title: MS analysis and molecular characterization of Botrytis cinerea protease Prot-2. Use in bioactive peptides production.
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.
authors.affiliation: 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.
authors.forename: Ferid
authors.initials: F
authors.lastname: Abidi
authors.forename: Nayssene
authors.initials: N
authors.lastname: Aissaoui
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Gaudin
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Chobert
authors.forename: Thomas
authors.initials: T
authors.lastname: Haertlé
authors.forename: Mohamed Nejib
authors.initials: MN
authors.lastname: Marzouki
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D000935:Antifungal Agents / Q000737:chemistry / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000302:isolation & purification / Q000494:pharmacology; D020171:Botrytis / Q000201:enzymology* / Q000235:genetics; D002176:Candida albicans / Q000187:drug effects; D002918:Chymotrypsin / Q000737:chemistry / Q000494:pharmacology; D052978:Disk Diffusion Antimicrobial Tests; D057075:Enzyme Assays; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D006868:Hydrolysis; D052981:Metarhizium / Q000201:enzymology; D009132:Muscles / Q000737:chemistry; D016366:Open Reading Frames; D010447:Peptide Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D010473:Perciformes; D020081:Phyllachorales / Q000201:enzymology; D013211:Staphylococcus aureus / Q000187:drug effects; D013302:Streptomyces / Q000201:enzymology
chemical_list: D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D023181:Antimicrobial Cationic Peptides; D005656:Fungal Proteins; D010447:Peptide Hydrolases; D002918:Chymotrypsin