• Journal of Microbiology and Biotechnology
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• 제23권2호
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• pp.167-176
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• 2013

A novel bioactive molecule produced by Bacillus thuringiensis subsp. kurstaki Bn1 (Bt-Bn1), isolated from a common pest of hazelnut, Balaninus nucum L. (Coleoptera: Curculionidae), was determined, purified, and characterized in this study. The Bt-Bn1 strain was investigated for antibacterial activity with an agar spot assay and well diffusion assay against B. cereus, B. weinhenstephenensis, L. monocytogenes, P. savastanoi, P. syringae, P. lemoignei, and many other B. thuringiensis strains. The production of bioactive molecule was determined at the early logarithmic phase in the growth cycle of strain Bt-Bn1 and its production continued until the beginning of the stationary phase. The mode of action of this molecule displayed bacteriocidal or bacteriolytic effect depending on the concentration. The bioactive molecule was purified 78-fold from the bacteria supernatant with ammonium sulfate precipitation, dialysis, ultrafiltration, gel filtration chromatography, and HPLC, respectively. The molecular mass of this molecule was estimated via SDS-PAGE and confirmed by the ESI-TOFMS as 3,139 Da. The bioactive molecule was also determined to be a heat-stable, pH-stable (range 6-8), and proteinase K sensitive antibacterial peptide, similar to bacteriocins. Based on all characteristics determined in this study, the purified bacteriocin was named as thuricin Bn1 because of the similarities to the previously identified thuricin-like bacteriocin produced by the various B. thuringiensis strains. Plasmid elution studies showed that gene responsible for the production of thuricin Bn1 is located on the chromosome of Bt-Bn1. Therefore, it is a novel bacteriocin and the first recorded one produced by an insect originated bacterium. It has potential usage for the control of many different pathogenic and spoilage bacteria in the food industry, agriculture, and various other areas.

• Journal of Biosystems Engineering
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• 제43권4호
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• pp.394-400
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• 2018

Purpose: This research evaluated five types of nanoparticles to develop a surface-enhanced Raman spectroscopy (SERS) method for the rapid detection of two Bacillus species (Bacillus cereus and Bacillus thuringiensis) that are commonly found on fresh produce, which can cause food poisoning. Methods: Bacterial concentrations were adjusted to a constant turbidity, and a total of $30{\mu}L$ of each Bacillus cell suspension was prepared for each nanoparticle. A point-scan Raman system with laser light source of wavelength 785 nm was used to obtain SERS data. Results: There was no qualitative difference in the SERS data of B. cereus and B. thuringiensis for any of the five nanoparticles. Three gold nanoparticles, stabilized in either citrate buffer or ethanol, showed subtle differences in Raman intensities of two Bacillus species at $877.7cm^{-1}$. Conclusions: Among the three types of nanoparticles, the gold nanoparticles stabilized in citrate buffer showed the lowest standard deviation, followed by gold nanoparticles stabilized in ethanol. This result supports the potential application of gold nanoparticles for SERS-based detection of B. cereus and B. thuringiensis.

• 한국미생물·생명공학회지
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• 제21권3호
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• pp.193-199
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• 1993

Effects of the five fermenation media on the growth and the production of endotoxin of twenty-five Bacillus thuringiensis isolates were investigated. Among the five different media, the growth and the production of endotoxin of B. thuringiensis were highest in the M4 and M5 media containing high amounts of nitrogen and carbohydrate. But the production of endotoxin was highest in the M1 and M3 media containing high amounts of nitrogen sources limiting carbohydrate. The average number of endotoxin and growth(O.D.) in the M1 and M3 medial were 4.4*107/ml and 2.42 in M1 medium and 1.3*107/ml and 1.46, respectively.

• 한국미생물·생명공학회지
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• 제33권2호
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• pp.154-158
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• 2005

Twenty-three Bacillus thuringiensis strains isolated from Korea were screened to detect the cry-type genes using PCR with 21 specific oligonucleotide primers. Eight strains contained distinct multiple crystal genes; cry1Aa2, cry1Ab1, cry1Ac1 and cry2Aa1. These results indicate that the strains coincided with the B. thuringiensis subsp. kurstaki strain. The other 15 strains were not recognised to the 21 specific primers.

