• Title/Summary/Keyword: isolation of enzymes

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Isolation and characterization of 4-chlorophenoxyacetic acid-degrading bacteria from agricultural soils

  • Chung, Min-Jae;Shin, Se-Young;Park, Yong-Keun;Min, Kyung-Hee;Ka, Jong-Ok
    • Journal of Microbiology
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    • v.35 no.2
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    • pp.117-122
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    • 1997
  • Several dominant 4-CPA-degrading bacteria were isoalted from agricultural soils. Most of the isolates were identified as Burkholderia species by fatty acid methyl ester (FAME) analysis, but they were idstinct in chromosomal patterns obtained by PCR amplification of repetitive extragenic palindromic (REP) sequences. These strains were generally restricted in their substrate utilization capabilities. The 4-CPA degradative enzymes were idnducible by 4-CPA and some isolates appeared to mineralize 4-CPA via formation of 4-chlorophenol and 4-chlorocatechol as intermediates during its biodegradation pathway. Plasmid DNAs were not detected from most of the isoaltes and their 4-CPA genes wer on the chromosomal DAN. The 4-CPA degradation patterns in axenic cultures and natural soils varied depending on the strains and soils. The inoculation of 4-CPA degraders much improved the removal of 4-CPA from the 4-CPA treated soils.

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Phylogenetic Analysis of Culturable Arctic Bacteria

  • Lee Yoo Kyung;Kim Hyo Won;Kang Sung-Ho;Lee Hong Kum
    • Proceedings of the Microbiological Society of Korea Conference
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    • 2003.05a
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    • pp.26-33
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    • 2003
  • We isolated and identifed culturable Arctic bacteria that have inhabited around Korean Arctic Research Station Dasan located at Ny-Alsund, Svalbard, Norway $(79^{\circ}N,\;12^{\circ}E)$. The pure colonies were inoculated into nutrient liquid media, genomic DNA was extracted, and phylogenetic analysis was performed on the basis of 16S rDNA sequences. Out of total 227 strains, 198 strains were overlapped or unidentified, and 43 bacteria were finally identified: 31 strains belonged to Pseudomonas, 7 strains Arthrobacter, two Flavobacterium sp., an Achromobacter sp., a Pedobacter sp., and a Psychrobacter sp. For isolation of diverse bacteria, we need more effective transport method than 3M petri-films, which were used for convenience of transportation that was restricted by volume. We also need to use other culture media than nutrient media. We expect these Arctic bacteria can be used for screening to develop new antibiotics or industrial enzymes that are active at low temperature.

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Isolation and Characterization of Pseudomonas putida N3 Degrading Naphthalene (Naphthalene을 분해하는 Pseudomonas putida N3의 분리 및 특성)

  • 고영희;하일호;배경숙
    • Microbiology and Biotechnology Letters
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    • v.16 no.3
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    • pp.199-204
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    • 1988
  • A strain capable of growth on naphthalene minimal medium was isolated from soil by selective enrichment culture and identified as Pseudomonas putida N3 according to its morphological and physiological characteristics. The optimum pH and temperature for growth of the isolate were 7.0 and 3$0^{\circ}C$, respectively. This strain was resistant to ampicillin, chloramphenicol, kanamycin and streptomycin but. sensitive to tetracycline and rifampicin. Of the naphthalene related compounds, 1, 5-dihydroxynaphthalene was more easily utilized than naphthalene due to its solubility. And catechol was degraded through meta-cleavage pathway. A 110 Kb plasmid which encodes for a single set of enzymes responsible for the degradation of naphthalene was obtained.

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Isolation and Fusion of Solanaceous Species Mesophyll Protoplast (가자과(茄子科)의 엽육조직(葉肉組織) 원형질체(原形質체体)의 분리(分離) 및 융합(融合))

