• Journal of Microbiology and Biotechnology
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• 제28권11호
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• pp.1859-1864
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• 2018

Synthesis of flavonoid glycoside is difficult due to diverse hydroxy groups in flavonoids and sugars. As such, enzymatic synthesis or biotransformation is an approach to solve this problem. In this report, we used stepwise biotransformation to synthesize two quercetin bisglycosides (quercetin 3-O-glucuronic acid 7-O-rhamnoside [Q-GR] and quercetin 3-O-arabinose 7-O-rhamnoside [Q-AR]) because quercetin O-rhamnosides contain antiviral activity. Two sequential enzymatic reactions were required to synthesize these flavonoid glycosides. We first synthesized quercetin 3-O-glucuronic acid [Q-G], and quercetin 3-O-arabinose [Q-A] from quercetin using E. coli harboring specific uridine diphopsphate glycosyltransferase (UGT) and genes for UDP-glucuronic acid and UDP-arabinose, respectively. With each quercetin 3-O-glycoside, rhamnosylation using E. coli harboring UGT and the gene for UDP-rhamnose was conducted. This approach resulted in the production of 44.8 mg/l Q-GR and 45.1 mg/l Q-AR. This stepwise synthesis could be applicable to synthesize various natural product derivatives in case that the final yield of product was low due to the multistep reaction in one cell or when sequential synthesis is necessary in order to reduce the synthesis of byproducts.

• Journal of Animal Science and Technology
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• 제44권5호
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• pp.633-642
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• 2002

1. ETEC F41 균주로부터 분리한 섬모항원의 분자량은 29.5 kDa으로 나타났으며, western blot을 통하여 섬모항원임을 확인하였다. 2. 분리한 섬모항원의 농도를 50 ${\mu}g$/$m\ell$, 200 ${\mu}g$/$m\ell$, 600 ${\mu}g$/$m\ell$로 조정 후 산란계에 접종하였다. 이 후 ELISA법을 이용하여 난황의 항체역가를 측정한 결과 최고치가 320,000(antigen 50${\mu}g$/$m\ell$), 450,000(antigen 200${\mu}g$/$m\ell$), 320,000(anti- gen 600${\mu}g$/$m\ell$)으로 나타났다. 3. F41난황항체와 K88, K99, 987P 섬모항원과의 교차반응을 ELISA법을 이용하여 조사해본 결과 난황항체를 30,000배 희석 시 교차반응이 없었다. 4. 실험실조건하에서 난황항체의 항원결합능력을 조사한 결과, 동결건조한 WSF을 2${\sim}$4 mg/$m\ell$ 첨가 시 균체의 농도가 $10^9$ CFU/$m\ell$에서 $10^5$ CFU/$m\ell$로 급격하게 감소하였다.

• 대한미생물학회지
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• 제22권3호
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• pp.241-250
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• 1987

A total 64 strains of Escherichia coli including 38 strains of urinary tract infection and 26 strains from other clincal sources were studied for several properties related to the virulence markers of organisms. Urinary isolates(76.3%) showed higher frequency of mannose resistant hemagglutination(MRHA) wi th human erythrocytes(A type, $Rh^+$) than the strains of control group isolated from other sources(34.6%). Seventeen strains(44.4%) of urinary isolates and 2 strains(7.7%) of control group showed hemolysis on blood agar plate. There was no significant difference in MIC's of 23 drugs between both groups of urinary isolates and control group. But they showed high frequency of resistance to ampicillin, carbenicillin, piperacillin, kanamycin, and trimethoprim, but were very susceptible to cefotaxime, moxalactam, ceftizidime, imipenem, and norfloxacine. Fourteen strains(36.8%) of urinary isolates and 10 strains(38.5%) of control group showed conjugally transferable resistance conferred to R plasmids. The urinary isolates carried one or more to 6(mean 3.4) plasmids of approximate molecular weight ranged 3.1 to 94 megadalton(Mdal) and strains of control group carried 2 to 5(mean 3.8) plasm ids of size ranged 3.6 to 130 Mdal. The size of conjugally transferable R plasmid identified with transconjugants ranged 32 to 130 Mdal.

