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MS Analysis and Molecular Characterization of Botrytis cinerea Protease Prot-2. Use in Bioactive Peptides Production

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Abstract

Prot-2 protease previously purified to homogeneity from Botrytis cinerea showed potentiality to be used in detergency and for production of bioactive peptides. To extend the characterization of Prot-2 protease, antifungal and antibacterial assays were performed in vitro using protein hydrolysates prepared from muscle of mackerel (Scomber scomborus) treated with this enzyme. The most active hydrolysate (degree of hydrolysis of 8 %) exhibited inhibition effect towards bacteria and phytopathogenic fungi, demonstrating that Prot-2 proteolysis generated bioactive peptides. Biochemical and molecular characterization of the purified Prot-2, by SDS-PAGE/Tryptic in gel-digestion and LC-MS/MS analysis, was investigated. The peptide amino acid sequence alignment search in database revealed a moderate homology between the determined amino acid sequence of Prot-2 protease and the known fungal trypsin/chymotrypsin in particular from Glomerella, Metarhizium and Streptomyces. From peptide sequence data obtained by mass spectrometry and sequences homologies, primers were defined and a cDNA fragment of 786 bp was amplified by RT-PCR. The cDNA nucleotide sequence analysis revealed an open reading frame coding for 262 amino acid residues. The deduced amino acid sequence of Prot-2 showed moderate identity with trypsin of Glomerella graminicola (74 %) and with chymotrypsin from Metarhizium anisopliae (71 %). Prot-2 exhibited a Ser protease homology and showed in addition the specific His motif of trypsin/chymotrypsin family.

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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, B. P. 676, 1080, Tunis Cedex, Tunisia

    Ferid Abidi, Nayssene Aissaoui & Mohamed Nejib Marzouki

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

    Jean-Marc Chobert & Thomas Haertlé

  3. UR 1268, INRA, Biopolymères Interactions Assemblages, Equipe Allergie, B. P. 71627, 44316, Nantes Cedex 3, France

    Jean-Charles Gaudin

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  1. Ferid Abidi
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  2. Nayssene Aissaoui
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Correspondence to Jean-Marc Chobert.

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Abidi, F., Aissaoui, N., Gaudin, JC. et al. MS Analysis and Molecular Characterization of Botrytis cinerea Protease Prot-2. Use in Bioactive Peptides Production. Appl Biochem Biotechnol 170, 231–247 (2013). https://doi.org/10.1007/s12010-013-0186-2

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