title: Purification and characterization of an extracellular protease from Penicillium chrysogenum Pg222 active against meat proteins.
abstract: 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 degrees C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45 degrees 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.
authors.affiliation: Higiene de los Alimentos, Facultad de Veterinaria, Universidad de Extremadura, Avenida de la Universidad s/n, 10071 Cáceres, Spain.
authors.forename: María J
authors.initials: MJ
authors.lastname: Benito
authors.forename: Mar
authors.initials: M
authors.lastname: Rodríguez
authors.forename: Félix
authors.initials: F
authors.lastname: Núñez
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Asensio
authors.forename: María E
authors.initials: ME
authors.lastname: Bermúdez
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Córdoba
mesh_terms: D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008460:Meat; D008461:Meat Products; D009124:Muscle Proteins / Q000378:metabolism*; D010408:Penicillium chrysogenum / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism; D020539:Sequence Analysis, Protein; D012965:Sodium Chloride; D013696:Temperature
chemical_list: D009124:Muscle Proteins; D012965:Sodium Chloride; D010450:Endopeptidases; D010447:Peptide Hydrolases; D000626:Aminopeptidases