• 한국식품위생안전성학회지
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• 제35권5호
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• pp.477-488
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• 2020

본 연구는 남부 3지역에서 시설재배 3농가 총 9농가를 선정하여 2019년과 2020년도 재배 중인 부추와 부추 재배 토양, 퇴비, 농업용수의 미생물 오염도를 조사하였다. 부추, 토양, 퇴비, 농업용수에서 위생지표세균(일반세균수, 대장균군, 대장균)과 B. cereus, S. aureus를 조사하였다. 2019년 채취해 온 시료의 오염도를 조사한 결과 A지역 일반세균수는 6.15-8.82 log CFU/g, 대장균군은 2.75-4.88 log CFU/g, 21.58-37.95 MPN/100 mL, B. cereus는 1.79-5.86 log CFU/g으로 검출되었다. B지역 일반세균수는 6.41-8.44 log CFU/g, 대장균군은 1.57-2.82 log CFU/g, B. cereus는 2.48-6.00 log CFU/g으로 검출되었다. C지역 일반세균수는 6.42-7.74 log CFU/g, 대장균군은 2.39-5.73 log CFU/g, 14.45-2419.6 MPN/100 mL, B. cereus는 1.48-5.56 log CFU/g으로 검출되었다. C지역 III농가 토양에서 대장균이 1.86 log CFU/g으로 검출되었다. 2020년 채취해온 시료의 오염도를 조사한 결과 A지역 일반세균수는 2.83-8.20 log CFU/g, 대장균군은 0.28-4.03 log CFU/g, B. cereus는 0.41-5.30 log CFU/g, S. aureus는 0.40-4.85 log CFU/g이 검출되었다. B지역 일반세균수는 0.84-7.25 log CFU/g, 대장균군은 3.13-3.56 log CFU/g, B. cereus는 1.69-2.82 log CFU/g, S. aureus는 2.44-3.78 log CFU/g이 검출되었다. C지역 일반세균수는 2.04-8.83 log CFU/g, 대장균군은 0.43-4.04 log CFU/g, B. cereus는 0.70-4.93 log CFU/g, S. aureus는 1.81-6.27 log CFU/g이 검출되었다. 부추는 토양과 퇴비로부터의 오염, 농업용수로부터의 오염, 농업용수로 오염된 토양과 퇴비로의 오염 등 다양한 경로를 통해 B. cereus가 오염될 수 있다. β-hemolysis 활성을 지니는 B. cereus가 부추와 재배환경시료로부터 분리되었기에 B. cereus의 오염 예방이 중요하다. 반면에 병원성 E. coli, E. coli O157:H7, L. monocytogenes, Salmonella spp.은 검출되지 않았다. B. cereus와 S. aureus의 오염을 줄이기 위해 농작물 재배 시 작물과 토양과 퇴비의 접촉을 최소화하기 위해 멀칭 재배와 농업용수의 관리, 사용 후 퇴비 관리 등 재배 환경을 관리하는 것이 미생물 오염도를 줄이는데 효과적일 것으로 판단된다. 또한 농산물로 인한 식중독 사고를 예방하기 위해 작물과 재배환경의 모니터링 연구와 작업자의 위생 관리에 대한 지속적인 연구가 필요하다.

• 대한수의학회지
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• 제49권1호
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• pp.39-44
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• 2009

Transmission of avian viruses both bird-to-bird and from birds to non-avian species is a major health concern. Newcastle disease virus (NDV) is an economically important avian virus that poses substantial risks to the poultry industry. Rapid and sensitive diagnostic methods, such as the enzymelinked immunosorbent assay (ELISA), are required to track such infections. To develop an ELISA for detecting anti-NDV antibody in avian sera, the nucleocapsid protein (NCP) gene of the NDV La Sota strain was cloned and expressed in Escherichia coli and the 513-amino acid recombinant NCP was purified by Ni-NTA affinity chromatography. To evaluate its ability to replace NDV whole virus antigen as a coating antigen, NCP-coated and whole NDV-coated ELISAs were tested and compared using a panel of NDV positive antisera from chickens. Results using purified NCP were highly correlated with those obtained using whole NDV (r= 0.927), demonstrating that recombinant NCP expressed in Escherichia coli is a suitable substitute antigen for whole NDV in a diagnostic ELISA.

