• Title/Summary/Keyword: Pseudomonas sp. P20

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Purification and Characterization of Two Alkaline Protease Produced by Pseudomonas sp. BK7

  • Lee, Eun-Goo;Park, Eun-Hee;Hyun, Hyung-Hwan
    • Journal of Microbiology and Biotechnology
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    • v.10 no.5
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    • pp.677-684
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    • 2000
  • Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enhanced by the increase of agitation speed. Two forms of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The $K_m$ value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0, and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both proteases were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were $50^{\circ}C$ and $45^{\circ}C$, respectively. About 56% of the original protease BK7-2 activity remained after being treated at $50^{\circ}C$ for 30 min but protease BK7-1 was rapidly inactivated at above $25^{\circ}C$. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.

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재조합균주 E. coli CK1092가 생산하는 2,3-Dihydroxybiphenyl Dioxygenase의 정제 및 특성

  • Park, Hyo-Nam;Kim, Young-Soo;Kim, Young-Chang;Kim, Chi-Kyung;Lim, Jai-Yun
    • Microbiology and Biotechnology Letters
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    • v.24 no.3
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    • pp.282-289
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    • 1996
  • 2,3-DHBP dioxygenase was purified from E. coli CK1092 carrying the pcbC gene, which was cloned from 4-chlorobiphenyl-degrading Pseudomonas sp. P20. Purification of this enzyme was done by acetone precipitation, DEAE- Sephadex A-25 ion exchange chromatography, and preparative gel electrophoresis. The molecular weight of subunit was 34 kDa determined by SDS-PAGE, and that of native enzyme was about 270 kDa. It suggests that this enzyme consist of eight identical subunits. This enzyme was specifically active against only 2,3-DHBP as a substrate with 18 $\mu$M of Km value, but not catechol, 3-methylcatechol, 4-methylcatechol and 4-chlorocatechol. The optimal pH and temperature of 2,3-DHBP dioxygenase were pH 8.0 and 40-60$\circ$C. The enzyme was inhibited by Cu$^{2+}$, Fe$^{2+}$ and Fe$^{3+}$ ions, and was inactivated by H$_{2}$0$_{2}$2 and EDTA. The lower concentrations of some organic solvents such as acetone and ethanol don't stabilize the activity of 2,3-DHBP dioxygenase. The enzyme was completely inactivated by adding the reagents such as N-bromosuccinimide, iodine and p- diazobenzene sulfonic acid.

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Isolation, Identification, and Culture Conditions of the Strain Producing Eicosapentaenoic Acid

  • Shin, Won-Cheol;Kim, Chang-Ho
    • Journal of Microbiology and Biotechnology
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    • v.4 no.4
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    • pp.338-342
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    • 1994
  • The bacterium producing EPA was isolated from the intestines of the marine fishes. The strains were studied for their identification and their culture conditions. The selected strain was gram negative, rod(0.7 $\times$ 2.4 $\mu m$ in size) and motile with a single polar flagellum. This strain was identified as a Pseudomonas sp. on the basis of its morphological, cultural and physiological characteristics. The strain showed a maximum productivity of phospholipid at $20^{\circ}C$ after 48 hours of culture time with an initia1 pH of 7.0 in the PYM-glucose medium. Under these culture conditions, the production of phospholipid was about 0.3 mg/ml and 0.06 mg/mg dry cell weight.

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Culture Conditions of E. coli CK1092 for the Production of 2,3-Dihydroxybiphenyl Dioxygenase (2,3-Dihydroxybiphenyl Dioxygenase 생산을 위한 E. coli CK1092의 배양조건)

  • Lee, Jung-Young;Kim, Youngsoo;Lee, Ki-Sung;Min, Kyung-Hee;Kim, Young-Chang;Kim, Chi-Kyung;Lim, Jai-Yun
    • Korean Journal of Microbiology
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    • v.34 no.1_2
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    • pp.20-25
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    • 1998
  • To obtain higher yield of 2,3-dihydroxybiphenyl(2,3-DHBP) dioxygenase by recombinant E. coli CK1092 carrying pcbC gene of Pseudomonas sp. P20, the environmental and physiological factors were investigated and the cultural conditions using jar fermentor were studied. E. coli CKl092 was grown in LB medium supplemented with 2% sucrose, as a basal medium. The effect of various metal ions on the enzyme production was investigated. In particular, the enzyme production increased in the presence of $Fe^{3+}$ and $Fe^{2+}$, and showed the maxium at the concentration of $10^{-5}M$. The enzyme production was increased by 55% in the medium containing $Fe^{3+}$ ($10^{-5}M$) ion. The optimal temperature and initial pH for cell growth were $37^{\circ}C$ and 7.0, respectively. In the culture using a fermentor at $37^{\circ}C$, the optimal conditions for the enzyme production were obtained at the initial pH 7.0, 1 v/v/m of aeration rate, 200 rpm of agitation speed. It was found that enzyme activity was higher when cultivated without pH control than with pH control.

