Glutamate decarboxylase, amylise and proteinase from selected Lactobacillus and Enterococcus strains

Abstract

Totally 116 strains of lactic acid bacteria isolated from various sources in Thailand were screened for glutamate decarboxylase, amylase, and/or proteinase abilities. Four rod-shaped of γ-aminobutyric acid (GABA) producing bacteria, two rod-shaped and three cocci in chains of starch hydrolyzing bacteria including two rod-shaped of proteinase producing bacteria were isolated. On the basis of the phenotypic characteristics, rpoA, pheS and 16S rDNA sequencing, phylogenetic analysis, and DNA-DNA similarity, the GABA producing strains, LSF8-13 was identified as Lactobacillus brevis, FS73-1, SEA62-2 and SR11-2 were identified as L. plantarum. The rod-shaped starch hydrolyzing strains SB2-3 and U3-1 were identified as L. plantarum. The starch hydrolyzing cocci in chains, FP 15-1 and N2-1A were the novel species in Enterococcus and N12-9 was E. gallinarum. The strain FP15-1 isolated from fermented tea leaves is proposed as Enterococcus camelliae sp. nov. The proteinase producing strains, SMC1 and SCR1 were E. faecalis and L. sakei respectively. A high GABA producing lactobacilli L13 isolated from a traditional Japanese pickle (senmaizuke) was polyphasic studied. This strain is belonged to a new species in Lactobacillus. Glutamate decarboxylase (GAD) encoding gene (gadB) of the strains L13, LSF8-13, FS73-1, SEA62-2 and SR11-2 were subjected to be comparatively studied. The core fragments of gadB were isolated from the isolates, using primers based on highly conserved regions of predicted gadB from genomic sequence of L. brevis ATCC367 and L. plantarum WCFS1. A full-length gadB of of the strain L13 was subsequently isolated by TAIL-PCR method. Nucleotide sequence analysis of L13 gadB revealed that the open reading frame (ORF) consisted of 1437 bases and encoded a protein of 479 amino acid residuse. The deduced amino acid showed 82.2, 51.5, and 51.3% identity to the predicted GAD of L. brevis ATCC 367, L. plantarum WCFS1 and that of the isolated LSF 8-13 gadB, respectively. The isolated gadB of the strain L13 and LSF8-13 was constructed in an expression vector pQE70, cloned and expressed in E. coli JM109. The expressed GADs in crude extract of the transformed E. coli were purified by Ni²⁺-HiTrap chelating HP affinity column. The purified GAD showed a single protein band on SDS-PAGE. The moledular weight of the purified L13 and LSF8-13 GADs were approximately 59 kDa. The recombinant GAD of L13 showed GAD activity significantly depended on a present of PLP in enzyme reaction while that of LSF8-13 had no activity. Deduced amino acids and 3D structures were comparatively analyzed. Addressed here, a novel glutamate decarboxylase gene from the novel species L13 was successfully identified, cloned, and expressed.