Applied Biochemistry and Biotechnology

, Volume 172, Issue 8, pp 3926–3938 | Cite as

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

  • M. PapagianniEmail author
  • D. Sergelidis


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.


Penicillium nalgiovense Extracellular enzyme Alkaline protease Purification Characterization 



The authors would like to thank Prof. E.M. Papamichael from the Department of Chemistry, University of Ioannina for his helpful suggestions regarding the methodologies followed in this work.


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© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Hygiene and Technology of Food of Animal Origin, School of Veterinary MedicineAristotle University of ThessalonikiThessalonikiGreece

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