• Title/Summary/Keyword: soil enzyme activity

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Immobilization of Bacillus sp. Strains, Catalase Producing Bacteria and Their Hydrogen Peroxide Removal Characteristics (카탈라제를 생산하는 고초균 (Bacillus sp.)의 고정화 및 과산화수소 분해 특성)

  • Han, Kyung-Ah;Jang, Yun-Hee;Rhee, Jong-Il
    • KSBB Journal
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    • v.25 no.6
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    • pp.520-526
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    • 2010
  • In this work we have investigated the production of catalase from Bacillus sp. strains, which were screened and identified from soil. These strains were cultivated in shaking flasks with tryptic soy broth (TSB) at $30^{\circ}C$ and 200 rpm. Effects of the temperature and pH on the stability of the native catalase and whole cell viability were studied in the temperature range of $25-60^{\circ}C$ and the pH range of 7-13. Korean natural zeolite was added to culture medium and mixed with microorganisms for 24 hours. The native catalase maintained its activity over $50^{\circ}C$. The enzyme acitiviy of the catalase from Bacillus flexus BKBChE-3 was highest among the Bacillus sp. strains studied. Bacillus flexus BKBChE-3 and immobilized Bacillus cells have survived under extreme conditions of over $50^{\circ}C$ and pH 12. 60 mL of 10.5 mM $H_2O_2$ solution were entirely removed within 1 hour with catalase produced from Bacillus sp. on the flask. When Bacillus cells were immobilized on Korean natural zeolite, colony forming unit of Bacillus flexus BKBChE-3 was increased and high efficiency of hydrogen peroxide removal was observed.

Purification and characterization of the chitinase from Bacillus subtilis JK-56 (Bacillus subtilis JK-56이 생산하는 chitinase isozyme의 정제와 특성 규명)

  • 전홍기;김낙원;정영기
    • Journal of Life Science
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    • v.12 no.1
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    • pp.77-86
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    • 2002
  • Chitin, a $\beta$-1,4 polymer of N-acetyl-D-glucosamine, is one of the most abundant organic compounds in nature. Chitinase (EC 3.2.1.14) is an enzyme that degrades chitin to chito-oligosaccharides, diacetyl rhitobiose and N-acetyl-D-glucosamine. An extracellular chitinase-producing bacterial strain was isolated from soil and named to as Bacillus subtilis JK-56. Optimum culture condition of B. subtilis JK-56 for the production of chitinase was 1% chitin, 0.5% polypepton, 0.1% KCl, 0.05% MnS $O_4$.4$H_2O$, 37$^{\circ}C$, initial pH 7.0 and 40 hour culture time. When B. subtilis JK-56 was grown in the optimum medium, one major active band and two minor active bands were detected by native-PAGE and active staining of the gel. Among them, the major band was purified from the culture supernatant by 70% ammonium sulfate precipitation and native-PAGE with BIO-RAD Model 491 Prep-Cell and named as Chi-56A. Its molecular weight was estimated to be 53kDa monomer and the isoelectric point (pI) was pH 4.3. The pH and temperature for the optimum activity of Chi-56A were pH 6.0 and $65^{\circ}C$, respectively. Chi-56A was stable up to $65^{\circ}C$ and in alkaline region. Its $K_{m}$ value for colloidal chitin was 17.33g/L. HPLC analysis of the reaction products confirmed that Chi-56A was an exo type chitinase.e.

Selection and Cultural Characteristics of Whole Chicken Feather-Degrading Bacterium, Bacillus sp. SMMJ-2 (Whole Chicken Feather-Degrading Keratinolytic Protease 생산균주의 분리 및 특성)

