• Journal of Microbiology and Biotechnology
• /
• v.17 no.5
• /
• pp.822-829
• /
• 2007

A gene coding for phosphoketolase, a key enzyme of carbohydrate catabolism in heterofermentative lactic acid bacteria(LAB), was cloned from a Lactobacillus paraplantarum C7 and expressed in Escherichia coli. The gene is 2,502 bp long and codes for a 788-amino-acids polypeptide with a molecular mass of 88.7 kDa. A Shine-Dalgarno sequence(aaggag) and an inverted-repeat terminator sequence are located upstream and downstream of the phosphoketolase gene, respectively. The gene exhibits an identity of >52% with phosphoketolases of other LAB. The phosphoketolase of Lb. paraplantarum C7(LBPK) contains several highly conserved phosphoketolase signature regions and typical thiamine pyrophosphate(TPP) binding sites, as reported for other TPP-dependent enzymes. The phosphoketolase gene was fused to a glutathione S-transferase(GST::LBPK) gene for purification. The GST::LBPK fusion protein was detected in the soluble fraction of a recombinant Escherichia coli BL21. The GST::LBPK fusion protein was purified with a yield of 4.32mg/400ml by GSTrap HP affinity column chromatography and analyzed by N-terminal sequencing. LBPK was obtained by factor Xa treatment of fusion protein and the final yield was 3.78mg/400ml. LBPK was examined for its N-terminal sequence and phosphoketolase activity. The $K_M\;and\;V_{max}$ values for fructose-6-phosphate were $5.08{\pm}0.057mM(mean{\pm}SD)$ and $499.21{\pm}4.33{\mu}mol/min/mg$, respectively, and the optimum temperature and pH for the production of acetyl phosphate were $45^{\circ}C$ and 7.0, respectively.

• Microbiology and Biotechnology Letters
• /
• v.21 no.1
• /
• pp.82-87
• /
• 1993

Fourteen samples of kimchies and soy bean pastes were used to isolate strictly anaerobic bacteria on complex BL agar and on a selective BS agar for bifidus bacteria. About $10^7$ ~ $10^8$ colonies per g sample were developed on BL agar under strictly anaerobic conditions, while BS agar supported the growth of $10^3$ ~ $10^6$ colonies per gram sample at the same condition. All colonies developed on BS agar at anaerobic conditions grew in aerobic conditions and did not show fructose6-phosphate phosphoketolase activity. Type cultures of Bifidobacterium did not grow in PYG medium containig more than 3% NaCI. From these results it is conduded that salted fermented food cannot support the growth of strictly anaerobes induding Bifidobactenum.

• Proceedings of the Korean Society for Food Science of Animal Resources Conference
• /
• 2005.10a
• /
• pp.283-285
• /
• 2005

• YAKHAK HOEJI
• /
• v.37 no.5
• /
• pp.483-489
• /
• 1993

Bifidobacterium bifidum, one strain of medical preparations being on the market for human intestinal disorder, is very sensitive to rifampicin. If this preparation is taken with rifampicin, its therapeutic effect can't be expected. To develope rifampicin resistant mutants, B. bifidum was treated with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG). All of thirty strains grown on the plates containing 10 $\mu\textrm{g}$/ml rifampicin were over 1, 000 times more resistant to rifampicin than parental strain and they were identified as B. bifidum by fructose-6-phosphoate phosphoketolase test. Three strains out of thirty, which produced almost same amount of organic acid as parental strain, were selected for further studies. They showed identical growth inhibition activity aganist E. coli compared with that of parental strain. And rifampicin was not inactivated.

