• KSBB Journal
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• 제10권3호
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• pp.343-348
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• 1995

LeI은 스테로이드를 통울에 투여하였을 때 분비가 촉진되어 항염증성 효과를 나타내는 calcium 의 존성 phospholipid 결합 단백질이다. S. cerevisiae는 대장균과 통물세포의 장점을 모두 가지고 있으므로 동물세포 유래의 이종 단백질의 분비 생산에 많이 이용되고 있다. 본 연구에서는 GAL10 promoter­p ppL-LCI유전자 LCI terminator로 구성된 pYGLPT5 로 LCI을 S. cerevisiae SEY2102에서 발현 분비시키고 각 분획으로 나누어 LCI양을 비교한 결과 protoplast 68.6 %. periplasmic 24 %, culture supernatant 7.4%로 분포하였다. pYGLPT5로 형질전환된 S. cereviswe 2102를 유가 배양한 결과, 최종적인 LCI의 생산량은 약 $500mg/\ell$ 였다. LCI은 N 말단 부근에 $CA^{++}$ 결합부위가 있으므로 이를 이용하여 hydroxylapatite column chromatography로 정 제하 였다. 배지로 분비된 34kDa LCI을 ultrafiltration 과 hydroxylapatite column chromatography 의 두 단계로 순도 99% 이상으로 정제할 수 있었다.

• 생명과학회지
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• 제6권2호
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• pp.79-86
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• 1996

A bacterium producing pullulanase was from soil, and was identified Bacillus cereus and named as Bacillus cereus JK36. The optimal culture conditions for the efficident production of pullulanase from B. cereus JK36 was obtained by cultivating with the medium composed of 1% pullulan, 1% teast extract, 1% bactopeptone, 0.1% NaH$_{2}$PO$_{4}$, 2H$_{2}$O, 0.02% MgSO$_{4}$\ulcorner7H$_{2}$O at 40$\circ$C, initial pH 6.5 for 70 hours. Using the culture supernatant as crude enzyme, the optimal pH and temperature of the pullulanase of this strain were 6.5 and 50$\circ$C. In effect of pH and temperature on the stability of the enzyme, the enzyme was stable in the range of pH6.0$\sim$9.5 and up to 40$\circ$C, respectively. The hydrolysis product on pullulan was mainly maltotriose.

• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1513-1517
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• 2006

One single lactic acid producing bacterium, isolated from kimchi, inhibited the growth and adherence of Helicobacter pylori to the human gastric epithelial cell line MKN-45. This isolate was identified as Lactobacillus plantarum and termed L. plantarum strain PL9011. The adherence of H pylori, in the presence of live or nonviable L. plantarum strain PL9011 (10-fold CFU), decreased to 14-20%. The spent culture supernatant of L. plantarum strain PL9011 resulted in the eradication of H pylori. This activity remained stable following neutralization and heat treatment, but not following pepsin treatment, thereby suggesting small peptides as the inhibitory factor. L. plantarum strain PL9011 did not produce any harmful metabolites or enzymes. The results obtained in this study suggest that the L. plantarum strain PL9011 may be a potential novel probiotic for the stomach.

• Asian-Australasian Journal of Animal Sciences
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• 제27권11호
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• pp.1562-1570
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• 2014

The effects of Lactobacillus mucosae (L. mucosae), a potential direct fed microbial previously isolated from the rumen of Korean native goat, on the rumen fermentation profile of brewers grain were evaluated. Fermentation was conducted in serum bottles each containing 1% dry matter (DM) of the test substrate and either no L. mucosae (control), 1% 24 h broth culture of L. mucosae (T1), or 1% inoculation with the cell-free culture supernatant (T2). Each serum bottle was filled anaerobically with 100 mL of buffered rumen fluid and sealed prior to incubation for 0, 6, 12, 24, and 48 h from which fermentation parameters were monitored and the microbial diversity was evaluated. The results revealed that T1 had higher total gas production (65.00 mL) than the control (61.33 mL) and T2 (62.00 mL) (p<0.05) at 48 h. Consequently, T1 had significantly lower pH values (p<0.05) than the other groups at 48 h. Ammonia nitrogen ($NH_3$-N), individual and total volatile fatty acids (VFA) concentration and acetate:propionate ratio were higher in T1 and T2 than the control, but T1 and T2 were comparable for these parameters. Total methane ($CH_4$) production and carbon dioxide ($CO_2$) were highest in T1. The percent DM and organic matter digestibilities were comparable between all groups at all times of incubation. The total bacterial population was significantly higher in T1 (p<0.05) at 24 h, but then decreased to levels comparable to the control and T2 at 48 h. The denaturing gradient gel electrophoresis profile of the total bacterial 16s rRNA showed higher similarity between T1 and T2 at 24 h and between the control and T1 at 48 h. Overall, these results suggest that addition of L. mucosae and cell-free supernatant during the in vitro fermentation of dried brewers grain increases the VFA production, but has no effect on digestibility. The addition of L. mucosae can also increase the total bacterial population, but has no significant effect on the total microbial diversity. However, inoculation of the bacterium may increase $CH_4$ and $CO_2$ in vitro.

