ORIGINAL PAPER Purification, characterization and in vitro cytotoxicity of the bacteriocin from Pediococcus acidilactici K2a2-3 against human colon adenocarcinoma (HT29) and human cervical carcinoma (HeLa) cells Karen I. Villarante • Francisco B. Elegado • Shun Iwatani • Takeshi Zendo • Kenji Sonomoto • Evelyn E. de Guzman Received: 13 January 2009 / Accepted: 13 August 2010 / Published online: 25 August 2010 Ó Springer Science+Business Media B.V. 2010 Abstract A bacteriocinogenic lactic acid bacterium (designated K2a2-3) isolated from the intestine of Philip- pine water buffalo was identified as Pediococcus acidilac- tici by 16S rRNA gene sequence analysis. The bacteriocin was purified by hydrophobic interaction chromatography, cation-exchange chromatography and reverse phase-high performance liquid chromatography. The purified protein has a molecular mass of 4,625.91 Da, quantified by elec- trospray ionization time-of-flight mass spectrometry. Based on a BLAST homology search of a partial sequence of 39 amino acid residues and the presence of the structural gene papA, detected through polymerase chain reaction, it was identified as very similar to pediocin PA-1. It was active against a wide spectrum of lactic acid bacteria and Listeria innocua. Partially-purified bacteriocin samples, conducted using pH-mediated bacteriocin extraction method, were found to be cytotoxic against human colon adenocarcinoma (HT29) and human cervical carcinoma (HeLa) cells in vitro, as determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] assay. Keywords Bacteriocin Á Purification Á Characterization Á Cytotoxicity Á Pediocin Á Pediococcus acidilactici Introduction Bacteriocins are ribosomally-synthesized bacterial peptides which can be used as natural preservatives and antibacte- rials in the food and pharmaceutical industries (Cleveland et al. 2001). Some bacteriocins such as colicin from Escherichia coli, pyocin from Pseudomonas aeruginosa, vibriocins from Vibrio cholerae and Vibrio eltor (Farkas- Himsley and Cheung 1975), nisin and gallidermin (Maher and McClean 2006; Murinda et al. 2003) have also been shown to have cytotoxic properties against cancer cells. Bacteriocin-producing lactic acid bacteria (LAB) have been isolated from several plant and food sources (Todorov and Dicks 2005; Banaay et al. 2004), feces of humans (Kang and Lee 2005) and gastrointestinal tracts of a few animals, including poultry (Chin et al. 2005) and water buffalo (Elegado et al. 2007). Pediococcus acidilactici K2a2-3 isolated from the intestines of Philippine water buffalo (Bubalus bubalis carabanensis) was found to have in vitro inhibitory activity against Listeria spp. (Elegado et al. 2007). To further understand and explore potential clinical applications of the bacteriocin produced by P. acidilactici K2a2-3, genetic identification, molecular characterization and cytotoxic assays on human colon adenocarcinoma (HT29) and human cervical carcinoma (HeLa) cell lines were done in this study. K. I. Villarante Á E. E. de Guzman Department of Biology, School of Science and Engineering, Loyola Schools, Ateneo de Manila University, Katipunan Avenue, Loyola Heights, 1108 Quezon City, Philippines F. B. Elegado (&) National Institute of Molecular Biology and Biotechnology, BIOTECH, University of the Philippines Los Ban ˜os, College, 4031 Laguna, Philippines e-mail: fbelegado@hotmail.com S. Iwatani Á T. Zendo Á K. Sonomoto Laboratory of Microbial Science and Technology, Division of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan 123 World J Microbiol Biotechnol (2011) 27:975–980 DOI 10.1007/s11274-010-0541-1