  • Park Sung-Min;Jung Hyuck-Jun;Yu Tae-Shick
    • Microbiology and Biotechnology Letters
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    • v.34 no.1
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    • pp.7-14
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    • 2006
  • Feather, generated in large quantities as a byproduct of commercial poultry processing, is almost pure keratin, which is not easily degradable by common professes. Four strains, SMMJ-2, FL-3, NO-4 and RM-12 were isolated from soil for production of extracellular keratinolytic protease. They were identified as Bacillus sp. based on their morphological and physiological characteristics. They shown high protease activity on 5.0% skim milk agar medium and produced a substrate like mucoid on keratin agar medium. Bacillus sp. SMMJ-2 had a faster production time for producing keratinolytic protease than other strains. This strain did not completely degrade whole chicken feather for five days in basal medium but completely degraded whole chicken feather when supplied with nitrogen source for 40hours in keratinolytic producing medium ($0.7%\;K_{2}HPO_{4},\;0.2%\;KH_{2}PO_{4},\;0.1%$ fructose, 1.2% whole chicken feather, $0.01%\;Na_{2}CO_3$, pH 7.0). When supplied with chicken feather as nitrogen source, keratinolytic protease activity was 89 units/ml/min. When soybean meal was used as nitrogen source, the keratinolytic protease production reached a maximum of 106 units/ml/min after 48 hours under $30^{\circ}C$, 180 agitation. To isolate the keratinolytic protease, the culture filtrate was precipitated with $(NH_4)_{2}SO_4$ and acetone. The recovery rate of keratinolytic protease was about 96% after treatment with 50% acetone. The enzyme was stable in the range of $30{\sim}50^{\circ}C$ and pH $6.0{\sim}12.0$.

Studies on the Germination Characteristics of Sesame (Sesamum indicum L.) (참깨의 발아특성(發芽特性)에 관(關)한 연구(硏究))

  • Kim, Choong Soo
    • Korean Journal of Agricultural Science
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    • v.10 no.1
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    • pp.28-60
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    • 1983
  • This study was carried out to define the effects of external factors including temperature, moisture, oxygen and light quality on the germination of sesame seeds and to investigate the change of major chemical constituents of seeds during germination. The results obtained are summarized as follows: 1. The average germination ratio was from 95.8% to 97.2% when it was tested every $5^{\circ}C$ intervals from $20^{\circ}C$ to $35^{\circ}C$ and no significant difference in germination ratio was found within $20^{\circ}C$ to $35^{\circ}C$. But the germination ratio dropped rapidly to 32.2% when seeds were germinated at $15^{\circ}C$ and the coefficient of variation become greater(77%) 2. The days required for germination ranged from 1.16 to 1. 64 at the temperatures of $35^{\circ}C$ to $25^{\circ}C$ and they were 3.07 and 10.4 at the temperatures of $20^{\circ}C$ and $15^{\circ}C$, respectively. 3. Considering the germination ratio and days needed, $15^{\circ}C$ was assumed to be the minimum temperature for germination practically and this temperature is recommended for testing low temperature tolerance of seed germination of sesame cultivars. 4. The varieties shown the highest low temperature tolerance were Shirogoma and Turkey. The next varieties shown some degree of low temperature germination were Suweon #29, Naebok and IS 58. The varieties with 70 to 80% of germination ratio were Maepo, Suweon #14, Kimpo, Moondeok, and Haenam. Among the 90 varieties tested, the varieties with comparatively high degree of low temperature tolerance were about 10%, and 70% of the low temperature tolerant varieties were domestic varieties. 