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Purification and Characterization of Laccase from Wood-Degrading Fungus Trichophyton rubrum LKY-7

  • Hyunchae Jung;Park, Chongyawl;Feng Xu;Kaichang Li
    • Proceedings of the Korea Technical Association of the Pulp and Paper Industry Conference
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    • 2001.04a
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    • pp.18-25
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    • 2001
  • A new wood-degrading fungus Trichophyton rubrum LKY-7 secretes a high level of laccase in a glucose-peptone liquid medium. The production of laccase by the fungus was barely induced by 2,5-xylidine. The laccase has been purified to homogeneity through three chromatography steps in an overall yield of 40%. The molecular mass of the purified laccase was about 65 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified laccase had the distinct blue color and had basic spectroscopic features of a typical blue laccase: two absorption maxima at 278 and 610 nm and a shoulder at 338 nm. The N-terminus of the laccase has been sequenced, revealing high homology to laccases from wood-degrading white-rot fungi such as Ceriporiopsis subvermispora. The enzyme had a "low" redox potential (0.5 V vs normal hydrogen electrode), yet it was one of the most active laccases in oxidizing a series of representative substrates/mediators. Compared with other fungal laccases, the laccase has a very low Km value with ABTS [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid] as a substrate and a very high Km value with violuric acid as a substrate. The laccase has the isoelectric point of 4.0. The laccase had very acidic optimal pH values (pH 3-4) while it was more stable at neutral pH than at acidic pH. The laccase oxidized hydroquinone faster than catechol and pyrogallol. The oxidation of tyrosine by the laccase was not detectable under the reaction conditions. The laccase was strongly inhibited by sodium azide and sodium fluoride. fluoride.

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Analysis of Antioxidative and Antimutagenic Activities of Ethanol Extracts from Pericarp and Seeds of Wild Grape (Vitis coignetiea) (머루 과피와 종자 에탄올 추출물의 항산화 활성 및 항돌연변이 활성 분석)

  • Won, Ji Hye;Kim, Meera
    • Journal of the East Asian Society of Dietary Life
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    • v.26 no.2
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    • pp.192-199
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    • 2016
  • The antioxidative activity and antimutagenic activity of the ethanol extracts from pericarp and seeds of wild grape (Vitis coignetiea) were analyzed in this study. The antioxidative activity of the extracts from wild grape was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. The antimutagenic activity of the extracts was evaluated on Salmonella typhimurium TA98 and TA100 by Ames test using 4-nitroquinoline 1-oxide (4-NQO) and sodium azide as mutagens. In the antioxidative activity determination, $IC_{50}$ values of the DPPH radical scavenging activity of the extracts from pericarp and seeds were 27.16 ppm and 7.61 ppm, respectively. Additionally, ABTS radical scavenging activities of pericarp and seed extract were 99.75% and 98.87% at 200 ppm, respectively. In the antimutagenic activity determination, pericarp extract at 5 mg/plate inhibited 72.6% and 74.3% of mutagenicity of S. typhimurium TA98 induced by 4-NQO and sodium azaid, respectively. Also, the mutagenicity inhibition rates of seed extract at 5 mg/plate were 77.8% and 74.5% in S. typhimurium TA100 induced by 4-NQO and sodium azaid, respectively. These results demonstrate that the ethanol extract from wild grape has remarkable antioxidant activity and antimutagenicity.

Evaluation on Early-maturing Korean Japonica Cultivars for High-temperature Tolerance during Grain Filling Stage (국내 육성 조생종 벼 품종들에 대한 등숙기 고온내성 평가)