• 한국식품과학회지
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• 제43권1호
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• pp.39-44
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• 2011

국내에 유통되고 있는 즉석섭취 편의식품에 대한 미생물 모니터링 검사를 실시하고자 식품품목별로 일반세균수, E. coli 정성 및 정량, S. aureus 정량, B. cereus group 정량, L. monocytogenes 정성 및 정량검사를 실시하였다. 즉석섭취 편의식품은 해산물함유, 육류함유, 빵류, 밥류, 샐러드류, 신선편의식품류로 유형을 분류하고 식품군별 미생물 오염도를 비교분석하였다. 일반 세균수에서는 대부분 3-5 log CFU/g의 오염분포를 보였으며, 가장 높은 오염도와 유의적인 차이를 보이면서 가장 높은 평균값(4.4 log CFU/g이상)를 보인 품목은 빵류, 밥류와 신선편의식품이었다. E. coli는 밥류 2건에서 식품기준 및 규격을 초과하였으며, S. aureus은 해산물함유 1건 및 빵류 1건에서 식품공전 기준 및 규격 이상을 초과하여 이들 식품군들의 식품위생관리가 더 요구되는 것으로 나타났다. 본 연구결과를 종합해 보면, 식중독균은 계절에 상관없이 E. coli, S. aureus 및 B. cereus group의 오염도를 꾸준히 보이고 있으며, 즉석섭취 편의식품의 미생물오염 관리는 계절에 상관없이 개인위생 및 환경위생에 지속적인 관리가 필요한 것으로 사료된다. 또한 즉석섭취 편의식품의 미생물적 안전성확보를 위해서는 제조업체의 생산단계부터 사용재료에 대한 정확한 분석, 운반과정에 대한 관리, 제조 및 유통단계에서의 위생관리 등 전반적인 위생관리가 적용되어할 것으로 판단된다.

• 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.

• 대한수의학회지
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• 제20권2호
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• pp.85-92
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• 1980

The antimicrobial drug susceptibility of 439 isolates of animal pathogens recovered from various clinical cases during 1978-79 has been investigated by the use of disk diffusion technique. The majority of 308 strains of Eschericihia coli were highly resistant to bacitracin, erythromycin, penicillin, streptomycin and tetracyclinon while only 0.3 per cent of them were resistant to gentamicin and 3.2 per cent to colistin. The percentages of strains resistant to ampicillin, carbenicillin, cephalothin, chloramphenicol and neomycin were 30.5%, 24.7%, 11:4%, 28.2% and 26.2% and repectively. However, none of E. coli cultures of ovine origin were resistant to ampicillin, carbenicillin, chloramphenicol, colistin, gentamicin, kanamycin, and neomycin. A total of 39 patterns of multipe drug1 resistance of 308 strains E. coli against 9 drugs in general use such as ampicillin, cephalothin, chloramphenicol, colistin, gentamicin, kanamycin, neomycin, streptomycin and tetracycline were observed and the most common multiple resistance patterns were SM, TC pattern (20.5%) and AM, CP, KM, NM, SM, TC pattern (9.7%). None of the 43 cultures of salmonella organism from pigs and chickens were resistant to ampicillin, carbenicillin, cephalothin, colistin, gentamicin and kanamycin; and the majority of the cultures were susceptible to chloramphenicol (90.0%), neomycin (97.7%) and tetracycline (93.0%). All the cultures were found to be resistant to bacitracin and penicillin and the rate of resistant strains to erythromycin and s treptomycin being 79.1% and 41.9% respectively. It was found that the majority of 63 cultures of staphylococcal isolates were resistant to lincomycin, penicillin, streptomycin and tetracycline. The percentages of 63 staphylococcal isolates susceptible to gentamicin, nitrofurantoin, cephalothin, ampicillin, methicillin, bacitracin and chloramphenicol were 98.4%, 98.4%, 95.2%, 93.7%, 93.7%, 92.1% and 92.1% respectively. The 25 cultures of streptococcal isolates were resistant in order of prevalence to streptomycin(88.0%), kanamycin(68.0%), gentamicin (44.0%), tetracycline (44.0%) and methicillin (40.0%) wihle the majority of them were sensitive to ampicillin, bacitracin, chloramphenicol and penicillin.