  • Kwon, Oh Sung;Kim, Dal Dng
    • Current Research on Agriculture and Life Sciences
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    • v.2
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    • pp.15-22
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    • 1984
  • This study was conducted to identify the enzyme treatment time, enzyme concentration and plant growth condition for isolation of potato mesopyll, it was also performed to determine the adquate sucrose molarity on purification of protoplasts and to investigate the incubation time, PEG concentration and DMSO effect for potato-petunia protoplast fusion. The results were summarized as follows: The optimal time of incubation in enzyme solution was 3 - 4 hours and high humidity and low light intensity made plants more effective to protoplast releasing enzymes. Our experimental results showed that the pectolyase Y-23 was an ideal agent for isolation from mesophyll cultured in vitro compared with macerozyme. The enzyme solution with 0.5 % macerozyme and 2 % cellulase was very effective and the purity of healthy protoplast was better in 0.4 and 0.5 M sucrose than in others. It was revealed that the rate of potato-petunia fusion according to the incubation time with PEG was effective at 30 min incubation and percentage of protoplast aggregation was increased by high molecular weight and concentration of PEG. Percentage of potato-petunia protoplast heteroplasmic aggregation was increased by 4 to 16 % in PEG 6000 compared with PEG 4000 and PEG 1500. Addition of 5 to 10 % DMSO to the PEG solution increased to the heteroplasmic aggregation of potato-petunia from 2 to 4 %.

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Plasma Activity of Lysosomal Enzymes in Active Pulmonary Tuberculosis (활동성 폐결핵 환자에서 혈중 리소솜 효소의 활성도)

  • Koh, Youn-Suck;Choi, Jeong-Eun;Kim, Mi-Kyung;Lim, Chae-Man;Kim, Woo-Sung;Chi, Hyun-Sook;Kim, Won-Dong
    • Tuberculosis and Respiratory Diseases
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    • v.42 no.5
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    • pp.646-653
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    • 1995
  • Background: The confirmative diagnosis of pulmonary tuberculosis(Tb) can be made by the isolation of Mycobacterium Tuberculosis(MTb) in the culture of the sputum, respiratory secretions or tissues of the patients, but positive result could not always be obtained in pulmonary Tb cases. Although there are many indirect ways of the diagnosis of Tb, clinicians still experience the difficulty in the diagnosis of Tb because each method has its own limitation. Therefore development of a new diagnostic tool is clinically urgent. It was reported that silica cause some lysosomal enzymes to be released from macrophages in vitro and one of these enzymes is elevated in workers exposed to silica dust and in silicotic subjects. In pulmonary Tb, alveolar macrophages are known to be activated after ingestion of MTb. Activated macrophages can kill MTb through oxygen free radical species and digestive enzymes of lysosome. But if macrophages allow the bacilli to grow intracellularly, the macrophages will die finally and local lesion will enlarge. Then it is assumed that the lysosomal enzymes would be released from the dead macrophages. The goal of this investigation was to determine if there are differences in the plasma activities of lysosomal enzymes, ($\beta$-glucuronidase(GLU) and $\beta$-N-acetyl glucosaminidase(NAG), among the groups of active and inactive pulmonary Tb and healthy control, and to see if there is any possibility that the plasma activity of GLU and NAG can be used as diagnostic indicies of active pulmonary Tb. Methods: The plasma were obtained from 20 patients with bacteriologically proven active pulmonary Tb, 15 persons with inactive Tb and 20 normal controls. In 10 patients with active pulmonary Tb, serial samples after 2 months of anti-Tb medications were obtained. Plasma GLU and NAG activities were measured by the fluorometric methods using 4-methylumbelliferyl substrates. All data are expressed as the mean $\pm$ the standard error of the mean. Results: The activites of GLU and NAG in plasma of the patients with active Tb were $21.52{\pm}3.01$ and $325.4{\pm}23.37$(nmol product/h/ml of plasma), respectively. Those of inactive pulmonary Tb were $24.87{\pm}3.78$, $362.36{\pm}33.92$ and those of healthy control were $25.45{\pm}4.05$, $324.44{\pm}28.66$(nmol product/h/ml of plasma), respectively. There were no significant differences in the plasma activities of both enzymes among 3 groups. The plasma activities of GLU at 2 months after anti-Tb medications were increased($42.18{\pm}5.94$ nmol product/h/ml of plasma) in the patients with active pulmonary Tb compared with that at the diagnosis of Tb(P-value <0.05). Conclusion: The results of the present investigation suggest that the measurement of the plasma activities of GLU and NAG in the patients with active pulmonary Tb could not be a useful method for the diagnosis of active Tb. Further investigation is necessary to define the reasons why the plasma activities of the GLU was increased in the patients with active pulmonary Tb after Tb therapy.