  • Cho, Seong-Woo;Jeung, Ji-Ung;Kang, Kyung-Ho;Kim, Hyun-Soon;Kim, Bo-Kyeong
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.60 no.2
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    • pp.146-152
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    • 2015
  • Early-maturing Korean Japonica cultivars and Jungmo1024 were used as plant materials to evaluate hightemperature tolerance during grain filling stage. National Institute of Crop Science (NICS) in Korea developed Jungmo1024, a mutant line from Namil (wild type) treated by using sodium azide (SA) as mutagen. To evaluate high-temperature tolerance, all cultivars were exposed to high-temperature (day $31.5{\pm}2.5^{\circ}C$/ night $27.5{\pm}1.3^{\circ}C$) and ordinary temperature (day $27.5{\pm}1.8^{\circ}C$/ night $24.7{\pm}1.6^{\circ}C$) during grain filling stage. In these conditions, we performed compared evaluation of grain shape such as length and width and grain quality by using a 1625 Cervitec grain inspector. High-temperature during grain filling stage caused decrease of grain shape. In grain shape such as length and width, the decrease rate of width (average 6.3%) was higher than the decrease rate of length (average 1.3%). Hence, high-temperature affected width of grain than length of grain. In addition, high-temperature showed a decided difference in rate of head rice between ordinary temperature (average 76.3%) and high-temperature (average 13.3%).As a result, Taebong, Ungwang, Manan, and Jungmo1024 seemed relatively a decent high-temperature tolerance than other cultivars. Especially, Jungmo1024 seemed remarkable rate of head rice (average $34.4{\pm}6.2%$) than other cultivars under high-temperature. It is considered that a genetic trait of Jungmo1024 can be useful to improve breeding for high-temperature tolerance.

The Types of Extended-Spectrum ${\beta}$-Lactamases Isolated from Suyeong Sewage Disposal Plant, Busan Environmental Corporation (부산 수영공공하수처리시설에서 분리된 광범위 항균제 베타락 탐 분해효소(Extended-Spectrum ${\beta}$-Lactamase, ESBL) 유형)

  • Kim, Gun-Do;Lee, Hun-Ku
    • Korean Journal of Microbiology
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    • v.46 no.1
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    • pp.38-45
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    • 2010
  • The study performed to identify the type of ESBL against strains which are producing extendedspectrum ${\beta}$-lactamases and isolated from sewage in Suyeong sewage disposal plant, Busan Environmental Corporation. By the standard activated sludge method, Suyeong sewage disposal plant purify living and lavatory sewage gathering from the northeast Busan and the facility purify total 550,000 tons of living sewage disposal a day. 14 strains were isolated by double disk synergy test and the third generation cepha-antibiotics test. Indole, methyl-red, Voges-Proskauer, Simmon's citrate, decarboxylasedihydrolase and sugar-fermentation tests identified as Klebsiella pneumoniae (n=4) and Escherichia coli (n=10). Plasmid-mediated transmission test against isolated 14 strains proved 11 strains transmitted resistance to recipient E. coli J53 (sodium $azide^R$, $ceftazidime^S$). 9 strains of conjugant were expressed ESBL genes transferred from parental strain but 2 conjugants did not expressed. The type of ESBL from each strain was determined by isoelectric focusing points, DNA and amino acids sequencing. The results indicated that the types of ESBL transmitted to recipient E. coli J53 were TEM-1, the parental TEM type and SHV-12 type.

Studies on coagulation of cheese curd by immobilized Mucor spp L42 milk clotting enzyme (고정화 Mucor SPP L42 응유효소를 이용한 치즈커드 형성에 관한 연구)

  • Park, Jong Lae
    • Current Research on Agriculture and Life Sciences
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    • v.6
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    • pp.157-162
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    • 1988
  • In order to study of practical purpose of immobilized Mucor spp L42 milk clotting enzyme on activated succimylamino-propyl glass beads with glutaraldehyde in continuous curd coagulation, acidified milk(pH5.6, $8^{\circ}C$) was treated through reactor packed with immobilized beads, and warmed at $30^{\circ}C$ and allowed to coagulation for the determination of enzyme stability, deactivation of milk clotting ability by continuous reaction, the beads treatment conditions, and contact time of milk and beads in reactors. The results obtained were summarized as follow ; 1) After 3 month's storage, activity of immobilized Mucor spp L42 milk clotting enzyme in 0.2M phosphate buffer(pH 4.6) with 0.06% sodium azide was only 80% of initial activity. 2) Milk clotting activity of the beads was decreased by continuouse exposure on acidified skim milk. Nitrogen accumulation on the beads paralled loss of the activity in initial reaction stage. 3) After 6 hours continuous treatment of the beads at 60 sec/ml surface time, the milk-clotting activity of the beads was about 70% of initial activity. 4) Bead reactor and shaking bed reactor were more effective than column reactor on continuouse skim milk coagulation.

