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Neutral Serine Protease from Penicillium italicum. Purification, Biochemical Characterization, and Use for Antioxidative Peptide Preparation from Scorpaena notata Muscle

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Abstract

In the present study, purification and properties of an extracellular neutral serine protease from the fungus Penicillium italicum and its potential application as an antioxidant peptides producer are reported. The protease was purified to homogeneity using ammonium sulfate precipitation, Sephacryl S-200 gel filtration, diethylaminoethanol (DEAE)-Sepharose ion exchange chromatography, and TSK-HPLC gel filtration with a 10.2-fold increase in specific activity and 25.8 % recovery. The purified enzyme appeared as single protein band with a molecular mass of 24 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the proteolytic activity were pH 7.0 and 50 °C, respectively. The enzyme was stable in the pH range of 6.0–9.0. The protease was activated by divalent cations such as Ca2+ and Mg2+. Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and relatively broad specificity. Scorpaena notata muscle protein hydrolysates prepared using purified serine protease (protease from P. italicum (Prot-Pen)) showed good in vitro antioxidative activities. The antioxidant activities of Scorpaena muscle protein hydrolyzed by Prot-Pen (SMPH-PP) were evaluated using various antioxidant assays: 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing power, ferrous chelating activity, and DNA nicking assay. SMPH-PP showed varying degrees of antioxidant activity and almost the same strongest protection against hydroxyl radical induced DNA breakage.

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Abbreviations

Prot-Pen:

Protease from Penicillium italicum

DPPH:

1,1-Diphenyl-2-picrylhydrazyl

EDTA:

Disodium ethylenediaminetetraacetate

BHA:

Butylated hydroxyanisole

FPLC:

Fast protein liquid chromatography

SMPH-PP:

Scorpaena muscle protein hydrolyzed by Prot-Pen

SMPH-FL:

Scorpaena muscle protein hydrolyzed by Flavourzyme

SMPH-CH:

Scorpaena muscle protein hydrolyzed by Chymotrypsin

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Acknowledgments

This work was supported by the financial project of LIP-MB Laboratory, INSAT, Carthage University, Ministry of Higher Education and Scientific Research of Tunisia. The authors acknowledge the support of Professor Mohamed Rabeh Hajlaoui, Laboratory of Plant Protection, National Institute for Agricultural Research, INRA, Tunisia (Rue Hedi Karray, 2049 Ariana, Tunisia).

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Authors and Affiliations

  1. Laboratory of Protein Engineering and Bioactive Molecules (LIP-MB), National Institute of Applied Sciences and Technology, University of Carthage, Centre Urbain Nord, B. P. 676, 1080, Tunis Cedex, Tunisia

    Ferid Abidi, Neyssene Aissaoui & Mohamed Nejib Marzouki

  2. UR 1268, Biopolymères Interactions Assemblages, Equipe Fonctions et Interactions des Protéines, INRA, B. P. 71627, 44316, Nantes Cedex 3, France

    Ferid Abidi, Jean-Marc Chobert & Thomas Haertlé

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  1. Ferid Abidi
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  2. Neyssene Aissaoui
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  3. Jean-Marc Chobert
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  5. Mohamed Nejib Marzouki
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Correspondence to Ferid Abidi.

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Abidi, F., Aissaoui, N., Chobert, JM. et al. Neutral Serine Protease from Penicillium italicum. Purification, Biochemical Characterization, and Use for Antioxidative Peptide Preparation from Scorpaena notata Muscle. Appl Biochem Biotechnol 174, 186–205 (2014). https://doi.org/10.1007/s12010-014-1052-6

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