Purification and amino acid sequence of a bacteriocins produced by Lactobacillus salivarius K7 isolated from chicken intestine

Pilasombut, K., Sakpuaram, T., Wajjwalku, W., Nitisinprasert, S., Swetwiwathana, A., Zendo, T., Fujita, K., Nakayama, J. and Sonomoto, K. Purification and amino acid sequence of a bacteriocin produced by Lactobacillus salivarius K7 isolated from chicken intestine Songklanakarin J. Sci. Technol., 2006, 28(Suppl. 1) : 121-131 A bacteriocin-producing strain, Lactobacillus K7, was isolated from a chicken intestine. The inhibitory activity was determined by spot-on-lawn technique. Identification of the strain was performed by morphological, biochemical (API 50 CH kit) and molecular genetic (16S rDNA) basis. Bacteriocin purification processes were carried out by amberlite adsorption, cation exchange and reverse-phase high performORIGINAL ARTICLE 1Ph.D. Student, Center for Agriculture Biotechnology, 2Ph.D.(Veterinary Science), Asst. Prof., Department of Veterinary Public Health, 3D.M.S.(Pathology), Assoc. Prof., Department of Pathology, Faculty of Veterinary Medicine, Kasetsart University, Kamphangsean Campus, Nakhon Pathom 73140, Thailand. 4D.Sc.(Microbiology), Assoc. Prof., Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Chatuchak, Bangkok 10900, Thailand. 5Ph.D.(Agricultural Science), Asst. Prof., Faculty of Agricultural Industry, King Mongkut's Institute of Technology Ladkrabang (KMITL), Lat Krabang, Bangkok, 10520 Thailand. 6Ph.D.(Applied Microbiology), 7Ph.D.(Applied Microbiology), Researcher, 8Ph.D.(Agriculture), Assoc. Prof., 9Ph.D.(Engineer), Prof., Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Corresponding email: [email protected] Received, 2 December 2004 Accepted, 21 November 2005 Songklanakarin J. Sci. Technol. Vol.28 (Suppl.1), 2006: Nutraceutical and Functional Food 122 Purification and amino acid sequence of bacteriocin produced Pilasombut, K., et al. ance liquid chromatography. N-terminal amino acid sequences were performed by Edman degradation. Molecular mass was determined by electrospray-ionization (ESI) mass spectrometry (MS). Lactobacillus K7 showed inhibitory activity against Lactobacillus sakei subsp. sakei JCM 1157T, Leuconostoc mesenteroides subsp. mesenteroides JCM 6124T and Bacillus coagulans JCM 2257T. This strain was identified as Lb. salivarius. The antimicrobial substance was destroyed by proteolytic enzymes, indicating its proteinaceous structure designated as a bacteriocin type. The purification of bacteriocin by amberlite adsorption, cation exchange, and reverse-phase chromatography resulted in only one single active peak, which was designated FK22. Molecular weight of this fraction was 4331.70 Da. By amino acid sequence, this peptide was homology to Abp 118 beta produced by Lb. salivarius UCC118. In addition, Lb. salivarius UCC118 produced 2-peptide bacteriocin, which was Abp 118 alpha and beta. Based on the partial amino acid sequences of Abp 118 beta, specific primers were designed from nucleotide sequences according to data from GenBank. The result showed that the deduced peptide was high homology to 2-peptide bacteriocin, Abp 118 alpha and beta.