• Journal of Microbiology and Biotechnology
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• 제6권5호
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• pp.331-335
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• 1996

The second $\beta$-Xylosidase gene (xylB) from Bacillus stearothermophilus was isolated from the genomic library, cloned into pBR322, and subsequently transferred into Escherichia coli HB101. Six out of 10, 000 transformants were selected from the selective LB medium supplemented with p-nitrophenyl-$\alpha$-L-arabinofuranoside (pNPAf) and ampicillin ($50\mu g$/ml) based on their ability to form a yellow ring around the colony. One of the clones was found to harbor the recombinant plasmid with 5.0 kb foreign DNA, which was identical to the $\alpha$-L-arabinofuranosidase gene (arfI) previously cloned in this lab, while the other five had 3.5 kb of the foreign DNA. Southern blotting experiments confirmed that the 3.5 kb insert DNA was from B. stearothermophilus chromosomal DNA. A zymogram with 4-methylumbelliferyl-$\alpha$-L-arabinofuranoside as the enzyme substrate revealed that the cloned gene product was one of the mutiple $\alpha$-L-arabinofuranosidases produced by B. stearothermophilus. Unlike the arfI gene product, the product of the gene on the insert DNA (xylB) showed an activity not only on pNPAf but also on oNPX suggesting that the cloned gene product could be a bifunctional enzyme having both $\alpha$-L-arabinofuranosidase and $\beta$-xylosidase activities.

• 한국미생물·생명공학회지
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• 제22권5호
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• pp.507-514
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• 1994

Acetyl xylan esterase was produced by E. coli HB101 harboring a recombinant plasmid pKMG6 which contained the estI gene of Bacillus stearothermophilus. The maximum production was observed when the E. coli strain was grown at 37$\circC for 12 hours in the medium containing 0.5% acetyl xylan, 1.0% tryptons, 1.0% sodium chloride, and 0.5% yeast extract. The esterase produced was purified to homogeneity using a combination of ammonium sulfate fractionation, DEAE Sepharose CL-6B ion exchange chromatography and Sephacryl S-200 gel filtration. The native enzyme had an apparent molecular mass of 60 kd and was composed of two identical subunits of 29 kd. The N-terminal amino acid sequence of the polypeptide was Ala-X-Leu-Gln- Ile-Gln-Phe-X-X-Gln. The acetyl esterase displayed a pH optimum of 6.5 and a temperature opti- mum of 45$\circC. The heavy metal ions such as Ag$^{++}$, Hg$^{++}$ and Cu$^{++}$ inhibited nearly completely the activity of the esterase, and no specific metal ion was found to be required for the enzyme activity. The enzyme readily cleaved MAS, $\beta$-D-glucose pentaacetate, $\alpha$-naphthyl acetate, $\rho$-nitrophenyl acetate as well as acetyl xylan, but had no activity on $\rho$-nitrophenyl propionate, $\beta$-nitrophenyl butyrate or $\beta$-nitrophenyl valerate. The Km and Vmax values for MAS were 2.87 mM and 11.55 $\mu$mole/min, respectively. Synergistic behavior was demonstrated with a combination of xylanase and esterase from B. stearothermophilus in hydrolyzing acetyl xylan.

• 한국물환경학회지
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• 제26권4호
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• pp.574-579
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• 2010

In this study the enzyme inhibition method using dehydrogenase which has been popularly used to estimate ecotoxicity was optimized. When three bacterial strains, Escherichia coli HB101, Enterobacter asburiae KCAD-4, and Aeromonas media KCAD-13, were compared, KCAD-4 was considered as the adequate strain to estimate toxicity because of its sensitivity and reproducibility. The optimal bacterial density was estimated as $5.4{\times}10^9CFU/mL$, at which the maximum sensitivity was observed. The phosphate buffer was suitable for the reaction solution. When the reaction times required for inhibition of enzyme activity by contact of toxicants and for reaction of damaged bacteria and substrate were tested, the optimal value was estimated as 20 min and 2 hrs, respectively. It is expected that the optimized conditions can be used to develop the standardized kits to estimate ecotoxicity of heavy metals in effluent from the industrial wastewater treatment facilities.

