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

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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 × 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.

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Acknowledgments

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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  1. Department of Hygiene and Technology of Food of Animal Origin, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, 54006, Greece

    M. Papagianni & D. Sergelidis

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  1. M. Papagianni
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  2. D. Sergelidis
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Correspondence to M. Papagianni.

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Papagianni, M., Sergelidis, D. Purification and Biochemical Characterization of a Novel Alkaline Protease Produced by Penicillium nalgiovense . Appl Biochem Biotechnol 172, 3926–3938 (2014). https://doi.org/10.1007/s12010-014-0824-3

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