Ja-Young Moon

Inhibition of 7-ethoxycoumarin O-deethylase activity in rat liver microsomes by naturally occurring flavonoids: structure-activity relationships

1. The inhibitory effects of several naturally occurring flavonoids and related compounds on cytochrome P450-dependent 7-ethoxycoumarin O-deethylase (ECOD) and the structure-activity relationships were studied in liver microsomes from rats treated with 3-methylcholanthrene (MC). 2. All the flavonoids (flavone, apigenin, chrysin, flavonol, fisetin, kaempferol, morin, myrisetin, quercetin, flavanone, hesperetin and naringenin) studied inhibited microsomal ECOD activity in the following order: flavones > flavonols > flavanones, were mixed type inhibitors and had Ki in the range of 0.17-4.5 microM. (+/-)-Catechin had no effect. 3. The double bond between C2 and C3 of the C ring, the keto group and hydroxyl group of this ring in the flavonoids seem to play major roles in inhibiting the ECOD activity. 4. The hydroxyl groups in the C5 and C7 positions of A ring in the flavone and the hydroxyl group in the C3 position of C ring in the flavonol classes, respectively, were important factors for the inhibition of the enzyme. 5. In a series of 3, 5, 7-trihydroxyflavones, the hydroxyl group at the C4 in the B ring was also an important factor for the inhibition of ECOD activity, but hydroxyl groups in other positions of the B ring had little effect on the inhibition. 6. We conclude that all the flavonoids studied inhibit ECOD activity by interfering with the binding of substrate to the active site and other site(s) of the enzyme and that their structural differences lead to different binding affinities at the active site and possibly to binding at other site(s) of the enzyme for the flavonoids.

Characterization and Purification of the Bacteriocin Produced by Bacillus licheniformis Isolated from Soybean Sauce

A bacteriocin-producing bacterium identified as Bacillus licheniformis was isolated from soybean sauce. Antibacterial activity was confirmed by paper disc diffusion method, using Micrococcus luteus as a test organism. The bacteriocin also showed antibacterial activities against Bacillus sphaericus, Lactobacillus bulgaricus, Lactobacillus plantarum, Paenibacillus polymyxa, and Pediococcus dextrinicus. Optimal culture conditions for the production of bacteriocin was attained by growing the cells in an MRS medium at a pH of 6.5～7.0 and a temperature of 37℃ for 36～48 hr. Solvents such as chloroform, ethanol, acetone, and acetonitrile had little effect on bacteriocin activity. However, about 50% of bacteriocin activity diminished with treatment of methanol and isopropanol at the final concentration of 50% at 25℃ for 1 hr. It was stable against a pH variation range from 3.0 and 7.0, but the activity reduced to 50% at a pH range from 9.0 to 11.0. Its activity was not affected by heat treatment at 100oC for 30 min and 50% of activity was retained after heat treatment at 100℃ for 60 min, showing high thermostability. The bacteriocin was purified to a homogeneity through ammonium sulfate precipitation, SP-Sepharose ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (HPLC). The entire purification protocol led to a 75-fold increase in specific activity and a 13.5% yield of bacteriocin activity. The molecular weight of purified bacteriocin was estimated to be about 2.5 kDa by tricine-SDS-PAGE.Characterization and Purification of the Bacteriocin Produced by Bacillus licheniformis Isolated from Soybean Sauce. Sung-Sub Jung 1 , Jung-I Choi 1 , Woo-Hong Joo 2 , Hyun-Hyo Suh 3 , Ae-Sil Na, Yong-Kweon Cho, Ja-Young Moon, Kwon-Chul Ha, Do-Hyeon Paik and Dae-Ook Kang*. Department of Biochemistry and Health Science, Changwon National University, Changwon 641-773, Korea, Interdisciplinary Program in Biotechnology, Graduate School, Changwon National University, Department of Biology, Changwon National University, Department of Environmental Engineering, Jinju National University, Jinju 660-758, Korea A bacteriocin-producing bacterium identified as Bacillus licheniformis was isolated from soybean sauce. Antibacterial activity was confirmed by paper disc diffusion method, using Micrococcus luteus as a test organism. The bacteriocin also showed antibacterial activities against Bacillus sphaericus, Lactobacillus bulgaricus, Lactobacillus plantarum, Paenibacillus polymyxa, and Pediococcus dextrinicus. Optimal culture conditions for the production of bacteriocin was attained by growing the cells in an MRS medium at a pH of 6.5~7.0 and a temperature of 37 o C for 36~48 hr. Solvents such as chloroform, ethanol, acetone, and acetonitrile had little effect on bacteriocin activity. However, about 50% of bacteriocin activity diminished with treatment of methanol and isopropanol at the final concentration of 50% at 25 o C for 1 hr. It was stable against a pH variation range from 3.0 and 7.0, but the activity reduced to 50% at a pH range from 9.0 to 11.0. Its activity was not affected by heat treatment at 100 o C for 30 min and 50% of activity was retained after heat treatment at 100 o C for 60 min, showing high thermostability. The bacteriocin was purified to a homogeneity through ammonium sulfate precipitation, SP-Sepharose ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (HPLC). The entire purification protocol led to a 75-fold increase in specific activity and a 13.5% yield of bacteriocin activity. The molecular weight of purified bacteriocin was estimated to be about 2.5 kDa by tricine-SDS-PAGE.