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Isolation and Identification of Agarose-degrading Bacterium, Pseudoalteromonas sp. GNUM08122 (아가로오스 분해세균인 Pseudoalteromonas sp. GNUM08122 분리 및 동정)

  • Kim, Yu-Na;Jeong, Yeon-Kyu;Kim, Mu-Chan;Kim, Sung-Bae;Chang, Yong-Keun;Chi, Won-Jae;Hong, Soon-Kwang;Kim, Chang-Joon
    • Microbiology and Biotechnology Letters
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    • v.40 no.1
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    • pp.1-9
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    • 2012
  • This study's aim was to isolate microorganisms producing agarase with a high activity, with possible applications in improving the performance of the pretreatment processes for bioethanol production. Marine algaes were collected from the south coast of Korea, from which three kinds of microorganisms were isolated. After a 4-day culture of these strains at $25^{\circ}C$, crude enzymes were obtained from culture supernatant or cell-free extract by ammonium sulfate precipitation and membrane dialysis. Agarase activity was observed in these crude enzymes. Notably higher specific activity was observed in the crude enzyme obtained from the culture supernatant rather than that from the cell-free extract. This indicates that a secreted enzyme has a much greater activity than a cellular enzyme. Crude enzymes from the GNUM08122 strain were inferred to have ${\alpha}$-agarase activity because release of p-nitrophenol was observed, possibly due to the cleavage of p-nitrophenyl-${\alpha}$-D-galactopyranoside. The 16S rRNA sequence of GNUM08122 showed a close relationship to Pseudoalteromonas issachenkonii KMM 3549 (99.8%) and Pseudoalteromonas tetraodonis IMA 14160 (99.7%), which led us to assign it to the genus Pseudoalteromonas. Biochemical and physiological study revealed that this strain can grow well at $40^{\circ}C$ under a wide range of pH (pH 4~8) in high-salt conditions (10% NaCl).

Isolation and Identification of a Marine Bacterium, Pseudomonas sp. BK1 Producing Extracellular Enzymes Capable of Decomposing Multiple Complex Polysaccharides (복합 다당류 분해 효소들을 생산하는 해양미생물 Pseudomonas sp. BK1의 분리 및 특성)

  • Kim, Beom-Kyu;Jeon, Beong-Sam;Cha, Jae-Young;Park, Jeong-Won;Kim, Sam-Woong;Kim, Ji-Yoon;Park, Yong-Lark;Cho, Young-Su;Song, Jae-Young
    • Journal of Life Science
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    • v.13 no.6
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    • pp.871-878
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    • 2003
  • A marine bacterium (strain BKl) that produces extracellular enzymes capable of decomposing complex polysac-charides, such as agar, chitin, carboxymethylcellulose, xylan and mannan, was isolated from the marine red alga Porphyra dentata. Strain BKl was gram-negative, aerobic, catalase- and oxidase-positive, polarly flagellated bacilli that produce gelatinase and urease, but not decarboxylases. The G+C content of the DNA was 51.6 mol%. The major isoprenoid quinone component was identified as an ubiquinone-8, and the major cellular fatty acids were C16:0, C16:1 w6c and C18:1 w7c. Comparative 16S rRNA sequence analysis placed strain BK1 with members of the genus Pseudomonas. On the basis of phenotypic and genotypic data, the strain BK1 was shown to be a member of the subgroup of Pseudomonas, and named as Pseudomonas sp. BK1.

