• 아시안잔디학회지
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• 제11권3호
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• pp.173-183
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• 1997

This study was conducted to develop an integrated disease management system against large patch disease. Attempts were made to estimate the effect of calcium hydroxide, silicate fertilizer and urea on the mycelial growth of Rhizoctonia sotani AG2-2 and the development of large patch in vitro and in vivo and to establish the most promising combination of fertilizer, fungicide and antagonistic microbes. 1.The mycelial growth of Rhizoctonia sotani AG2-2 were completely inhibited at 2,000, 1,000 and 3,000ppm concentration by calcium hydroxide, silicate fertilizer and urea, respectively. Inhibition effect of silicate fertilizer was the highest, but that of urea was the lowest compared with other treatments. 2.Treatment of calcium hydroxide at rate of l00g /$m^2$ was the most effective, and control effect appeared from 30 days after treatment in spring, which was better in autumn than in spring. Silicate fertilizer treated at rate of l00g /$m^2$, 200g /m$^2$, in spring and 50g /$m^2$ in autumn were very effective. Urea at rate of 30g /$m^2$ was more effective than 60g /m$^2$ and 120g /m$^2$. 3.The efficacies of mepronil and toclofos-methyl, applied twice in spring and once in autumn, were 83.8% in spring, which persisted to 70% in autumn compared with untreated plot. 4.The efficacies of Trichoderma harzianum were the highest by 55% in spring, but those of Bacillus amyloliquefaciens BL-3 and Peudomonas putida were the highest by 80% in autumn among other antagonists tested, when two organisms were applied twice in spring and once in autumn.

• Journal of Microbiology and Biotechnology
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• 제27권1호
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• pp.9-18
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• 2017

Nine bacilli with fibrinolytic activities were isolated from doenjang, a traditional Korean fermented soy food. Among them, RSB34 showed the strongest activity and was identified as Bacillus amyloliquefaciens by 16S rRNA and recA gene sequencing. During growth on LB up to 96 h, RSB34 showed the highest fibrinolytic activity ($83.23mU/{\mu}l$) at 48 h. Three bands of 23, 27, and 42 kDa in size were observed when the culture supernatant was analyzed by SDS-PAGE and 27 and 42 kDa bands by fibrin zymography. The gene encoding the 27 kDa fibrinolytic enzyme AprE34 was cloned by PCR. BLAST analyses confirmed that the gene was a homolog to genes encoding AprE-type proteases. aprE34 was overexpressed in Escherichia coli BL21(DE3) using pET26b(+). Recombinant AprE34 was purified and examined for its properties. The $K_m$ and $V_{max}$ values of recombinant AprE34 were $0.131{\pm}0.026mM$ and $16.551{\pm}0.316{\mu}M/l/min$, respectively, when measured using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. aprE34 was overexpressed in B. subtilis WB600 using pHY300PLK. B. subtilis transformants harboring pHYRSB34 (pHY300PLK with aprE34) showed higher fibrinolytic activity than B. amyloliquefaciens RSB34.

• The Plant Pathology Journal
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• 제17권3호
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• pp.141-148
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• 2001