• 한국미생물·생명공학회지
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• 제42권4호
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• pp.324-330
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• 2014

한천을 분해하는 해양미생물을 한천을 유일한 탄소원으로 하는 인공 해수 한천 배지를 이용하여 제부도 개펄에서 분리하였다. SH-1으로 명명한 분리된 균주는 그람음성균이며 한 개의 극성 편모를 가지는 균이었다. 16S rRNA 유전자의 염기서열의 유사성 분석 결과 분리된 균주는 Neiella marina J221 [9]과 가장 높은 상동성을 보였다(96.5%). 분리 균주는 $28-37^{\circ}C$에서 생장하였지만 $42^{\circ}C$에서는 생장하지 못하였고 한천분해효소의 활성은 $37^{\circ}C$보다 $28^{\circ}C$에서 높은 활성을 보였다. 또한 SH-1균주는 1-5% NaCl (w/v)를 포함하는 배양액에서 생장이 가능했으며 3%의 농도에서 가장 좋은 생장을 보였고 한천을 분해하는 효소의 활성도 3% 염분농도의 배양액에서 가장 높았다. 48시간 배양한 세포배양액을 농축하여 조효소액을 준비하여 효소의 적정 pH와 적정 온도를 조사한 결과 pH 7.0에서와 $40^{\circ}C$에서 최적 효소 활성을 보였다. 조효소액을 사용하여 zymogram 분석을 실시한 결과 분자량 15, 35, 52 KD 크기의 3개 이상의 한천 분해효소를 생산하는 것으로 보인다. 박막크로마토그라피(TLC) 분석 결과 아가로스를 분해하여 네오올리고당을 생성하는 ${\beta}$-agarase 를 생산하는 것으로 추정된다.

• 미생물학회지
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• 제45권4호
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• pp.362-367
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• 2009

대청호의 한 지류인 소옥천으로부터 고효율 조류억제 세균을 분리하기 위하여 176균주를 분리 스크린하였으며, 이 중 AJ1이 가장 높은 조류성장억제능을 나타내었다(지름 50.0 mm 성장억제환). 조류성장억제능이 높았던 AJ1 균주 동정을 위하여 형태학적, 생리 생화학적, 16S rRNA gene sequence 분석, 지방산 분석을 수행하였으며 그 결과, Pseudomonas aeruginosa 로 판별되었다. AJ1 배양액을 원심분리한 후 상등액을 조류배양액에 첨가 시, 상등액에 의한 조류성장억제능이 나타남에 따라 세포 외 물질 분비에 의한 것을 확인할 수 있었다. 가장 높은 조류성장억 제능[60.2(${\pm}$1.3)%]은 탄소원으로 mannitol을 사용하고, 온도 $30^{\circ}C$, pH 8에서 배양할 때 보였다. 또한, AJ1 균주의 배양기간 및 투여시기에 따른 조류성장억제능 평가 결과, 조류 성장 초기 단계에 조류성장억제균을 투여하였을 때 조류성장억제능이 높게 나타났고, 균주의 배양기간에 따른 조류성장억제능은 연관성이 나타나지 않았다. 상등액 접종량에 따른 조류성장억제능은 상등액의 접종량이 높아질수록 M. aeruginosa의 제거량은 증가하였으며, 고농도(40ml/L)로 적용하였을 때 80.3(${\pm}$8.6)%의 가장 높은 M. aeruginosa 제거효율을 보여주었다. 제거 속도의 경우 상등액 접종량이 낮아질수록 M. aeruginosa 제거율이 높아지는 경향을 확인하였으며, 저농도(10 ml/L)로 적용시 $8.2{\mu}g$ chl-a/supernatant ml/day로 가장 높게 나타났다. 본 연구 결과, AJ1 균주의 현장 적용 시 M. aeruginosa의 제어에 효율적일 것으로 사료된다.

• 대한미생물학회지
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• 제21권1호
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• pp.151-161
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• 1986