• 농약과학회지
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• 제13권4호
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• pp.315-324
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• 2009

배추좀나방(Plutella xylostella L.)의 효과적인 방제를 위해서 현재 상용화되고 있는 친환경 농자재인 Bacillus thuringiensis subsp. kurstaki($30{\times}10^8\;cfu/mg$)와 Neem oil(0.5% azadirachtin)을 사용하였다. 두 약제를 추천농도로 처리했을 때 배추좀나방 1. 2령 유충에서 B. thuringiensis는 2일만에 100%의 사충률을 보였으며, Azadirachtin은 7일까지 지효적 효과를 보였다. 배추좀나방 1, 2, 3, 4령 유충에 대한 B. thuringiensis의 $LC_{50}$ 값은 각각 $2.8{\times}10^4$, $3.1{\times}10^4$, $3.4{\times}10^4$, $1.5{\times}10^5\;cfu/ml$이였으며, Azadirachtin은 2.7, 3.9, 4.7, 7.1 ppm이였다. B. thuringiensis와 Azadirachtin를 배추좀나방 성충에 처리하였을 경우에 산란수는 무처리와 비교하여 Azadirachtin 25 ppm에서 57.5개의 감소를 보였다. 또한 알의 부화율은 무처리와 비교하여 B. thuringiensis는 큰 차이가 없었고, Azadirachtin 50 ppm에서 45.4%의 부화율 감소를 나타냈다. 배추좀나방은 B. thuringiensis와 Azadirachtin을 혼합처리할 경우, 각각의 처리구에서 각각을 단독으로 처리하였을 때보다 높은 방제효과를 나타내었다. 포트시험의 경우에서도 B. thuringiensis와 Neem oil의 혼합처리 사용은 배추좀나방 알, 유충, 번데기, 성충의 친환경적인 방제에 효과를 나타냈다.

• 한국잠사곤충학회지
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• 제28권1호
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• pp.43-53
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• 1986

해충의 미생물적 방제를 위한 미생물살충제 중 특히 주목되어지는 Bacillus thruingiensis살충제를 개발함에 있어서 우리나라와 같은 양잠국가에서는 누에에 대한 피해가 우려되므로 익충인 누에에 대해서는 비교적 저독성이고 목적해충에 대해서는 맹독성인 균주를 선발하기 위한 기초자료를 얻고자 plasmid영동상에 의해 확인된 3종의 B.t.균주 kurstaki. aizawai, dendrolimus를 이용하여 Renograffin 이중층법에 의해 분리된 내독소 단백질 및 포자와 내독소 단백질 혼합물 각각을 누에와 흔불나방에 대해 독성을 검정하여 아래와 같은 연구결과를 얻었다. 1. plasmid의 전기영동상을 분석한 결과 kurastaki가 150-1.5Md 사이에 10개, dendrolimus가 75-33Md 사이에 4개, aizawai가 62-4.2Md사이에 6개 band가 나타났다. 2. 누에와 흔불나방에 대한 독성검정결과는 아래와 같다. i)dendrolimus가 누에에 있어서는 가장 저독성이고 목적해충인 흰불나방에 대해서는 kurstaki와 대등한 우수한 살충력을 나타내었다. ii)공시충에 따라 균주간에 독성차이가 크게 나타났으며 kurstaki가 공시충에 따른 독성차이가 가장 작고 비교적 독성이 높은 것으로 나타났다.