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Cloning and Characterization of a Cellulase Gene from a Plant Growth Promoting Rhizobacterium, Bacillus subtilis AH18 against Phytophthora Blight Disease in Red-Pepper (고추역병을 방제하는 PGPR균주 Bacillus subtilis AH18의 항진균성 Cellulase 유전자의 Cloning 및 효소 특성 조사)

  • Woo, Sang-Min;Jung, Hee-Kyoung;Kim, Sang-Dal
    • Microbiology and Biotechnology Letters
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    • v.34 no.4
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    • pp.311-317
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    • 2006
  • Using PCR amplification, we cloned a cellulase gene (ce/H) from the Bacillus subtilis AH18 which has plant growth-promoting activity and antagonistic ability against pepper blight caused by Phytophthora capsici. The 1.6 kb PCR fragment contained the full sequence of the cellulase gene and the 1,582 bp gene deduced a 508 amino acid sequence. Similarity search in protein database revealed that the cellulase of B. subtilis AH18 was more than 98% homologous in the amino acid sequence to those of several major Bacillus spp. The ce/H was expressed in E. coli under an IPTG inducible lac promoter on the vector, had apparent molecular weight of about 55 kDa upon CMC-SDS-PAGE analysis. Partially purified cellulase had not only cellulolytic activity toward carboxymethyl-cellulose (CMC) but also insoluble cellulose, such as Avicel and filter paper (Whatman No. 1). In addition, the cellulase could degrade a fungal cell wall of Phytophthora capsici. The optimum pH and temperature of the ce/H coded cellulase were determined to be pH 5.0 and $50^{\circ}C$. The enzyme activity was activated by $AgNO_3$ or $CoCl_2$. However its activity was Inhibited by $HgC1_2$. The enzyme activity was activated by hydroxy urea or sodium azide and inhibited by CDTA or EDTA. The results indicate that the cellulase gene, ce/H is an antifungal mechanism of B. subtilis AH18 against phytophthora blight disease in red-pepper.

Production and Properties of Laccase from Coriolus versicolor (Coriolus versicolor에 의한 Laccase 생산(生産) 및 성질(性質)에 관한 연구(硏究))

  • Hong, Jai-Sik;Kim, Myung-Kon;Kim, Yun-Hi;Lee, Jong-Bae
    • The Korean Journal of Mycology
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    • v.15 no.2
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    • pp.99-170
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    • 1987
  • The production and properties of laccase(E.C.1.10.3.2) from Coriolus versicolor were studied. The results were as follows; The nutritional optimum conditions for laccase production were 1% indulin At, 0.3% peptone 0.1% $KH_2PO_4$, 0.02% $MgSO_4$, 0.1 mg% $CuSO_4$.and 0.005 mg% thiamine HCI. The optimum temperature and pH of laccase production were $25^{\circ}C$ and 5.0, and respectively, and the cultural period was 20 days. The optimum pH and temperature for the activity were 4.6 and $40^{\circ}C$, respectively. The enzyme was almost stable under the temperature of $40^{\circ}C$ and within the pH range of 4.0-5.0. The enzyme was stable at $40^{\circ}C$ for 30 min. $Cu^{++}$, $Fe^{++}$ and $Ca^{++}$ activated the enzyme activity, but $Mn^{++}$ and $Hg^{++}$ were inhibited. The enzyme was totally inhibited by 1 mM sodium azide and 1 mM potassium cyanide, and partly inhibited by EDTA and hydroxyamine.

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The Types of Extended-Spectrum $\beta$-Lactamase (ESBL) Produced by Enteric Bacteria, Klebsiella pneumoniae and Escherichia coli Isolated from Sewage of Wastewater Treatment Plant at Minragdong in Busan, Korea (부산 민락동 오수처리장에서 분리된 장내세균 Klebsiella pneumoniae와 Escherichia coli가 생성한 광범위 베타 락탐(Extended-Spectrum $\beta$-Lactamase, ESBL) 분해효소의 유형)