• YAKHAK HOEJI
• /
• v.48 no.1
• /
• pp.20-26
• /
• 2004

Twelve strains of Bifidobacterium spp. were isolated from the feces of healthy Korean 20∼30 years. The identification of genera from isolates were performed by the microscopic observation and fructose-6-phosphate phosphoketolase (F6PPK) activity which is the key enzyme to distinguish the Bifidobacterium spp. from other anaerobic bacteria. To determine the antibacterial resistance patterns, minimum inhibitory concentration (MIC) of several antibiotics (including anti-tuberculosis agents) was determined. Five of the isolate!, showed the high degree of resistance to vancomycin. To investigate the genetic diversity between isolates and type strain of Bifidobacterium spp. from KCTC, we peformed the RAPD-fingerprinting. Using a total set of four primers, it is possible to distinguish the isolates and Bifidobacterium spp. from KCTC. Thus, Bifidobacterium strains isolated from our samples may be a new species or strains of Bifidobacteriurn genera, and have the potential as a probiotics.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1993.04a
• /
• pp.143-143
• /
• 1993

현재 시판되고 있는 정장용 생균 제제에 함유되어있는 정장 균주의 하나인 Bifidobacterium bifidum은 항결핵제 중 rifampicin에 감수성으로 rifampicin과 병용 투여시 본래의 정장 효과를 기대할 수없다. 따라서, rifampicin에 내성인 돌연변이 균주를 얻기 위해 B. bifidum을 N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)로 처리하여 rifampicin에 내성인 30 종의 균주를 선별하였고, rifampicin에 대한 Minimal Inhibitory Concentration (MIC)를 측정해 본 결과 내성이 1,000 배 이상 상승하였다. 균주 동정을 위하여 fructose-6-phosphate phosphoketolase test를 실시해 본 결과 Bifidobacterium임이 확인되었다. 이들 내성 균주들의 유기산 생산량을 측정하여 그 생산량이 모균주와 가장 유사한 3 종의 균주를 선발하였다. 이들에 대하여 Escherichia coli 생육 억제능을 시험해 본 결과 E. coil 생육 억제능이 모균주와 유사하였다. 또, rifampicin을 함유한 배지에서 돌연변이 균주를 배양시킨 경우 rifampicin이 안정한 상태로 잔존한 것을 알 수 있었다. 이것으로 보아 돌연변이 균주들은 rifampicin을 분해 또는 변형시키는 효소를 생산하지 않는다고 볼 수 있다. 이상의 결과로 본 연구에서 개발한 돌연변이 균주들, 즉 B. bifidum RFRll, RFR21 그리고 RFR61은 rifampicin에 내성이면서 모균주와 동일한 생화학적 특성을 갖는 정장 균주로 여겨진다.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1994.04a
• /
• pp.252-252
• /
• 1994

현재 시판되고 있는 정장용 생균 제제에 함유되어있는 정장균주의 하나인 Bifidohacterium bifidum은 항결핵제 중 rifampicin에 감수성으로 rifimpicin과 병용 투여시 본래의 정장 효과를 기대할 수 없다. 따라서, rifampicin에 내성인 돌연변이 균주를 얻기 위해 B. bifidum을 N-methyl-N'-nitro-N-nitroso- -guanidine(MNNG)로 처리하여 rifampicin에 내성인 30종의 균주를 선별 하였고, rifampicin에 대한 Minimal Inhibitory Concentration(MIC)를 측정해 본 결과 내성이 1,000배 이상 상승하였다. 또한 rifampicin에 내성인 균주 RFR61을 자연 돌연변이시켜 ofloxacin에도 내성인 돌연변이 균주 20종을 선별 하였고, MIC를 측정한 결과 내성이 4배 이상 증가하였다. 또, fructose-6-phosphate phosphoketolase test를 실시해 본 결과, 모두 Bifidobacterium임이 확인되었다. 유기산 생성량을 측정하여 모균주의 유기산 생성량과 가장 유사한 3균주, B. bifidum RFRll, RFR21, RFR61 그리고 OFR9을 선별하였다. 이 네 균주의 E. coli 생육 억제능을 측정한 결과 모두 모균주와 유사한 E. coli 생육 억제능을 가지고 있었다. Rifampicin 내성균주들에 대하여 내성 유지 시험을 한 결과 복귀 돌연변이에 의해 내성이 소실될 가능성은 없는 것으로 여겨진다. 마지막으로, 내성 균주에 의한 rifampicin 불활성화 여부를 알아 본 결과 rifampicin이 불활성 화되지 않음을 알 수 있었다. 이상의 결과를 통해 본 연구실에서 개발한 B. bifidum RFR11, RFR21 그리고 RFR61 균주들은 rifampicin에 내성이며, B.bifidum OFR9은 rifampicin과 ofloxacin에 이중 내성을 갖는 균주로서 모균주와 유사한 생화학적 특성을 갖는 우수한 정장 세균으로 여겨진다.