• 대한한방부인과학회지
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• 제19권4호
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• pp.17-32
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• 2006

Purpose : The aim of this study is to investigate the antibiotic effects of 14 herbs among blood-activating stasis-dispelling medicinals on vaginal microorganisms. Methods : Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Candida albicans, and Gardnerella vaginalis were used for vaginal pathogenic microorganisms. Lactobacillus gasseri, Streptococcus spp. and Escherichia coli HB101 were used for vaginal normal flora. The blood-activating stasis-dispelling medicinals, Mucunae Caulis, Salviae Radix, Persicae Semen, Myrrha, Zedoariae Rhizoma, Achuranthis Radix, Leonuri Herba, Melandrii Herba, Gleditsiae Spina, Lycopi Herba, Scirpi Rhizoma, Caesalpiniae Lignum, Corydlais Tuber and Polygoni Cuspidati Radix were used in this study. In vitro antibiotic activities were observed by optical density and colony test. Results : The optical density and colony test showed that Gleditsiae Spina, Scirpi Rhizoma, Corydlais Tuber, Polygoni Cuspidati Radix and Melandrii Herba of herbs among blood-activating stasis-dispelling medicinals had antimicrobial effect. Gleditsiae Spina had antimicrobial susceptibility and selective toxicity in Gardnerella vaginalis and MRSA. Scirpi Rhizoma had antimicrobial susceptibility and selective toxicity in Staphylococcus aureus and MRSA. Corydlais Tuber had antimicrobial susceptibility and selective toxicity in MRSA. Polygoni Cuspidati Radix had antimicrobial susceptibility and selective toxicity in Gardnerella vaginalis, Staphylococcus aureus and MRSA. Melandrii Herba had antimicrobial susceptibility and selective toxicity in Gardnerella vaginalis. Conclusion : According to the above results, we could suggest that Gleditsiae Spina, Scirpi Rhizoma, Corydlais Tuber, Polygoni Cuspidati Radix and Melandrii Herba of herbs among blood-activating stasis-dispelling medicinals be available to antimicrobial agent of vaginal pathogenic microbial species in vitro.

• 대한한방부인과학회지
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• 제19권4호
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• pp.33-46
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• 2006

Purpose : This study was conducted to investigate effects of 7 herbs among medicinals resolving dampness with aroma on vaginal microorganisms. Methods : Staphylococcus aureus, Methicillin- resistant Staphylococcus aureus, Candida albicans and Gardnerella vaginalis were used for vaginal pathogenic microorganisms. Lactobacillus gasseri, Streptococcus spp. and Escherichia coli HB101 were used for vaginal flora. Medicinals resolving dampness with aroma, Pogostemonis Herba, Amomi Cardamomi Fructus, Amomi Semen, Atractylodis Rhizoma, Ammomi Tsao-ko Fructus, Alpiniae Katsumadaii Semen and Magnoliae Cortex were used in this study. In vitro antimicrobial activities were observed by optical density and colony test. Results : The optical density showed that Alpiniae Katsumadaii Semen and Magnoliae Cortex among medicinals resolving dampness with aroma had antimicrobial effects on pathogenic vaginal microorganisms (Methicillin-resistant Staphylococcus aureus). Pogostemonis Herba, Ammomi Tsao-ko Fructus had antimicrobial effects on Gardnerella vaginalis. The colony test showed that Alpiniae Katsumadaii Semen, Magnoliae Cortex among medicinals resolving dampness with aroma had antimicrobial effects on Methicillin-resistant Staphylococcus aureus. Pogostemonis Herbs, Alpiniae Katsumadaii Semen had antimicrobial effects aganist Gardnerella vaginalis. The optical density showed that Magnoliae Cortex among medicinals resolving dampness with aroma had antimicrobial effect on normal vaginal microorganisms (Streptococcus spp.). The colony test showed that medicinals resolving dampness with aroma had no antimicrobial effect on normal vaginal microorganisms. Conclusion : From this study, we could suggest that Pogostemonis Herba, Ammomi Tsao-ko Fructus, Alpiniae Katsumadaii Semen, Magnoliae Cortex of medicinals resolving dampness with aroma are available to antimicrobial agent of pathogenic vaginal microorganisms in vitro.