Characterization of Bacteriocin Produced from Isolated Strain of Bacillus sp.

As an effort to find a potential biopreservative, we isolated bacterial strains producing bacteriocin from fermented foods. A strain was finally selected and characteristics of the bacteriocin were investigated. The selected strain was identified as Bacillus subtilis E9-1 based on the 16S rRNA gene analysis. The culture supernatant of B. subtilis E9-1 showed antimicrobial activity against Gram-positive bacteria. Subtilisin A, α-chymotrypsin, trypsin and proteinase K inactivated the antimicrobial activity, which means its proteinaceous nature, a bacteriocin. The bacteriocin activity was fully retained at the pH range from 2.0 to 8.0 and stable at up to 100°C for 60 min. Solvents such as ethanol, isopropanol and methanol had no effect on the antimicrobial activity at the concentration of 100% but acetone and acetonitrile reduced the activity at up to 100% concentration. Cell growth of four indicator strains was dramatically decreased in dose-dependent manner. Listeria monocytogenes was the most sensitive, but Enterococcus faecium was the most resistant. Bacillus cereus and Staphylococcus aureus showed the medium sensitivity. The bacteriocin showed its antimicrobial activity against B. cereus and L. monocytogenes via bactericidal action. The number of viable cells of L. monocytogenes started to reduce after addition of bacteriocin to the minced beef. The bacteriocin was purified through acetone concentration, gel filtration chromatography and RP-HPLC. The whole purification step led to a 6.82 fold increase in the specific activity and 6% yield of bacteriocin activity. The molecular weight of the purified bacteriocin was determined to be 3.3 kDa by MALDI-TOF/TOF mass spectrometry.

Prototypes of Panaxadiol and Panaxatriol Saponins Suppress LPS-mediated iNOS/NO Production in RAW264.7 Murine Macrophage Cells

This study was performed to investigate the modulatory effects of two prototypes of Panax ginseng saponin fractions, 20(S)-protopanaxadiol saponins (PDS) and 20(S)-protopanaxatriol saponins (PTS), on the induction of inflammatory mediators in lipopolysaccharide (LPS)-treated RAW264.7 murine macrophage cells. For this purpose, RAW264.7 cells were treated with LPS (10 μg/ml) before, after, or simultaneously with PDS or PTS (150 μg/ml), and the released level of nitric oxide (NO) and expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were evaluated. When RAW264.7 cells were treated with LPS and ginseng saponin fractions simultaneously for 24 hr, PTS, compared to PDS, more strongly attenuated the NO production induced by LPS treatment. When the cells were pretreated with LPS for 2 hr followed by PDS or PTS treatment for 24 hr, both ginseng saponins strongly reduced NO release. The pretreatment of RAW264.7 cells with PDS or PTS for 2 hr followed by LPS treatment for 24 hr significantly attenuated the LPS-induced production of NO. PTS showed stronger inhibitory potency to NO generation than PDS. Our western blot experiment showed that both PDS and PTS (150 μg/ml) also significantly down-regulated the expressions of iNOS and COX-2 induced by LPS treatment. Our results suggest that both PDS and PTS possess strong protective effects against LPS-stimulated inflammation and that their protective effects are mediated by the suppression of NO synthesis via down-regulation of pro-inflammatory enzymes, iNOS, and COX-2 in the RAW264.7 cells.

Purification and Characterization of the Bacteriocin Produced by Lactococcus sp. KD 28 Isolated from Kimchi