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Biodegradation of Crude Oil and Petroleum Products by Crude Oil-degrading Microorganism (미생물을 이용한 원유 및 원유제품의 분해 특성)

  • 정선용;오경택;박귀환;이정일;이중기
    • KSBB Journal
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    • v.17 no.3
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    • pp.247-254
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    • 2002
  • Two kinds of crude oil-degrading microorganisms from soil and one kind from sea were isolated and named strain Al32, strain F722 and strain OM1, respectively. These microorganism were identified Acinetobacter sp., Pseudomonas aeruginosa and Acinetobacter calcoaceticus, respectively. The optimum cultivation temperature of Acinetobacter sp. A132 and P. aeruginosa F722 was $35^{\circ}C$ and optimum growth pH was 8 and 9, respectively. The growth was the highest at 2.0% (w/v) substrate concentration when crude oil was only carbon source. The growth of A. calcoaceticus OM1 isolated from sea was the highest at 3.0% (w/v) of crude oil. In inspection of crude oil degradability, strain Al32 showed 5.49 g/L.day with Eleuthera (OMAN), 2.0% (w/v). P. aeruginosa F722 showed 1.19 g/L g/L.day with L-Zakum (AFRICA). In case of kerosene $nC_9\simnC_{20}$ and diesel $nC_9\simnC_{28}$, A. calcoaceticus OM1 was degraded 95% and 75%, respectively, for 7 days culture, and P. aeruginosa F722 was 80% after 10 days.

Effect of Hot Water and Lime-Sulfur Mixture Treatment for Disinfection of Seeds for Organic Lettuce (유기농 상추 재배를 위한 온탕침지와 석회유황합제의 종자소독 효과)

  • Kim, Min-Jeong;Shim, Chang-Ki;Ko, Byeong-Gu;Kim, Ju;Park, Jong-Ho;Yoon, Ji-Young
    • Journal of agriculture & life science
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    • v.53 no.3
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    • pp.27-39
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    • 2019
  • The purpose of this study was to investigate the effect of hot water treatment and pH corrected lime sulfur combination treatment on the fungicidal and bacterial disinfection effects and germination rate of organic lettuce seeds. Among the followers, Alternaria sp. was infected 53.3% and Aspergillus sp. and Cladosporium sp. Infected 14.5% and 5.4%, respectively. Bacteria were isolated only Pseudomonas sp., and infected with 16.5%. In order to investigate the effect of disinfection on the temperature of hot water (45℃, 50℃, 55℃ and 60℃). The seed germination rate sharply decreased with increasing temperature and treatment time. The germination effect and germination rate of the follower were highest when hot water treatment was carried out for 20 minutes in hot water at 50℃. In the case of combined treatment of 50℃ hot water for 10 min and 0.4% pH adjusted lime sulfur mixture, showed the highest sterilization effect and germination rate with 100% and 97.6%, respectively. The results of this study can contribute to the development of technology to sterilize not only seed surface but also fungi and bacteria inside of seed.

Isolation and Characterization of Odor Treatment Bacteria (악취제거용 균주의 분리 및 특성)

  • Jeong Gwi-Taek;Lee Gwang-Yeon;Lee Kyoung-Min;Lee Hye-Jin;Ryu Hwa-Won;Kim Doman;Chough Sung-Hyo;Kim Si-Wouk;Cha Jin-Myoung;Jang Young-Seon;Park Don-Hee
    • KSBB Journal
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    • v.20 no.5 s.94
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    • pp.345-349
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    • 2005
  • The microorganism for odor gas removal was isolated from sewage and contaminated soil. This was characterized as Pseudomonas sp. TKC by morphological, biochemical/physiological, and cultural characteristics analysis of the isolates. The optimum conditions for isolates growth were as follows; substrate concentration 500 ppm, initial medium pH 7.0, incubation temperature $30^{\circ}C$, agitation speed 150 rpm, and MSM medium containing 3 g/L $(NH_4)_2SO_4$.

