Antibacterial activity of a cell wall hydrolase from Lactobacillus paracasei NRRL B-50314 produced by recombinant Bacillus megaterium.

ABSTRACT

The cell-free supernatant (CFS) from Lactobacillus paracasei NRRL B-50314 culture has been previously reported as containing antibacterial activity against a wide variety of Gram-positive bacteria. The CFS protein gel slice corresponding to antibacterial activities was subjected to trypsin digestion and ion trap MASS (Gel/LC-MS/MS) analysis. BlastP search of the resulted IQAVISIAEQQIGKP sequence led to a hypothetical cell-wall associated hydrolase (designated as CWH here) from Lactobacillus paracasei ATCC 25302. Further analyses of CWH revealed that the IQAVISIAEQQIGKP belongs to a highly conserved region of the NlpC/P60 superfamily. The L. paracasei NRRL B-50314 CWH gene, cloned in pStrepHIS1525CWH477, was introduced into Bacillus megaterium MS 941. The production of CWH477 protein was induced by xylose. The CWH477 protein was purified by using NiNTA column, and elution fraction E2 showed highest antibacterial activity. This study and bioinformatics analyses suggested that the antibacterial activity of CWH could originate from its cell wall degrading enzymatic function.