The bacterial strain isolated from Kimchi showed antibacterial activity against Micrococcus luteus IAM 1056. The selected strain was identified as Lactococcus lactis by 16S rRNA nucleotide sequence analysis and named as Lactococcus sp. KD 28. The treatment of culture supernatant with proteinase K removed antibacterial activity, indicating its proteinaceous nature, a bacteriocin. This bacteriocin was sensitive to hydrolytic enzymes such as α-chymotrypsion, trypsin, proteinase K, lipase, α-amylase and subtilisin A. The bacteriocin was highly thermostable and resistant to heating at 80°C for up to an hour but 50 % of the total activity was remained at 100°C for 30 min. The pH range from 2.0 to 8.0 had no effect on bacteriocin activity and it was not affected by solvents such as acetonitrile, isopropanol, methanol, chloroform and acetone up to 50% concentration. The bacteriocin showed antibacterial activity against M. luteus IAM 1056, Lactobacillus delbrueckii subsp. lactis KCTC 1058, Enterococcus faecium KCTC 3095, Bacillus cereus KCTC 1013, B. subtilis KCTC 1023, Listeria ivanovii subsp. ivanovii KCTC 3444, Staphylococcus aureus subsp. aureus KCTC 1916, B. megaterium KCTC 1098 and B. sphaericus KCTC 1184. The bacteriocin was purified through ammonium sulfate concentration, SP-Sepharose chromatography and RP-HPLC. The molecular weight was estimated to be about 3.4 kDa by tricine-SDS-PAGE analysis.The bacterial strain isolated from Kimchi showed antibacterial activity against Micrococcus luteus IAM 1056. The selected strain was identified as Lactococcus lactis by 16S rRNA nucleotide sequence analysis and named as Lactococcus sp. KD 28. The treatment of culture supernatant with proteinase K removed antibacterial activity, indicating its proteinaceous nature, a bacteriocin. This bacteriocin was sensitive to hydrolytic enzymes such as α-chymotrypsion, trypsin, proteinase K, lipase, α-amylase and subtilisin A. The bacteriocin was highly thermostable and resistant to heating at 80℃ for up to an hour but 50 % of the total activity was remained at 100℃ for 30 min. The pH range from 2.0 to 8.0 had no effect on bacteriocin activity and it was not affected by solvents such as acetonitrile, isopropanol, methanol, chloroform and acetone up to 50% concentration. The bacteriocin showed antibacterial activity against M. luteus IAM 1056, Lactobacillus delbrueckii subsp. lactis KCTC 1058, Enterococcus faecium KCTC 3095, Bacillus cereus KCTC 1013, B. subtilis KCTC 1023, Listeria ivanovii subsp. ivanovii KCTC 3444, Staphylococcus aureus subsp. aureus KCTC 1916, B. megaterium KCTC 1098 and B. sphaericus KCTC 1184. The bacteriocin was purified through ammonium sulfate concentration, SP-Sepharose chromatography and RP-HPLC. The molecular weight was estimated to be about 3.4 kDa by tricine-SDS-PAGE analysis.

Optimization of a Medium for the Production of Cellulase by Bacillus subtilis NC1 Using Response Surface Methodology

Hee-Jong Yang 1 , Chang-Su Park 2 , Ho-Yeon Yang 3 , Su-Ji Jeong 1 , Seong-Yeop Jeong 1 , Do-Youn Jeong 1 , Dae-Ook Kang, Ja-Young Moon and Nack-Shick Choi* Microbial Institute for Fermentation Industry (MIFI), Sunchang 595-804, Korea 2 Department of Food Science and Technology, Catholic University of Daegu, Hayang 712-702, Korea RealBioTech Co., Ltd., Gongju-si, Chungnam 314-853, Korea Department of Biochemistry and Health Science, Changwon National University, Changwon 641-773, Korea

The Signal Sequence of Sporulation-Specific Glucoamylase Directs the Secretion of Bacterial Endo-1,4-β-D-Glucanase in Yeast

The sporulation-specific glucoamylase (SGA) of Saccharomyces diastaticus is known to be produced in the cytoplasm during sporulation. For the purpose of proving that SGA has secretory potential, we constructed a hybrid plasmid, pYESC25, containing the promoter and the putative signal sequence of the SGA fused in frame to the endo-1,4- -D-glucanase (CMCase) gene of Bacillus subtilis without its own signal sequence. The recipient yeast strain of S. diastaticus YIY345 was transformed with the hybrid plasmid. CMCase secretion from S. diastaticus harboring pYESC25 into culture medium was confirmed by the formation of yellowish halos around transformants after staining with Congo red on a CMC agar plate. The transformant culture was fractionated to the extracellular, periplasmic, and intracellular fraction, followed by the measurement of CMCase activity. About 63% and 13% enzyme activity were detected in the culture supernatant (extracellular fraction) and periplasmic fraction, respectively. Furthermore, ConA-Sepharose chromatography, native gel electrophoresis, and activity staining revealed that CMCase produced in yeast was glycosylated and its molecular weight was larger than that of the unglycosylated form from B. subtilis. Taking these findings together, SGA has the potential of secretion to culture medium, and the putative signal sequence of SGA can efficiently direct bacterial CMCase to the yeast secretion pathway.

Study on Pandoraea sp. BCNU 315 Isolated from Soil

Bacteria that antagonize plant pathogenic fungi were isolated from the sediment soil at the Ansan industrial estate. One isolate of them showed growth inhibition of Rhizoctonia solani, Botrytis cenerea, and Fusarium oxysporum. This strain was identified as Pandoraea sp. based on phenotypic and phylogenetic characteristics and termed Pandoraea sp. BCNU 315. Tryptone as nitrogen source and sucrose as carbon source were found to be most effective for the microbial growth. In addition, the optimum temperature and pH for microbial growth were and pH 7.0, respectively. The substances generated from Pandoraea sp. BCNU 315 were purified and analyzed by column chromatography, HPLC, GC-MS and NMR. As a result, one compound was determined to be indole, another compound was predicted as cyclopentadecaheptene. Detailed structural clarification of the all of the rest six compounds from Pandoraea sp. BCNU 315 has to be accompanied in the further studies.