  • Lee, Hun-Ku
    • Korean Journal of Microbiology
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    • v.45 no.2
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    • pp.163-169
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    • 2009
  • This study was performed to investigate the type of extended-spectrum $\beta$-lactamases (ESBL) produced by bacteria isolated from the sewage of wastewater treatment plant at Minragdong, Suyong-gu in Busan. The facility is located at sushi restaurants and guides its drain water to the wastewater treatment plant at Yonghodong, Nam-gu in Busan. Samples were collected on January, 2009. A total of 19 strains were selected as potential ESBL positive strains through a double disk synergy test. On the basis of the results from biochemical tests including indole, methyl-red, Voges-Proskauer, Simmon's citrate, decarboxylase-dihydrolase and sugar-fermentation tests, the 19 strains were identified with 16 strains of Escherichia coli and 3 strains of Klebsiella pneumoniae. Out of 19 strains, 4 transconjugants against Escherichia coli J53, which is sodium azide resistant recipient strain, were obtained. The plasmids isolated from transconjugants were used for PCR analysis. The type of each extended-spectrum $\beta$-lactamase (ESBL) produced by the strains was determined on the basis of isoelectric focusing analysis and DNA sequencing. The results indicated that the types of ESBL from Klebsiella pneumoniae were SHV-12 (3 strains), and Escherichia coli was SHV-12/TEM-1 (1 strain), respectively.

Evaluation of Fermentation Ability of Microbes for Corn Silage Inoculant (옥수수 사일리지용 미생물의 발효능력 평가)

  • Kim, Jong-Geun;Ham, Jun-Sang;Chung, Eui-Soo;Seo, Sung;Park, Hyung-Soo
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.30 no.4
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    • pp.333-342
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    • 2010
  • Corn is very important forage in Korea. The great part of them is utilized as silage. Generally, it contains a lot of grains that is feed of animal. This experiment was conducted to evaluation of fermentation ability of microbes for corn silage inoculant. Good lactic acid bacteria were isolated from good corn silage by plating MRS agar containing 0.02% sodium azide, and assessed by growing and acid producing ability in MRS broth. Six lactic acid bacteria were selected, and were found to be Gram positive, rods and catalase negative and were identified to be lactobacillus plantarum (C3-2, B13-1, CC9-1), Lactobacillus fermentum (C11-4), Lactobacillus paracasei (B14-1), and Leuconostoc lactis (A3-1) on the basis of the biochemical characteristics and utilization of substrates. Corn was ensiled at ripen stage following treatment with selected five lactic acid bacteria, two commercial inoculant, and no additive (control). After 2 month, B13-1 and CC9-1 bacteria inoculated silage were lower pH and higher lactic acid content than others treatments. The Flieg's score and grade of B13-1 and CC9-1 bacteria treated silage were higher than commercial inoculant. According to this experiment, lactobacillus plantarum B13-1 and CC9-1 strain were recommendable for good inoculant of corn silage.

Antimicrobial Activity against Food Hazardous Microorganisms and Antimutagenicity against Salmonella serotype Typhimurium TA100 of an Ethanol Extract from Sanguisorba officinalis L. (지유 에탄올 추출물의 식품 위해성 세균에 대한 항균 활성 및 Salmonella serotype Typhimurium TA100에 대한 항돌연변이 활성 효과)

  • Kim, Se-Ryoung;Won, Ji-Hye;Kim, Mee-Ra
    • Korean journal of food and cookery science
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    • v.27 no.4
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    • pp.17-26
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    • 2011
  • This study was performed to analyze the antibacterial activity against food hazardous microorganisms and antimutagenic effects of Sanguisorba officinalis L. ethanol extracts on Salmonella Typhimurium TA100. The antibacterial activity was evaluated by paper disc diffusion assay, minimum inhibition concentration (MIC), and optical density of the culture with the ethanol extract for 24 hr. Antibacterial activity was tested with seven microorganisms including Escherichia coli, Escherichia coli O157:H7, Pseudomonas aeruginosa, Salmonella Typhimurium, Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus. The paper disc diffusion assay showed distinct clear inhibition zones around the discs treated with the extract for five microorganisms, except Escherichia coli and Escherichia coli O157:H7. MIC values were 0.625-2.5 mg/mL for these five strains that showed clear zones. The time-kill assay was consistent with the results from the paper disc diffusion assay and MIC test. Additionally, antimutagenicity of the extract was determined using the Ames test. The ethanol extract at 5 mg/plate inhibited 72.42% and 89.85% of mutagenicity induced by 4-nitroquinoline 1-oxide and sodium azide, respectively. These results demonstrate that the ethanol extract from S. officinalis L. has remarkable antibacterial activity and antimutagenicity.