Postharvest decay of onion bulbs was examined by inspecting the commercial packages in the market or in storage. Bulb rot incidence was unexpectedly high, and onion bulbs with 1st quality grade were rotten most severely by 51%, followed by 32% for 2nd and 21% for 3rd grades. This indicates that larger bulbs had higher incidences of bulb rots. Major pathogens associated with basal and neck rots were Fusarium oxysporum and Aspergillus sp. or Botrytis allii, respectively, of which basal rot was most prevalent and damaging during storage. Among the epiphytic microorgani는 from onion plants, several Bacillus and Paenibacillus spp. and previously selected Pseudomonas putida and Trichoderma harzianum had inhibitory efficacy against bulb rot pathogens. Among these B. amyloliquefaciens BL-3, Paenibacillus polymyxa BL-4, and P. putida Cha 94 were highly inhibitory to conidial germination of F. oxysporum and B. allii. P. putida Cha 94, B. amyloliquefaciens BL-3, P. polymyxa BL-4, and T. harzianum TM were applied in the rhizoplane of onion at transplanting. Initially antagonist populations decreased rapidly during the first one month. However, among these antagonists, rhizoplane population densities of BL-3, Cha 94, and TM were consistently high thereafter, maintaining about 10$^4$-10$^{5}$ cells or spores per gram of onion root up to harvest time. The other bacterial antagonist BL-4 survived only for two months. TM was the most effective biocontrol agent against basal rot, with the number of rotten bulbs recorded at 4%, while that of the control was 16%. Cha 94 was effective for the first 20 days, but basal rot increased thereafter and had about the same control efficacy as that of BL-3 and BL-4. When the antagonists were applied to the topping areas of onion bulbs at harvest, TM was the most effective in protecting the stored onion bulbs from neck rotting. The second effective antagonist was BL-3. TM and BL-3 completely suppressed the neck rot in another test, suggesting that biocontrol of postharvest decay of onion using these microorganisms either at the time of transplanting or at harvesting may be promising.

• The Plant Pathology Journal
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• 제15권3호
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• pp.162-167
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• 1999

Integration of microbial antagonists with fungicides was tried to control the gray mold caused by Botrytis cinerea on pepper in greenhouse conditions and to reduce fungicide uses. All of the selected bacterial antagonists, Bacillus amyloliquefaciens BL3, Paenibacillus polymyxa BL4, and Pseudomonas putida Cha94, completely inhibited the conidial germination of B. cinerea until 30 days after treatment. However, bacterial colonization of pepper phylloplane was poor in BL4, while the other bacterial isolates and the fungal antagonist Trichoderma harzianum TM colonized well on the phylloplane, maintaining the population density of 104-105 cfu/g until 15 days after microbial treatments. Out of 13 kinds of selected fungicides used for gray mold diseases, polyoxin B and BKF 1995 showed the most discriminatory activity on the fungal growth between B. cinerea and TM. TM grew readily on the media containing those fungicides, while B. cinerea showed poor or no mycelial growth on them. The selected fungicides and antagonists alone reduced incidence of gray mold on pepper, showing disease indices of about 2.4 to 3.0, while its was increased up to 4.2 in the untreated control. Alternate treatments with the antagonists and 2-fold diluted fungicides inhibited the disease incidence as much as the antagonists or fungicides alone, and reduced the secondary inoculum more than the single treatments. This suggests that integration of antagonists and fungicides may be an efficient way to reduce fungicide sprays with reliable control efficacy of the disease. However, there was not much difference in the early and mid-term disease progress among the treatments and the untreated control, probably due to extremely favorable environmental conditions for the disease development in this experiment.

• 한국식품영양과학회지
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• 제46권3호
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• pp.279-288
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• 2017

