Details

Autor(en) / Beteiligte

• Papagianni, M
• Sergelidis, D

Titel

Purification and Biochemical Characterization of a Novel Alkaline Protease Produced by Penicillium nalgiovense

Ist Teil von

• Applied biochemistry and biotechnology, 2014-04, Vol.172 (8), p.3926-3938

Ort / Verlag

New York: Springer-Verlag

Erscheinungsjahr

2014

Link zum Volltext

Quelle

SpringerNature Journals

Beschreibungen/Notizen

• 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 × 10²) (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, Mn²⁺ and Zn²⁺, 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.