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Applied and Environmental Microbiology, July 2002, p. 3532-3536, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3532-3536.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins

María J. Benito,,{dagger} Mar Rodríguez, Félix Núñez, Miguel A. Asensio,* María E. Bermúdez, and Juan J. Córdoba

Higiene de los Alimentos, Facultad de Veterinaria, Universidad de Extremadura, 10071 Cáceres, Spain

Received 5 November 2001/ Accepted 10 April 2002

An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.

* Corresponding author. Mailing address: Higiene de los Alimentos, Facultad de Veterinaria, Universidad de Extremadura, Avda. de la Universidad s/n, 10071 Cáceres, Spain. Phone: 34 927 257124. Fax: 34 927 257110. E-mail: masensio{at}unex.es.

{dagger} Present address: Nutrición y Bromatología, Escuela de Ingenierías Agrarias, Universidad de Extremadura, 06071 Badajoz, Spain.

Applied and Environmental Microbiology, July 2002, p. 3532-3536, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3532-3536.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.





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Copyright © 2002 by the American Society for Microbiology. All rights reserved.