5. At $12^{\circ}C$ the Shirogoma was the only variety which showed over 50% of germination ratio, 71.4% of the varieties showed less than 20% of germination ratio. When the temperature was raised to $27^{\circ}C$ 18 days after placement at $12^{\circ}C$ all the varieties showed over 90% of germination ratio within 2days. 6. The amounts of water imbibition needed for seed germination were 0.48 to 0.62 times of the seed dry weight at $25^{\circ}C$ and were significantly different among sesame cultivars. About 63% of water required for germination was imbibed in 2 hours after placement of seeds under the germination condition. 7. Under saturated moisture condition the average germination ratio was 0.42%. In the soil of which water potential was -0.4bar 64.8% of the seeds germinated and the most adequate soil water potential for sesame seed germination was about -0.4 to -5.5 bar. The germination ratio decreased as the soil water potential declined below -5.5 bar. 8. Six out of 10 varieties were not influenced by 5% of oxygen in air germination chamber, while varieties such as Yecheon, PI 158073, IS 103 and Euisangcheon showed 64 to 91% of germination under the 5% oxygen content. Under anaerobic condition, cotyledones were not emerged but only hypocotyl was emerged and elongated. The germination ratio of IS 103 decreased significantly under anaerobic condition. 9. When the seeds were dried for 24 hours after 12 hours imbibition of water, the seeds of Cheongsong did not lose their germination ability and 27.5% was germinated but Suweon #9 and Early Russian failed to germinate. However, the germination ratio of IS 103 decreased when the seed were dried 24 hours after 4 hours imbibition of water and the germination ability of IS 103 was maintained even though the seeds were dried for 24 hours after 24 hours imbibition of water. 10. During germination, sugar content of sesame seed increased rapidly and activity of ${\alpha}$-amylase increased gradually while starch content decreased significantly. The rates of increase in sugar content and enzyme activity and decrease in starch content were significantly lower at $15^{\circ}C$ compared with those at $25^{\circ}C$. 11. During germination of sesame seeds, lipid content in the seeds dropped rapidly and the activity of alkaline lipase increased significantly at early stage of germination. The rate of decrease in lipid content and increase in emzyme activity was lower at $15^{\circ}C$ than at $25^{\circ}C$. 12. Four out of 6 varieties were not affected in germination by light wave length. But Suweon #8 was inhibited in germination by 600-650nm. and IS 103 by 600 to 650nm and 500 to 550nm of light wave length. Suweon #8 showed high germination ratio under 650 to 760 nm and 500 to 560nm, and IS 103 under 400 to 470nm and complete darkness. 13. The germination ratios increased significantly in the seeds of which 1000 grain weight is heavier. When the seeds were placed at soil 4cm deep, Cheongsong and Early Russian failed to emerge their cotyledones, but Suweon #9 and IS 103 showed 32.5 and 50% cotyledone emergence, respectively. The extracts from sesame plant and soil where the sesame was cultivated previously did not affect in the-germination of sesame seeds. 14. The covering by black or transparent polyethylene films increased germination ratio compared with uncovered seeds. The covering was effective in shortening the days needed for germination and in improving the early seedling growth, number of capsules per plant and grain yield. Difference was not so seizable between the two polyethylene films but the transparent film appeared somewhat more effective than the black one. 15. Simcheon, Cheongsong. Suweon #9. PI 158073 and IS 103 showed lower rate of water absorbtion by seed during germination and Suweon #8, Suweon #26, Orotall and Euisangcheon showed high increase in seed weight after water absorbtion by seed.