Isolation and Characteristics of Microorganisms Producing Extracellular Enzymes from Jeju Traditional Fermented Soybean Paste (Doenjang) (제주전통된장으로부터 세포외효소 분비능이 우수한 미생물의 분리 및 특성)

  • Oh, You-Sung;Park, Ji-Eun;Oh, Hyun-Jeong;Kim, Jung-Hyon;Oh, Myung-Cheol;Oh, Chang-Kyung;Oh, Young-Ju;Lim, Sang-Bin
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.39 no.1
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    • pp.47-53
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    • 2010
  • Bacteria strains with high activities of extracellular enzymes (protease, fibrinolytic enzyme, amylase, cellulase, and lipase) were isolated from Jeju traditional fermented soybean paste (Doenjang), and characterized by 16S rRNA gene sequence analysis and physiological properties. Protease activities were higher in JR14, JR19, JR25, JR32, JR38, JR47, and JR64 than Bacillus subtilis KCCM 12027 (standard strain). Amylase activities were shown in JR6, JR25, JR38, JR56 and JR81, while not in KCCM12027. Cellulase activities were higher in JR6, JR14, JR48, and JR65 than those of other isolated strains and KCCM 12027 whereas lipase activities were the higher in JR-14 and JR-48. Thrombolytic activity in JR19 with high hemolysis activity were 192% compared with that of plasmin as a positive control. Zymogram analysis indicated that the thrombolytic active strains had 4~5 bands in the molecular weight range of 25~75 kDa. Gene sequence analysis of rRNA revealed that the isolated stains had 99% homology with Bacillus species, and the thrombolytic active stain JR19 was B. stratosphericus $41KF2a^T$.

Isolation and Characterization of Yam-Putrefactive Psychrotrophic Bacteria from Rotted Yam (생마 저온부패 원인세균의 분리 및 부패균의 특성)

  • Ryu Hee-Young;Kim Young-Sook;Park Sang-Jo;Lee Bong-Ho;Kwon Soon-Tae;Sohn Ho-Yong
    • Microbiology and Biotechnology Letters
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    • v.34 no.2
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    • pp.109-114
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    • 2006
  • Yam has been recognized as healthy food due to its various biological activities, such as anti-obesity, antimicrobial, anticancer and immuno-stimulation activities, and its consumption has been increased during last decades. In this study, to investigate low-temperature, long-term storage of yam and to develop processed yam products, yam-putrefactive psychrotrophic bacteria were isolated from rotted yam and identified based on BBL identification system, fatty acid analysis in cell membrane and 16S rDNA sequence analysis. The putrefaction activity of isolated thirteen bacteria was evaluated using yam-slices (NaOCl-treated, autoclaved yam and without treatment), and YAM-10 and YAM-12 were identified as major psychrotrophic putrefactive bacteria. Both YAM-10 (Pseudomonas cepacia) and YAM-12 (Pseudomonas rhodesiae) bacteria grew well at 4$\sim$12$^{\circ}C$ and showed strong activity of polymer degrading enzymes, especially amylase, carboxy methyl cellulase and xylanase, at 20$^{\circ}C$. But they failed to grow at acidic pH (<5) or alkaline pH (>10). Our results suggested that the control of psychrotrophic Pseudomonas sp. by pH change and inhibition of polymer degrading enzymes, such as amy-lase, are necessary to long-term storage of yam.

Isolation and Characterization of Wild Yeasts for Improving Liquor Flavor and Quality (주류의 풍미 및 품질 향상을 위한 야생 효모의 분리 및 특성분석)

  • Baek, Seong Yeol;Lee, You Jung;Kim, Jae Hyun;Yeo, Soo-Hwan
    • Microbiology and Biotechnology Letters
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    • v.43 no.1
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    • pp.56-64
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    • 2015
  • It has been known for some time to the wine industry that non-Saccharomyces yeasts play an important role in increasing volatile components through the secretion of extracellular enzymes. The objective of this study was to investigate what types of enzymes are produced by 1,007 non-Saccharomyces yeast strains isolated from Korean fermented foods. Among 1,007 yeast strains, the 566, 45 and 401 strains displayed β-glucosidase, glucanase and protease activity, respectively. In addition, the 563 and 610 strains possessed tolerances against cerulenin and TFL, and the 307 strain was tolerant to 15% ethanol. Yeasts producing harmful biogenic amines and hydrogen sulfide were excluded from further study, and eventually 12 yeast strains belonging to the genera Wickerhamomyces, Hanseniaspora, Pichia, Saccharomyces were identified, based on the 26S rRNA gene sequences. Among the 12 strains, the 9 and 5 strains possessed glucose and ethanol tolerance, respectively. Yeasts belonging to the genus Saccharomyces produced more than 8% alcohol, but non-Saccharomyces yeasts produced only 3% alcohol.