Optimal Culture Conditions and Isolation of a ι-Carrageenase-producing Marine Bacterium

  • Shim Hang-Sun;Lim Su-Jin;Choi Min-Jung;Kim Jong-Oh;Kim Seok-Ryel;Kim Hyeung-Rak
    • Fisheries and Aquatic Sciences
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    • v.9 no.2
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    • pp.57-63
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    • 2006
  • A bacterial strain capable of hydrolyzing carrageenan was isolated from the coast of Busan in Korea. The isolated strain (HS5322) is aerobic, gram-negative, rod-shaped, and motile. Comparison of the 16S rDNA of the isolate with that of known Pseudomonas sp. showed that sequence similarity was at most 95%, implying that the isolate is a new Pseudomonas species. The organism grew optimally at NaCl concentrations of 2.0 to 2.5%. The optimum temperature and pH for carrageenase production in a 72-h flask culture containing 1% carrageenan was $20^{\circ}C$ and pH 8.5, respectively. Of the synthetic substrates tested, the highest enzyme activity was obtained with p-nitrophenyl ${\beta}$-D-galactopyranoside.

Applicability of Spent Mushroom Media as Horticultural Nursery Media (버섯재배 후 탈병배지의 원예용 상토재료 이용성 검토)

  • Lee, Chan-Jung;Cheong, Jong-Chun;Jhune, Chang-Sung;Kim, Seung-Hwan
    • Korean Journal of Soil Science and Fertilizer
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    • v.42 no.2
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    • pp.117-122
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    • 2009
  • This study was carried out to investigate applicability of Spent Mushroom Media(SMM) as horticultural nursery media. After the mushroom has been harvested, the SMM contains a lot of organic material, different microorganism and high density of mushroom hypha. The pH, phosphate and exchangeable cation concentrations of SMM of Flammulina velutipes were higher than those of any other treatment. The CEC and $NH_4-N$ were the highest in SMM of bottle-cultivated oyster mushroom (Pleurotus ostreatus). Bacteria and fungi showed the highest density in SMM of Flammulina velutipes. Most dominant bacteria were Microbacterium sp., Rhodococcus sp. and Agrobacterium sp. in SMM of Flammulina velutipes and Bacillus sp., Pseudomonas sp., Curtobacterium sp. and Microbacterium sp. in that of Pleurotus eryngii. The SMM contained high density of mushroom hypha that inhibited germination of seed and growth of young seedlings. Therefore, composting process of the SMM is indispensible to decline of vitality of mushroom hypha. The SMM of Flammulina velutipes with 0~30% vermiculite showed high germination rate in red pepper and chinese cabbage seeds. SMM of Pleurotus eryngii with 20% vermiculite showed 100% germination rate in red pepper seeds, but chinese cabbage seeds nearly failed to germinate with 30% vermiculite. The growth of red pepper was increased according to increasing mixture ratio of vermiculite. Accordingly, we concluded that SMM of Flammulina velutipes contained 0~30% of vermiculite can be used to horticultural growth bed for red pepper.

Studios on the Processing of Low Salt Fermented Sea Foods 3. Changes of Microflora during Fermentation of Low Salted Sardine (저염수산발효식품의 가공에 관한 연구 3. 저염정어리젓의 미생물상의 변화)

  • CHA Yong-Jun;CHUNG Su-Yeol;HA Jae-Ho;JEONG In-Cheol;LEE Eung-Ho
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.16 no.3
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    • pp.211-215
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    • 1983
  • The changes of microflora during fermentation of low salted sardine were observed. The viable cell count in the low salt fermented sardine with $8\%\;or\;10\%$ salt showed lower than that of control ($20\%$ salt) during the fermentation period and it was considered that the microbial growth was controlled by adding ethanol, sorbitol and lactic acid. Among 48 strains isolated, 7 genus of bacteria and 1 genus of yeast were identified during the fermentation of sardine. The changes of microflora also occurred during fermentation depending on the salt levels in the product. Brevibacterium, Pseudomonas, Flavobacterium and Baciilus were detected at early stage of fermentation and they disappeared after 50 days fermentation from the product with $20\%$ salt and Halobacterium, Micrococcus, Pediococcus and Torulopsis were isolated, whereas Brevibacterium, Micrococcus and Pediococcus were isolated from the product with $8\%\;or\;10\%$ salt.

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