• 한국응용곤충학회지
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• 제49권2호
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• pp.151-158
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• 2010

토양에서 분리한 구형의 내독소단백질을 가지는 18개의 균주 중, 이집트숲모기와 지하집모기에 90%이상의 살충활성을 보이는 Bacillus thuringiensis subsp. israelensis CAB199 균주를 선발하였다. 이 균주는 습지에 발생한 이나도미집모기에도 높은 살충효과를 나타내었다. 모기유충의 행동패턴은 주로 호흡을 위해 수면에서 주로 활동하고 있어, Bti 균주를 유효성분으로 하는 제제가 수면 위에 장시간 부유 할 수 있는 제형을 선발하고자 하였다. 수화제와 액상수화제의 두 가지 형태의 제형을 만들어 실내에서의 살충효과 및 습지에서의 방제효과를 확인한 결과, 실내에서의 경우에는 액상수화제 제형이 실험한 3종의 모기유충에 대해 속효적이며 높은 살충율을 보여주었고, 습지에서도 이나도미집모기에 대하여 가장 좋은 활성을 나타내었다.

• International Journal of Industrial Entomology and Biomaterials
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• 제6권1호
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• pp.81-85
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• 2003

To verify importance of the intervening sequence between the ribosome binding site (RBS) and the initiation codon for expression of Bacillus thuringiensis $\delta$-endotoxin, the pProMu, containing SphI and NcoIsites between RBS and the initiation codon of the cry1Ac gene, and the deletion derivatives of pProMu were constructed and transformed into the B. thuringiensis subsp. kurstaki $Cry^{-B}$ strain. The pProMu-ΔSphIhad identical six bases of intervening sequence to pProAc though the arrangement of sequence was different. Other mutants containing pProMu had 1 or 10 or 14 bases between RBS and the initiation codon. Among deletion mutants, only ProMu-ΔSphI/CB only produced 130 kDa typical bipyramidal crystals like those seen for ProAc/CB. However, ProMu/CB, $ProMu-{\Delta}NcoI$, and ProMu-ΔSphI＋NcoIdid not produce Cry1Ac crystals. In conclusion, the results suggest that 6-base intervening sequence was important for expression of cry1-type class gene. Furthermore, spacing effect of the intervening sequences may play an important role in expression of individual crystal proteins in B. thuringiensis without doubt.

• 한국미생물·생명공학회지
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• 제13권1호
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• pp.51-57
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• 1985

Bacillus thuringiensis subspecies중 HD-1, HD-9, HD-73과 finitimus의 내독소와 플라스미드분리에 대하여 얻은 실험결과는 다음과 같다. 1. 밀도구배에 의한 ${\delta}$-내독소 정제는 NaBr, CsCl, Renografin-76으로 행하여 밀도구배 재료간 독소 수율은 Renografin-76>CsCl>NaBr순으로 독소와 아포의 분리가 양호하였고 그 순도는 약 98%정도였다. 2. 정제한 내독소는 SDS-PAGE, 현미경 관찰, 생물검정으로 조사하였고, SDS-PAGE에 의한 하위단위체의 분자량은 약 66,000과 130,000달돈 이었다. 3. 내독소의 결정단백질의 외부형태는 finitimus를 제외한 3균주는 모두 이중피라마드형이었다. 4. 담배나방 유충에 대한 살충실험 결과 $0.1{\mu}g/ml$의 내독소 농도에서 HD-1은 60%, HD-73은 100% 치사율을 보였다. 5. B. thuringiensis의 플라스미드 크기는 약 0.5~120Kb 정도였고 그 수는 HD-1, HD-9, HD-73에서 각각 12, 3, 11개 였고, finitimus 균주에는 플라스미드가 없었다.

• Journal of Microbiology and Biotechnology
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• 제22권6호
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• pp.788-792
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• 2012

We report the computational structural simulation of the Cry1Ab19 toxin molecule from B. thuringiensis BtX-2 based on the structure of Cry1Aa1 deduced by x-ray diffraction. Validation results showed that 93.5% of modeled residues are folded in a favorable orientation with a total energy Z-score of -8.32, and the constructed model has an RMSD of only $1.13{\AA}$. The major differences in the presented model are longer loop lengths and shortened sheet components. The overall result supports the hierarchical three-domain structural hypothesis of Cry toxins and will help in better understanding the structural variation within the Cry toxin family along with facilitating the design of domain-swapping experiments aimed at improving the toxicity of native toxins.