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Some promoters were isolated and characterized from the genome of Leuconostoc mesenteroides SY2, an isolate from kimchi, a Korean traditional fermented vegetable. Chromosomal DNA of L. mesenteroides SY2 was digested with Sau3AI and ligated with BamHI-cut pBV5030, a promoter screening vector containing a promoterless cat-86 . Among E. coli transformants (TFs) resistant against Cm (chloramphenicol), 17 were able to grow in the presence of 1,000 μg/ml Cm and their inserts were sequenced. Transcription start sites were examined for three putative promoters (P04C, P25C, and P33C) by primer extension. Four putative promoters were inserted upstream of a promoterless α-amylase reporter gene in pJY15α. α-Amylase activities of E. coli TFs containing pJY15α (control, no promoter), pJY03α (pJY15α with P03C), pJY04α (with P04C), pJY25α (with P25C), and pJY33α (with P33C) were 66.9, 78.7, 122.1, 70.8, and 99.3 U, respectively. Cells harboring pJY04α showed 1.8 times higher activity than the control. Some promoters characterized in this study might be useful for construction of foodgrade expression vectors for Leuconostoc sp. and related lactic acid bacteria.

A gamma-aminobutyric acid (GABA)-producing microorganism was isolated from jeot-gal (anchovy), a Korean fermented seafood. The isolate, A156, produced GABA profusely when incubated in MRS broth with monosodium glutamate (3% (w/v)) at 37°C for 48 h. A156 was identified as Lactobacillus sakei by 16S rRNA gene sequencing. The GABA conversion yield was 86% as determined by GABase enzyme assay. The gadB gene encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was located immediately upstream of gadB. The operon structure of gadCB was confirmed by RT-PCR. gadB was overexpressed in Escherichia coli BL21(DE3) and recombinant GAD was purified. The purified GAD was 54.4 kDa in size by SDS-PAGE. Maximum GAD activity was observed at pH 5.0 and 55°C and the activity was dependent on pyridoxal 5'-phosphate. The Km and Vmax of GAD were 0.045 mM and 0.011 mM/min, respectively, when glutamate was used as the substrate.