• 대한한방부인과학회지
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• 제19권3호
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• pp.175-190
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• 2006

Purpose : This study was conducted to investigate the in vitro inhibitory effects of heat-clearing medicinal on common bacterias in gynecology. Methods : The heat-clearing medicinals ( Trichosanthis Radix, Sophorae Fructus, Phragmitis Rhizoma, Buddleiae Flos, Bambusae Folium, Anemarrhenae Rhizoma, Celosiae Semen, Gardeniae Fructus, Prunellae Spica, Sophorae Radix, Dictamni Radicis Cortex, Coptidis Rhizoma, Gentianae Scabrae Radix, Scutellariae Radix, Phellodendri Cortex) were used in this study. Staphylococcus aureus, MRSA, Candida albicans and Gardnerella vaginalis were used for vaginal pathogenic microorganisms. Streptococcus spp., Escherichia coli HB101, Lactobacillus gasseri were used for normal vaginal florae. We evaluated antibiotic effect by the optical density and the colony test. Results : The optical density and colony test showed that Celosiae Semen, Prunellae Spica, Scutellariae Radix and Phellodendri Cortex of herbs among heat-clearing medicinal had antimircobial effect. Celosiae Semen and Prunellae Spica had antimicrobial susceptibility and selective toxicity in MRSA. Scutellariae Radix and Phellodendri Cortex had antimicrobial susceptibility and selective toxicity in Gardnerella vaginalis. Conclusion : According to the above results, we could suggest that Celosiae Semen, Prunellae Spica, Scutellariae Radix and Phellodendri Cortex among heat-clearing medicinal be available to antimicrobial agent of vaginal pathogenic microbial species in vitro.

• 대한한방부인과학회지
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• 제20권2호
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• pp.124-138
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• 2007

Purpose: This study was conducted to investigate the antimicrobial effects of the interior warming herbs on vaginal microbes. Methods: Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Candida albicans and Gardnerella vaginalis were used for vaginitis-induced microbes. Lactobacillus gasseri, Streptococcus spp. and Escherichia coli HB101 were used for normal vaginal florae. And herbs for warming the interior (Zingiberis Rhizoma, Alpiniae Officinari Rhizoma, Aconiti Tuber, Anethi Fructus, Evodiae Fructus, Cinnamomi Cortex Spissus, Caryophylli Flos, Aconiti Tube, Zanthoxyli Pericarpium, Piperis Longi Fructus, Piperis Nigri Fructus) were used in this study. Antimicrobial activities were tested by the change of optical densities (OD) and colony test in vitro. Results: In the results of vaginitis-induced microbes, Anethi Fructus was decreased the OD values on MRSA and Gardnerella vaginalis and Aconiti Tuber was decreased on MRSA. There were no viable MRSA and Gardnerella vaginalis colony forming against Evodiae Fructus, Staphylococcus aureus and MRSA colony forming against Piperis Longi Fructus, Staphylococcus aureus colony forming against Piperis Nigri Fructus and MRSA colony forming against Zanthoxyli Pericarpium. In the results of normal vaginal florae, Zingiberis Rhizoma was decreased the OD values on Streptococcus spp. and all normal vaginal florae were showed viable colony forming against all experimental herbs. Conclusion: According to these results, we can suggest that some kinds of interior warming herbs have antimicrobial effects on vaginal microbes. So there might be needed to make furthermore studies to seek the herbs which have selective antimicrobial effect on pathologic vaginal microbes.

• 대한한방부인과학회지
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• 제19권2호
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• pp.127-140
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• 2006

Purpose : This study was conducted to investigate the antimicrobial effects of Astringent medicinals against vaginal microbe. Methods : Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Candida albicans and Gardnerella vaginalis were used for vaginitis-induced microbe. Lactobacillus gasseri, Streptococcus spp. and Escherichia coli HB101 were used for normal vaginal florae. And Astringent medicinals (Rubi Fructus, Tritici Immatri Semen, Corni Fructus, Nelumbinis Semen, Mume Fructus, Schizandrae Fructus, Ailanthi Radicis Cortex, Galla Rhois, Myristicae Semen, Terminariae Fructus, Rosae laevigatae Fructus, Ephedrae Radix) were used in this study. Antimicrobial activities were tested by optical density and colony test in vitro. And then wee valuated the antimicrobial effects in comparison with optical density and colony test. Results : The optical density and colony test showed that Terminariae Fructus and Galla Rhois among Astringent medicinals had the antimicrobial effects. Teminariae Fructus had the antimicrobial susceptibility and selective toxicity against Candida albicans and Gardnerella vaginalis of vaginitis-induced microbe. Galla Rhois had the antimicrobial susceptibility and selective toxicity against Staphylococus aureus, Methicillin-resistant Staphylococus aureus, Candida albicans and Gardnerella vaginalis of vaginitis-induced microbe. Conclusion : According to these results, we can suggest that Terminariae Fructus and Galla Rhois among Astringent medicinals be available to the antimicrobial agent of vaginitis-induced microbe in vitro.