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Brief Introduction of Research Progresses in Control and Biocontrol of Clubroot Disease in China

  • He, Yueqiu;Wu, Yixin;He, Pengfei;Li, Xinyu
    • 한국균학회소식:학술대회논문집
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    • 2015.05a
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    • pp.45-46
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    • 2015
  • Clubroot disease of crucifers has occurred since 1957. It has spread to the whole China, especially in the southwest and nourtheast where it causes 30-80% loss in some fields. The disease has being expanded in the recent years as seeds are imported and the floating seedling system practices. For its effective control, the Ministry of Agriculture of China set up a program in 2010 and a research team led by Dr. Yueqiu HE, Yunnan Agricultural University. The team includes 20 main reseachers of 11 universities and 5 institutions. After 5 years, the team has made a lot of progresses in disease occurrence regulation, resources collection, resistance identification and breeding, biological agent exploration, formulation, chemicals evaluation, and control strategy. About 1200 collections of local and commercial crucifers were identified in the field and by artificiall inoculation in the laboratories, 10 resistant cultivars were breeded including 7 Chinese cabbages and 3 cabbages. More than 800 antagostic strains were isolated including bacteria, stretomyces and fungi. Around 100 chemicals were evaluated in the field and greenhouse based on its control effect, among them, 6 showed high control effect, especially fluazinam and cyazofamid could control about 80% the disease. However, fluzinam has negative effect on soil microbes. Clubroot disease could not be controlled by bioagents and chemicals once when the pathogen Plasmodiophora brassicae infected its hosts and set up the parasitic relationship. We found the earlier the pathogent infected its host, the severer the disease was. Therefore, early control was the most effective. For Chinese cabbage, all controlling measures should be taken in the early 30 days because the new infection could not cause severe symptom after 30 days of seeding. For example, a biocontrol agent, Bacillus subtilis Strain XF-1 could control the disease 70%-85% averagely when it mixed with seedling substrate and was drenching 3 times after transplanting, i.e. immediately, 7 days, 14 days. XF-1 has been deeply researched in control mechanisms, its genome, and development and application of biocontrol formulate. It could produce antagonistic protein, enzyme, antibiotics and IAA, which promoted rhizogenesis and growth. Its The genome was sequenced by Illumina/Solexa Genome Analyzer to assembled into 20 scaffolds then the gaps between scaffolds were filled by long fragment PCR amplification to obtain complet genmone with 4,061,186 bp in size. The whole genome was found to have 43.8% GC, 108 tandem repeats with an average of 2.65 copies and 84 transposons. The CDSs were predicted as 3,853 in which 112 CDSs were predicted to secondary metabolite biosynthesis, transport and catabolism. Among those, five NRPS/PKS giant gene clusters being responsible for the biosynthesis of polyketide (pksABCDEFHJLMNRS in size 72.9 kb), surfactin(srfABCD, 26.148 kb, bacilysin(bacABCDE 5.903 kb), bacillibactin(dhbABCEF, 11.774 kb) and fengycin(ppsABCDE, 37.799 kb) have high homolgous to fuction confirmed biosynthesis gene in other strain. Moreover, there are many of key regulatory genes for secondary metabolites from XF-1, such as comABPQKX Z, degQ, sfp, yczE, degU, ycxABCD and ywfG. were also predicted. Therefore, XF-1 has potential of biosynthesis for secondary metabolites surfactin, fengycin, bacillibactin, bacilysin and Bacillaene. Thirty two compounds were detected from cell extracts of XF-1 by MALDI-TOF-MS, including one Macrolactin (m/z 441.06), two fusaricidin (m/z 850.493 and 968.515), one circulocin (m/z 852.509), nine surfactin (m/z 1044.656~1102.652), five iturin (m/z 1096.631~1150.57) and forty fengycin (m/z 1449.79~1543.805). The top three compositions types (contening 56.67% of total extract) are surfactin, iturin and fengycin, in which the most abundant is the surfactin type composition 30.37% of total extract and in second place is the fengycin with 23.28% content with rich diversity of chemical structure, and the smallest one is the iturin with 3.02% content. Moreover, the same main compositions were detected in Bacillus sp.355 which is also a good effects biocontol bacterial for controlling the clubroot of crucifer. Wherefore those compounds surfactin, iturin and fengycin maybe the main active compositions of XF-1 against P. brassicae. Twenty one fengycin type compounds were evaluate by LC-ESI-MS/MS with antifungal activities, including fengycin A $C_{16{\sim}C19}$, fengycin B $C_{14{\sim}C17}$, fengycin C $C_{15{\sim}C18}$, fengycin D $C_{15{\sim}C18}$ and fengycin S $C_{15{\sim}C18}$. Furthermore, one novel compound was identified as Dehydroxyfengycin $C_{17}$ according its MS, 1D and 2D NMR spectral data, which molecular weight is 1488.8480 Da and formula $C_{75}H_{116}N_{12}O_{19}$. The fengycin type compounds (FTCPs $250{\mu}g/mL$) were used to treat the resting spores of P. brassicae ($10^7/mL$) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A260) and at 280 nm (A280) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be lyzed by the FTCPs of XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol. In the five selected medium MOLP, PSA, LB, Landy and LD, the most suitable for growth of strain medium is MOLP, and the least for strains longevity is the Landy sucrose medium. However, the lipopeptide highest yield is in Landy sucrose medium. The lipopeptides in five medium were analyzed with HPLC, and the results showed that lipopeptides component were same, while their contents from B. subtilis XF-1 fermented in five medium were different. We found that it is the lipopeptides content but ingredients of XF-1 could be impacted by medium and lacking of nutrition seems promoting lipopeptides secretion from XF-1. The volatile components with inhibition fungal Cylindrocarpon spp. activity which were collect in sealed vesel were detected with metheds of HS-SPME-GC-MS in eight biocontrol Bacillus species and four positive mutant strains of XF-1 mutagenized with chemical mutagens, respectively. They have same main volatile components including pyrazine, aldehydes, oxazolidinone and sulfide which are composed of 91.62% in XF-1, in which, the most abundant is the pyrazine type composition with 47.03%, and in second place is the aldehydes with 23.84%, and the third place is oxazolidinone with 15.68%, and the smallest ones is the sulfide with 5.07%.

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