• Journal of Microbiology and Biotechnology
• /
• v.14 no.5
• /
• pp.919-923
• /
• 2004

The sk10 isolated from kimchi was identified as W. kimchii on the basis of l6s-rDNA sequencing. Studies were made to analyze the metabolic flux shift of the sk10 on glucose under aerobic growth conditions. The sk10 produced 38.2 mM acetate, 16.3 mM ethanol, and 33.2 mM lactate under aerobic conditions, but 2.4 mM acetate, 48.0 mM ethanol, and 44.1 mM lactate under anaerobic conditions. The NADH peroxidase (NADH-dependent hydrogen peroxidase) activity of sk10 grown under aerobic conditions was 11 times higher than that under anaerobic conditions. Under the low ratio of $NADH/NAD^+$, the metabolic flux toward lactate and ethanol was shifted to the flux through acetate kinase without NADH oxidation. The kinds of enzymes and metabolites of sk10 were close to those in the pathway of Leuconostoc sp., but the metabolites produced under aerobic growth conditions were different from those of Leuconostoc sp. The stoichiometric balance calculated using the concentrations of metabolites and substrate was about 97%, coincident with the theoretical values under both aerobic and anaerobic conditions. From these results, it was concluded that the metabolic flux of W. kimchii sk10 was partially shifted from lactate and ethanol to acetate under aerobic conditions only.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.2
• /
• pp.388-394
• /
• 2005

In order to investigate the distribution of dominant Bifidobacterium species in intestinal microflora of Korean adults and seniors, SDS-PAGE profiles of whole cell proteins were used for the identification of bifidobacteria. To confirm the reliability of SDS-PAGE, the Bifidobacterium species identified by SDS-PAGE of whole cell proteins were validated by using 16S rDNA sequencing analysis. The results of SDS­PAGE corresponded well with those determined by the analysis of 16S rDNA sequencing. Based on the analysis of SDS-PAGE patterns on unidentified fecal strains which showed positive in fructose-6-phosphate phosphoketolase activity, B. adolescentis, B. longum, and B. bifidum were identified in the feces of adults, and B. adolescentis, B. longum, B. bifidum, B. breve, and B. dentium were identified in those of seniors. In most of the fecal samples tested, the predominant Bifidobacterium species consisted of only a few species, and differences in the distribution and numbers of Bifidobacterium species were observed between adults and seniors. B. adolescentis and B. longum were found to be the most common species in feces of adults, but not in seniors. Accordingly, the distribution and abundance of bifidobacteria in the human intestinal microflora varied depending on the age of hosts.

• Food Science of Animal Resources
• /
• v.38 no.4
• /
• pp.806-815
• /
• 2018

This study was performed to isolate some strains of Bifidobacterium breve from fecal materials of neonates and to screen them for the biotransformation activity of converting linoleic acid into conjugated linoleic acid (CLA). Fecal samples were collected from twenty healthy neonates between 14 and 100 days old, and four hundred colonies were randomly selected from a Bifidobacterium selective transoligosaccharide medium. A duplex polymerase chain reaction technique was developed for the rapid and accurate molecular characterization of the B. breve strains that have been reported to show the species-specific characteristic of CLA production. They are identified by 16S ribosomal DNA, fructose-6-phosphate phosphoketolase encoding genes (xfp), and rapid pulsed field gel electrophoresis. Thirty-six isolates were identified as B. breve, and just two of the 12 neonates were harboring B. breve strains. Each isolate showed different CLA-producing ability in the spectrophotometric assay. All of the positive strains from the primary spectrophotometric assay were confirmed for their CLA-producing activities using gas-chromatographic analysis, and their conversion rates were different, depending on the strain isolated in this study. Some strains of B. breve were successfully isolated and characterized based on the CLA-producing activity, and further studies are necessary to characterize the enzyme and the gene responsible for the enzyme activity.