INTERNATIONAL JOURNAL OF AGRICULTURAL TECHNOLOGY

Volume 16, No. 04, Month JULY, Year 2020, Pages 1013 - 1036

Cloning and expression of the antimicrobial peptide from lactobacillus reuteri kub-ac5 and its characterization

Sobanbua, S., Tangthong, J., Suveatwatanakul, A., Nakphaichit, M., Keawsompong, S. and Nitisinprasert, S.

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Lactobacillus reuteri KUB-AC5 was isolated from chicken intestine and played an important role as probiotic in Salmonella growth inhibition and promoting the growth of broiler chicken. Gene coding for antimicrobial peptide from the strain KUB-AC5 (AMP-AC5) and characterization of its product were cloned. Genomic library cloning of gene coding for AMPAC5 based on the size of homogeneous peptide of 4721.95 Dalton purified by amberlite adsorption-desorption, gel filtration chromatography, reversed phase HPLC and cation exchange chromatography, was successfully cloned into pNZ307 and expressed in Escherichia coli DH5α to obtain the recombinant clone E. coli ACE-C46. Its recombinant plasmid pACEC46 was subsequently subcloned into pSIP609/L. plantarum TLG02 to obtain an active recombinant clone L. plantarum ACLP-C46-F2.1 which had recombinant plasmid containing an open reading frame I-C46-F2.1 (153 nucleotides). It deduced amino acid sequence of “YMLYKFLAGLFHTSIDSIYWSVTFIAPALALITYIVCWPDS” (ID number 2253028) which showed no similarity to bacteriocin. The AMP from both the wild type and the recombinant strain exhibited similar characters in stability at wide pH range of 2-9, high temperature up to 121°C and inhibition spectrum against both G+ and G- bacteria but not to lactic acid bacteria including closely related specie of L. reuteri resulting in a potential single AMP produced by the wild type. Furthermore, overexpression of kac5 into the wild type provided the recombinant L. reuteri ACLR-C46-F2.1 which exhibited higher inhibitory activities than the wild type for 1.6 folds. The novel AMP named KAC5 would be promising for food and feed safety uses in the future.

Keywords

Antimicrobial peptide KAC5, Lactobacillus reuteri KUB-AC5, kac5 gene cloning, Food grade vector system

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