In order to develop sensitive and sepcific assay methods for E. coli heat labile enterotoxin(LT) hybridoma cell lines secreting LT specific monoclonal antibody were obtained. LT was purified from cell lysate of E. coli O15H11. The steps included disruption of bacteria by French pressure, DEAE Sephacel ion exchange chromatography, Sephadex G200 gel filtration, and second DEAE Sephacel ion exchange chromatography, successively. Spleen cells from Balb/c mice immunized with the purified LT and $HGPRT^{(-)}$ plasmacytomas, $P3{\times}63Ag8.V653$ were mixed and fused by 50% (w/v) PEG. Hybrid cells were grown in 308 wells out of 360 wells, and 13 wells out of them secreted antibodies reacting to LT. Among these hybridoma cell 1G8-1D1 cell line was selected since it had produced high-titered monoclonal antibody continuously. By using culture supernatant and ascites from 1G8-1D1 cells the monoclonal antibody was characterized, and an assay system for detecting enterotoxigenic E. coli was established by double sandwich enzyme-linked immunosorbent assay (ELISA). The following results were obtained. 1. Antibody titers of culture supernatant and ascites from 1G8-1D1 hybridoma cells were 512, and 102, 400, respectively by GM1-ELISA and its immunoglobulin class was IgM. 2. The maximum absorption ratio of 1G8-1D1 cell culture supernatant to LT was 90% at $300\;{\mu}g/ml$ of LT concentration. LT concentration shown at 50% absorption ratio was $103.45{\mu}g$ and the absorption ratio was decreased with tile reduction of LT concentration. This result suggests that monoclonal antibody from 1G8-1D1 hybridoma cell bound with LT specifically. 3. The reactivities of 1G8-1D1 cell culture supernatant to LT and V. cholerae enterotoxin(CT) were 0.886 and 0.142(O.D. at 492nm) measured by the GM1-ELISA, indicating 1G8-1D1 monoclonal antibody reacted specifically with LT but not with CT. 4. The addition of 0.1ml of ascites to 0.6mg and 0.12mg of LT decreased the vascular permeability factor to 41% and 44% respectively, but it did not completely neutralize LT. 5. By double sandwich ELISA using monoclonal antibody, as little as 75ng of the purified LT per ml could be detected. 6. The results by assay of detecting LT in culture supernatants of 14 wild strains E. coli isolated from diarrhea patients by the double sandwich ELISA were almost the same level as those by reverse passive latex agglutination.

• Journal of Microbiology
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• 제40권1호
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• pp.26-32
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• 2002

From the culture supernatant of the psychrotrophic strain of Bacillus amyloliquefaciens an extracellular serine protease was purified to apparent homogeneity by successive purification steps using QAE-Sephadex, SP-Sephadex and Sephacryl S-100 column chromatography. The pretense is monomeric, with a relative molecular mass of 23,000. It is inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride, but not by EDTA. The enzyme is most active at pH 9-10 and at $45^{\circ}C$, although it is unstable at $60^{\circ}C$.

• 한국식물병리학회지
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• 제11권3호
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• pp.258-264
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• 1995

Production and some properties of chitinolytic enzymes were investigated by 80% ammonium sulfate precipitates (crude enzymes) from culture supernatant of antagonistic bacteria, Chromobacterium violaceum strain C-61 and strain C-72, Aeromonas hydrophila, Aeromonas caviae, and Serratia marcescens. The maximum production of chitinase was obtained from the 3-day culture at 28$^{\circ}C$ in C. violaceum stains, the 6-day culture in S. marcescens, and the 2-day culture in A. hydrophila and A. caviae. In the optimum culture periods, chitinase activity of C. violaceum strains C-61 was 1.5, 5.5, 12.0 and 11.3 times higher than those of strain C-72, S. marcescens, A. hydrophila and A. caviae, respectively. However, N,N'-diacetylchitobiase activity was 3.2 times higher in S. marcescens than in C. violaceum strain C-61, and that of Aeromonas spp.was very low. On gels containing glycol chitin, chitinase of C. violaceum strains showed four isoforms of 54-, 52-, 50- and 37-kDa, whereas there were four isoforms of 58-, 52-, 48- and 38-kDa in S. arcescens, three isoforms of 70-, 58- and 54-kDa in A. hydrophila and six isoforms of 90-, 79-, 71-, 63-, 58- and 38-kDa in A. caviae. The chitinase of C. violaceum strain C-61 was most active at pH 7.0 and at 5$0^{\circ}C$ and was stable in ranges of pH 5.0~10.0 for 2 hours and of 0~5$0^{\circ}C$ for 30 min.

• 한국식품과학회지
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• 제32권2호
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• pp.417-423
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• 2000

김치에서 분리한 Lactobacillus plantarum KLAB21 균주에 있어서 MNNG와 NQO에 대한 항돌연변이원성 물질 생산을 위한 최적 조건을 조사하였다. 탄소원으로 glucose를 첨가시 가장 높은 항돌연변이 효과를 나타내었으며, 질소원으로서는 yeast extract와 bactopeptone첨가시 항돌연변이 효과가 우수하였다. 탄소원으로 glucose의 농도를 2% 첨가시 MNNG와 NQO에 대한 항돌연변이 효과가 가장 우수하였으며, 질소원으로서 yeast extract는 1%와 bactopeptone의 농도는 MNNG인 경우 1%, NQO인 경우 1.5%에서 가장 우수한 항돌연변이 효과를 나타내었다. 항돌연변이원성 물질 생산을 위한 최적 배양 조건은 초기 pH, 배양 온도, 배양 속도가 각각 7.0, $37^{\circ}C$ 및 150 rpm이었다. 상기의 최적 조건에서 36시간 배양시 가장 높은 항돌연변이 효과를 나타내었는데 S. typhimurium TA100과 TA98을 이용한 경우 항돌연변이 효과가 각각 98.4%와 57.3%이었다.