본 연구에서는 발효콩 유지가 비만 억제 및 예방, 3T3-L1 지방전구세포의 성장 및 분화 억제 효과를 관찰하기 위해 실험을 실시하였다. 3T3-L1 지방전구세포에 발효콩 유지를 처리하여 세포생존율을 측정한 결과, NF군 $100{\mu}g/mL$에서 3T3-L1 지방전구세포의 생존율을 유의적으로 감소시켜 세포독성이 나타났다. 분화를 유도한 다음 세포 내 triglyceride 함량을 측정한 결과, 비발효콩 유지(NF) 처리군보다 발효콩 유지처리군에서 triglyceride 함량 저해 효과가 높게 나타났으며, 특히 BS, LBA, BLO군 순으로 높게 나타났다. 또한, BS군, LBA군, BLO군 순으로 지방세포분화 관련 유전자인 $PPAR{\gamma}$의 mRNA 발현을 농도 의존적으로 감소시켰으며, $PPAR{\gamma}$ 유전자와 상관관계에 있는 $C/EBP{\alpha}$ 유전자도 농도 의존적으로 감소시켰다. 그리고 adiponectin 유전자의 발현은 농도 의존적으로 증가시켰다. 고지방식이로 비만을 유도한 C57BL/6J 생쥐를 이용하여 항비만 활성을 관찰한 결과, 총 체중증가량은 대조군에 비해 4주째에 감소하는 경향이 나타났다. 혈액 내 지질농도를 측정한 결과 중성지방, 총콜레스테롤, LDL-콜레스테롤 함량은 LBA, BLO군에서 유의적으로 낮게 나타났으며(P<0.05), HDL-콜레스테롤 함량은 대조군보다 증가하였으나 유의적인 차이는 나타나지 않았다. 동맥경화 지수인 AI(atherogenic index)는 대조군보다 감소시키는 경향을 보였다. 비만관련 호르몬인 adiponectin의 농도는 SO, BS, LBA, BLO군에서 유의적으로 증가하였고(P<0.05), insulin 농도는 유의적으로 감소하였다(P<0.05). Leptin은 대조군보다 감소하였으나 유의적인 차이는 나타나지 않았다. 따라서 이러한 결과를 종합하여 볼 때 발효콩 유지가 3T3-L1 지방전구세포의 mRNA 단계에서부터 지방분화를 억제하여 항비만 활성을 가지고 있는 것으로 추정되고, 고지방식이로 비만이 유도된 C57BL/6J 생쥐에서 총체중을 감소시키고, 혈액 내 지질농도인 triglyceride와 총콜레스테롤, LDL-콜레스테롤 및 HDL-콜레스테롤과 비만관련 호르몬인 adiponectin, insulin, leptin을 조절하여 항비만 활성을 가지고 있는 것으로 확인되었다. 이 결과로 발효콩 유지가 항비만 생리활성을 나타내는 새로운 식품 소재로서 이를 활용한 기능성 식품 개발의 가능성이 있다고 생각된다.

• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.1070-1083
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• 2015

A gene (GT-SM3B) encoding a thermostable secreted oligoendopeptidase (GT-SM3B) was cloned from the thermophile Geobacillus thermoleovorans DSM 15325. GT-SM3B is 1,857 bp in length and encodes a single-domain protein of 618 amino acids with a 23-residue signal peptide having a calculated mass of 67.7 kDa after signal cleavage. The deduced amino acid sequence of GT-SM3B contains a conservative zinc metallopeptidase motif (His⁴⁰⁰-Glu⁴⁰¹-X-XHis⁴⁰⁴). The described oligopeptidase belongs to the M3B subfamily of metallopeptidases and displays the highest amino acid sequence identity (40.3%) to the oligopeptidase PepF_Ba from mesophilic Bacillus amyloliquefaciens 23-7A among the characterized oligopeptidases. Secretory production of GT-SM3B was used, exploiting successful oligopeptidase signal peptide recognition by Escherichia coli BL21 (DE3). The recombinant enzyme was purified from the culture fluid. Homodimerization of GT-SM3B was determined by SDS-PAGE. Both the homodimer and monomer were catalytically active within a pH range of 5.0–8.0, at pH 7.3 and 40℃, showing the K_m, V_max, and k_cat values for carbobenzoxy-Gly-Pro-Gly-Gly-Pro-Ala-OH peptidolysis to be 2.17 ± 0.04 × 10^-6 M, 2.65 ± 0.03 × 10^-3 µM/min, and 5.99 ± 0.07 s^-1, respectively. Peptidase remained stable at a broad pH range of 5.0–8.0. GT-SM3B was thermoactive, demonstrating 84% and 64% of maximum activity at 50℃ and 60℃, respectively. The recombinant oligopeptidase is one of the most thermostable M3B peptidase, retaining 71% residual activity after incubation at 60℃ for 1 h. GT-SM3B was shown to hydrolyze a collagenous peptide mixture derived from various types of collagen, but less preferentially than synthetic hexapeptide. This study is the first report on an extracellular thermostable metallo-oligopeptidase.