Search

The aprE2 gene with its prosequence from Bacillus subtilis CH3-5 was overexpressed in Escherichia coli BL21(DE3) by using plasmid pET26b(+). After IPTG induction, active and mature AprE2 was produced when cells were grown at 20°C, whereas inactive and insoluble enzyme was produced in a large amount when cells were grown at 37°C. The insoluble fraction was resuspended with 6 M guanidine-HCl and dialyzed against 2 M Tris-HCl (pH 7.0) or 0.5 M sodium acetate (pH 7.0) buffer. Then active AprE2 was regenerated and purified by a Ni-NTA column. Purified AprE2 from the soluble fraction had a specific activity of 1,069.4 ± 42.4 U/mg protein, higher than that from the renatured insoluble fraction. However, more active AprE2 was obtained by renaturation of the insoluble fraction. AprE2 was most stable at pH 7 and 40°C, respectively. The fibrinolytic activity of AprE2 was inhibited by PMSF, but not by EDTA and metal ions. AprE2 degraded Aα and Bβ chains of fibrinogen quickly, but not the γ-chain. AprE2 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-pNA. The Km and kcat/Km of AprE2 was 0.56 mM and 3.10 × 10(4) S(-1) M(-1), respectively.

A 4661 bp cryptic plasmid, pFMBL1, was isolated from Leuconostoc mesenteroides SY2, an isolate from Kimchi, and characterized. Nucleotide sequence analysis revealed two open reading frames, orf1 and orf2. orf2 was 453 bp in size and its translation product had 58% identity with a putative protein possibly involved in the replication of pTXL1, a cryptic plasmid from L. mesenteroides ssp. mesenteroides Y110. RNA transcript from orf2 was detected but not from orf1 or intergenic region. Minimum 3.5 kb fragment encompassing orf1 and orf2 was required for the replication of pFMBL1 and employed for the construction of Escherichia coli-Leuconostoc shuttle vector, pSJ33E. L. mesenteroides SY1 (another Kimchi isolate), Leuconostoc ssp., and Lactobacillus brevis were successfully transformed with pSJ33E, and the transformation efficiencies were ranged between 1.1 × 101 and 4 × 105 transformants/μg DNA. No single-stranded DNA intermediate was detected from L. mesenteroides SY1 cells harboring pSJ33E, indicating that pFMBL1 probably replicated via θ-type mechanism. pSJ33E was stably maintained in L. mesenteroides SY1 in the absence of erythromycin (Em, 5 μg/ml) and after 1 month of daily subculturing in MRS broth without selective pressure, three percent of cells still retained pSJ33E.

Lactic acid bacteria (LAB) were isolated from Kimchi, a Korean traditional fermented vegetable food. LAB accumulating GABA (γ-aminobutyric acid) in the culture media were screened by TLC analysis. One isolate, GU240, produced the highest amount of GABA among the 3,000 isolates and identified as a Lactobacillus zymae strain. Glutamate decarboxylase (GAD) gene was cloned and over-expressed in E. coli BL21(DE3) using pET26b(+). The recombinant GAD was purified by using a Ni–NTA column. Its size was 53 kDa by SDS-PAGE. Maximum GAD activity was at pH 4.5 and 41 °C and the activity was dependent on pyridoxal 5′-phosphate. Km and Vmax of LzGAD were 1.7 mM and 0.01 mM/min, respectively, when glutamate was used as a substrate.

Bacillus subtilis CH3-5 was isolated from cheonggukjang prepared according to traditional methods. CH3-5 secreted at least four different fibrinolytic proteases (63, 47, 29, and 20 kDa) into the culture medium. A fibrinolytic enzyme gene, aprE2, encoding a 29 kDa enzyme was cloned from the genomic DNA of CH3-5, and the DNA sequence determined. aprE2 was overexpressed in heterologous B. subtilis strains deficient in extracellular proteases using a E. coli-Bacillus shuttle vector. A 29 kDa AprE2 band was observed and AprE2 seemed to exhibit higher activities towards fibrin rather than casein.

A Lactobacillus paraplantarum strain producing a bacteriocin was isolated from kimchi using the spot-on-the lawn method and named L. paraplantarum C7. The bacteriocin, paraplantaricin C7, was found to inhibit certain Lactobacillus strains, including L. plantarum, L. pentosus, and L. delbrueckii subsp. lactis. It also inhibited Enterococcus faecalis, yet did not inhibit most of the other LAB (lactic acid bacteria) tested. The maximum level of paraplantaricin C7 activity was observed under the culture conditions of 25 degrees C and a constant pH of 4.5. Paraplantaricin C7 retained 90% of its activity after 10 min of treatment at 100 degrees C and remained stable within a pH range of 2-8. Based on a culture supernatant, paraplantaricin C7 was purified by DEAE-Sephacel column chromatography and C18 reverse-phase HPLC. SDS-PAGE and activity staining were then conducted using the purified paraplantaricin C7, and its molecular mass determined to be about 3,800 Da. The 28 N-terminal amino acids from the purified paraplantaricin C7 were determined, and the structural gene encoding paraplantaricin C7, ppnC7, was cloned by PCR using degenerate primers based on the N-terminal amino acid sequence. The nucleotide sequences for ppnC7 and other neighboring orfs exhibited a limited homology to the previously reported plantaricin operon genes. Paraplantaricin C7 is a novel type II bacteriocin containing a double glycine leader sequence.