Purification, characterization and amino acid sequencing of divergicin M35: a novel class IIa bacteriocin produced by Carnobacterium divergens M35

Abstract

Carnobacterium divergens M35, isolated from a commercial sample of frozen smoked mussels, produces a new bacteriocin, divergicin M35, a class IIa bacteriocin. Divergicin M35 is sensitive to pronase-E, alpha-chymotrypsin and proteinase K, but not to trypsin and withstands thermal treatments up to 121 degrees C for 30 min. Divergicin M35 was extracted from the culture supernatant of C. divergens M35 using an SP-Sepharose cation-exchange column, desalted and purified on a C18 Sep-Pack column and further purified by reverse phase-high pressure liquid chromatography. This procedure allowed the recovery of 10% of the bacteriocin present in the culture supernatant with purity higher than 99%. Divergicin M35 had a molecular mass of 4518.75 Da as determined by mass spectrometry, a pI value of 8.3 and positive net charge (+3). The amino acid sequence of divergicin M35 was found to consist of 43 amino acid with four cysteine residues (Cys10, 15, 25, 43) and showed 80.5% homology with divercin V41 (80.5%) and 80.0% with bavaricin MN. Divergicin M35 showed powerful antilisterial activity, especially against Listeria monocytogenes and was also active against carnobacteria but not against strains of Lactococcus, Lactobacillus, Enterococcus, Bifidobacteria and Escherichia. Divergicin M35 production began in late exponential phase and reached a maximum activity of 65,000 AU/ml in early stationary phase. Initial broth pH, Tween 80 and acetate did not affect C. divergens M35 growth or divergicin production. This bacteriocin may be a potential tool for inhibiting L. monocytogenes in seafood products that do not usually undergo an adequate heat treatment.