• Animal cells and systems
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• 제3권2호
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• pp.221-228
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• 1999

In order to find a biochemical marker for late Iysosomes, we characterized two cDNAs which were cloned by using a monoclonal antibody (mAb) against Iysosomes in Amoeba proteus as a probe. The two cDNAs, a 1.3-kb cDNA in pBSK-Iys45 and a 1.6-kb cDNA in pBSK-Iys60, were found to encode proteins homologous to pepstatin-insensitive carboxyl proteinases (PICPs). E. coli transformed with pBSK-Iys45 produced two immunopositive polypeptides (45 and 43 kDa) and the cDNA in 1274 bases encoded a 44,733-Da protein (Lys45) of 420 amino acids containing one site for a core oligosaccharide. On the other hand, E. coli transformed with pBSK-Iys60 produced several polypeptides (64, 54, 45, 41, and 37 kDa) reacting with the mAb. The cDNA contained 1629 bases and encoded a 59,231-Da protein (Lys60) of 530 amino acids containing two sites for asparagine-linked core oligosaccharides. These two cDNAs showed identities of 60.3% in nucleotide sequences and 23.6% in amino acid sequences. Lys45 and Lys60 appeared to share XXEFQK as a common antigenic domain. The amino acid sequence of the Lys45 protein showed 17.4% identity and 40.9% similarity to that of PICP from Pseudomonas sp. 101. On the other hand, Lys60 showed a 24.3% identity and 51.9% similarity with human Iysosomal PICP in the amino acid sequence. A putative active center for serine protease, GTS＊xxxxxFxG, was found to be conserved among PICP homologues. The two PICPs are the first reported enzymatic markers for late Iysosomes.

• 한국식품영양과학회지
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• 제44권8호
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• pp.1144-1149
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• 2015

식용 표고버섯을 acetone, ethyl acetate 및 ethanol 등 여러 용매로 추출하여 각 추출용매에 따른 표고버섯 추출물의 항산화 및 항균 활성을 측정하였다. 추출물은 최소 농도인 98 mg/mL에 맞추어 모든 실험을 진행하였다. 표고버섯의 폴리페놀 함량은 유의적 차이는 보이지 않았으나 극성이 큰 ethanol 추출물이 acetone 추출물과 ethyl acetate 추출물보다 다소 높게 조사되었으며, ABTS 라디칼 소거능에서도 유사한 경향을 확인하였다. 반면에 플라보노이드 함량과 DPPH 라디칼 소거능은 ethyl acetate와 acetone 추출물이 ethanol 추출물보다 다소 높게 조사되었다. 폴리페놀과 플라보노이드 함량은 낮았지만 DPPH 라디칼 소거능에서 항산화 활성을 확인할 수 있었으며, 특히 ABTS 라디칼 소거능은 86.8~98.5%로 표준물질 항산화제인 ascorbic acid보다 좀 더 높은 항산화 활성을 확인하였다. 표고버섯의 다재 내성 관련 항균 활성은 ethyl acetate 추출물이 6종의 다제 내성 균주인 3종의 그람 양성균 Bacillus subtilis, Staphylococcus aureus 및 Micrococcus luteus와 3종의 그람 음성균 Escherichia coli, Pseudomonas aeruginosa 및 Enterobacter cloacae 등 모두에서 항균 활성을 가지고 있는 것을 확인하였다. 특히 B. subtilis에 대하여 가장 높은 항균활성을 보였으며, P. aeruginosa, M. luteus, Ent. cloacae 및 B. subtilis에서는 acetone, ethyl acetate 및 ethanol 추출물 모두에서 항균 활성을 확인할 수 있었다.