• 한국수산과학회지
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• 제35권2호
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• pp.115-121
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• 2002

R plasmid의 분포와 새로운 conjugation 방법 개발을 위하여, 한국의 남해안과 동해안의 양식현장의 어류질병관련 세균으로부터 항균제 다제 내성균을 분리하였다. 분리균 134종에서 10균주가 chlorarnphenicol, tetracycline, streptomycin, ampicillin, colistin, nalidixic acid, oxolinic acid, kanamycin 등에 대한 다제내성의 특성을 가지고 있었으며 이중 V. damsela JE1 한 균주는 chloram-phenicol과 tetracycline에 대한 내성전달 특성을 갖는 R plasmid 와 ampicillin과 kanamycin에 대한 유전자를 chromosome 등에 함유하고 있었다. 분리된 어병세균의 다제내성 특성이 R plasmid에 기인하는 것인지의 확인은 다양한 장내세균의 선택배지로 최근 개발된 CC 배지에서 각기 다른 균주들이 나타내는 증식여부 또는 집락 색깔의 차이를 이용하여 donor, recipient 그리고 transconjugant를 구별할 수 있는 방법을 적용하였다. Conjugation의 최적화를 위해 여러 가지 조건을 비교한 결과, V. damsela JE1의 R plasmid의 전달은 filter mating법을 사용하는 것이 broth mating법보다 약 100배 이상의 높은 성공률을 보여 주었으며 이때 사용온도는 30$^{\circ}C$ 이상, mating 시간은 24시간에서 가장 높은 전달 빈도를 나타내었다. R plasmid를 함유한 V. damsela JE1이 분리된 4개월 후, 동일 양식장의 동일 그룹의 넙치 장내세균에서 분리한 다제내성균 Sphingomonas sp. 3균주는 V. damsela JE1에서 분리된 것과 같은 크기의 R plasmid를 함유하고 있었으며, 그 특성 또한 V. damsela JE1의 R plasmid와 마찬가지로 다제 내성 중 Cm과 Tc에 대한 유전자만이 recipient로 전달되어 장내 세균총이 R plasmid의 reservoir로서의 역할을 하고 있음을 확인하였다.

• Current Research on Agriculture and Life Sciences
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• 제4권
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• pp.36-41
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• 1986

대장균(大腸菌)과 효모(酵母)의 셔틀 벡터인 YIp, YRp, YEp를 S. cerevisiae MC 16, DBY747에 형질전환(形質轉換)시켰다. 이들 벡터들을 대장균(大腸菌)에 형질전환(形質轉換)시켰을 때 그 빈도(頻度)가 YIp5, YIp26, YEp13, YRp7에서 각각 $5.1{\times}10^{-4}$, $1.5{\times}10^{-3}$, $3{\times}10^{-3}$, $1.3{\times}10^{-3}$으로 나타났다. YEp13을 MC16과 DBY747에 Ito 법(法)으로 형질전환(形質轉換)시켰을 때 MC16, DBY747에서의 빈도(頻度) 각각 $3.3{\times}10^{-4}$, $1.2{\times}10^{-4}$으로 나타났다. DBY747을 수용세포(受容細胞)로 하여 YRp7과 YIp26을 환상(環狀)으로 각각 형질전환(形質轉換)시켰을 때 그 빈도(頻度)는 각각 $3{\times}10^{-6}$, $6{\times}10^{-8}$이하로 나타났다. DBY747을 수용세포(受容細胞)로 하여 YEp13과 YIp26을 선상(線狀)으로 절단하여 $Li^+$ 처리(處理)한 S. cerevisiae에 cotransformation을 하였을 때 YIp26+YEp13 ($Leu^+$, $Ura^+$)의 cotransformant는 $1{\times}10^{-5}$ 빈도(頻度)가 나왔으며 YIp26과 YEp13에 대한 각각의 빈도(頻度)는 $10^{-7}$ 이하, $5{\times}10^{-5}$ 빈도(頻度)로 나타났다. 또 YIp5와 YEp13, YIp26과 YRp7을 원형질체화(原形質體化)한 S. cerevisiae DBY747에 cotransformation 하였을 때 빈도(頻度)는 YIp5+YEp13은 $4{\times}10^{-6}$으로 나오고 YIp26+YRp7에서 $1.5{\times}10^{-6}$으로 나타났다.