title: Purification and biochemical characterization of a novel alkaline protease produced by Penicillium nalgiovense.
abstract: Penicillium nalgiovense PNA9 produces an extracellular protease during fermentation with characteristics of growth-associated product. Enzyme purification involved ammonium sulfate precipitation, dialysis, and ultrafiltration, resulting in 12.1-fold increase of specific activity (19.5 U/mg). The protein was isolated through a series of BN-PAGE and native PAGE runs. ESI-MS analysis confirmed the molecular mass of 45.2 kDa. N-Terminal sequencing (MGFLKLLKGSLATLAVVNAGKLLTANDGDE) revealed 93 % similarity to a Penicillium chrysogenum protease, identified as major allergen. The protease exhibits simple Michaelis-Menten kinetics and K m (1.152 mg/ml), V max (0.827 mg/ml/min), and k cat (3.2 × 10(2)) (1/s) values against azocasein show that it possesses high substrate affinity and catalytic efficiency. The protease is active within 10-45 °C, pH 4.0-10.0, and 0-3 M NaCl, while maximum activity was observed at 35 °C, pH 8.0, and 0.25 M NaCl. It is active against the muscle proteins actin and myosin and inactive against myoglobin. It is highly stable in the presence of non-ionic surfactants, hydrogen peroxide, BTNB, and EDTA. Activity was inhibited by SDS, Mn(2+) and Zn(2+), and by the serine protease inhibitor PMSF, indicating the serine protease nature of the enzyme. These properties make the novel protease a suitable candidate enzyme in meat ripening and other biotechnological applications.
authors.affiliation: Department of Hygiene and Technology of Food of Animal Origin, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, 54006, Greece, mp2000@vet.auth.gr.
authors.forename: M
authors.initials: M
authors.lastname: Papagianni
authors.forename: D
authors.initials: D
authors.lastname: Sergelidis
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004549:Elastin / Q000378:metabolism; D010450:Endopeptidases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D009211:Myoglobin / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D010407:Penicillium / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D059748:Proteolysis; D017421:Sequence Analysis; D012965:Sodium Chloride / Q000494:pharmacology; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D008670:Metals; D009211:Myoglobin; D011480:Protease Inhibitors; D013501:Surface-Active Agents; D012965:Sodium Chloride; D004549:Elastin; D010450:Endopeptidases; C087822:alkaline protease; D009218:Myosins