italy
Search for more papers by this authorPatrick F. Fox
Department of Food Chemistry, University College Cork, Cork, Ireland
Search for more papers by this authorEmanuele Smacchi
Istituto di Industrie Agrarie (Microbiologia), Facoltà di Agraria, Università degli Studi di Perugia, S. Costanzo, 06126 Perugia, Italy
Search for more papers by this authorCorresponding Author
Marco Gobbetti
Istituto di Produzioni e Preparazioni Alimentari, Facoltà di Agraria, Università degli Studi di Bari, Via Napoli 25, 71100 Foggia, Italy
*Corresponding author. Istituto di Industrie Agrarie (Microbiologia), Facoltà di Agraria, Università degli Studi di Perugia, S. Costanzo, 06126 Perugia, Italy. Tel.: +39 (75) 32387; Fax: +39 (75) 32387, E-mail address: [email protected]Search for more papers by this authorRosalba Lanciotti
Istituto di Produzioni e Preparazioni Alimentari, Facoltà di Agraria, Università degli Studi di Bari, Via Napoli 25, 71100 Foggia, Italy
Search for more papers by this authorPatrick F. Fox
Department of Food Chemistry, University College Cork, Cork, Ireland
Search for more papers by this authorAbstract
An extracellular proline iminopeptidase, with a molecular mass of about 53 kDa, was purified from Arthrobacter nicotianae 9458 and characterized. The enzyme had temperature and pH optima of 37°C and 8.0, respectively, was completely inactivated by heating for 1 min at 80°C and showed highest activity on Pro-pNA. The proline iminopeptidase was characterized by activity at low temperature, NaCl concentrations up to 7,5% and by high sensitivity to pH values 6.0, serine enzyme inhibitor PMSF and divalent cations, Fe2+, Sn2+, Cu2+, Zn2+, Hg2+, Co2+ and Ni2+. The extracellular proline iminopeptidase from A. nicotianae 9458 was able to hydrolyze proline-containing peptides at the pH, temperature and NaCl concentration typical of the surface of smear-ripened cheeses and may contribute to proteolysis of these cheeses during ripening.
References
- [1] Kunji, E.R.S., Mierau, I., Hagting, A., Poolman, B., Konings, W.N. (1996) The proteolytic system of lactic acid bacteria. Antonie van Leeuwenhoek 70, 187–221.
- [2] Fox, P.F., McSweeney, P.L.H. (1996) Proteolysis in cheese during ripening. Food Rev. Int. 12, 457–509.
- [3] Bhowmik, T., Marth, E.H. (1990) Role of Micrococcus and Pediococcus species in cheese ripening: a review. J. Dairy Sci. 73, 859–866.
- [4] Fernandez-Espla, M.D., Fox, P.F. (1997) Purification and characterization of X-prolyl dipeptidyl aminopeptidase from Propionibacterium shermanii NCDO 853. Int. Dairy J. 7, 23–29.
- [5] Panon, G. (1990) Purification and characterization of a proline iminopeptidase from Propionibacterium shermanii 13673. Lait 70, 439–452.
- [6]
Eliskases-Lechner, F.,
Ginzinger, W. (1995) The bacterial flora of surface-ripened cheeses with special regard to coryneforms.
Lait
75, 571–584.
10.1051/lait:1995644 Google Scholar
- [7] El-Erian, A. (1969) Bacteriological studies on Limburger cheese. Ph.D. Thesis, Agricultural University, Wageningen, The Netherlands.
- [8] Bockelmann, W., Hoppe-Seyler, T., Lick, S., Heller, K.J. (1998) Analysis of casein degradation in Tilsit cheeses. Kieler Milchwirtschaftl. Forsch. Ber. 50, 105–113.
- [9] Bockelmann, W., Fuehr, C., Martin, D., Heller, K.J. (1997) Color development by red-smear surface bacteria. Kieler Milchwirtschaftl. Forsch. Ber. 49, 285–292.
- [10] Bockelmann, W., Krusch, U., Engel, G., Klijn, N., Smith, G., Heller, K.J. (1997) The microflora of Tilsit cheese. Part 1. Variability of the smear flora. Nahrung 41, 208–212.
- [11] Ottogalli, G. (1991) Microbiologia lattiero-casearia. Clesav, Città Studi, Milan.
- [12] Valdes-Stauber, N., Scherer, S., Seiler, H. (1997) Identification of yeasts and coryneform bacteria from the surface microflora of Brick cheeses. Int. J. Food Microbiol. 34, 115–129.
- [13] Marcellino, N., Benson, D.R. (1992) Scanning electron and light microscopic study of microbial succession on Bethlehem St. Nectaire cheese. Appl. Environ. Microbiol. 58, 3448–3454.
- [14] Bjare, U., Hofsten, B.V., Rydèn, A.-C. (1979) Cell bound proteolytic enzymes of Arthrobacter. Biochim. Biophys. Acta 220, 134–136.
- [15] Michotey, V., Blanco, C. (1994) Characterization of an endoserine protease secreted by Arthrobacter aureus. Appl. Environ. Microbiol. 60, 341–343.
- [16] Hofsten, B.V., Vankley, H., Eaker, D. (1965) An extracellular proteolytic enzyme from a strain of Arthrobacter. II. Purification and chemical properties of the enzyme. Biochim. Biophys. Acta 110, 585–598.
- [17] Smacchi, E., Fox, P.F., Gobbetti, M. (1999) Purification and characterization of two extracellular proteinases from Arthrobacter nicotianae 9458. FEMS Microbiol. Lett. 170, 327–333.
- [18] El-Soda, M., Desmazeaud, J. (1982) Les peptide-hydrolases des lactobacilles du groupe Thermobacterium. I. Mise en evidence de ces activities chez Lactobacillus helveticus, L. acidophilus, L. lactis et L. bulgaricus. Can. J. Microbiol. 28, 1181–1187.
- [19] Doi, E., Shibata, D., Matoba, T. (1981) Modified colorimetric ninhydrin methods for peptide assays. Anal. Biochem. 118, 173–182.
- [20] Box, G.E.P., Hunter, W.G. and Hunter, J.S. (1978) Statistics for Experimenters. Wiley, New York.
- [21] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
- [22] Hemme, D., Richard, J. (1986) Utilization of L-methionine and production of methanetiol by bacteria isolated from raw milk Camembert cheese. Lait 66, 135–142.
- [23] Varmanen, P., Rantanen, T., Palva, A. (1996) An operon from Lactobacillus helveticus composed of a proline iminopeptidase gene (pepI) and two genes coding for putative members of the ABC transporter family of proteins. Microbiology 142, 3459–3468.
- [24] Rattray, F.P., Bockelmann, W., Fox, P.F. (1995) Purification and characterization of an extracellular proteinase from Brevibacterium linens ATCC 9174. Appl. Environ. Microbiol. 61, 3454–3456.
- [25] Hayashi, K., Cliffe, A., Law, B. (1990) Purification and preliminary characterization of five serine proteinases produced by Brevibacterium linens. Int. J. Food Sci. Technol. 25, 180–187.
- [26] Gobbetti, M., Lowney, S., Smacchi, E., Battistotti, B., Damiani, P., Fox, P.F. (1997) Microbiology and biochemistry of Taleggio cheese during ripening. Int. Dairy J. 7, 509–517.
- [27] Eliskases-Lechner, F., Ginzinger, W. (1994) Tilsit cheese without smear?. Lebensm. Milchwir. 115, 174–177.
