• Journal of Life Science
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• v.17 no.5 s.85
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• pp.658-663
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• 2007

Bacillus thuringiensis is a well-known species of entomophathogenic bacteria that is widely used as a biopesticide against many insect pests. It produces parasporal crystals ($\delta$-endotoxin) and endospores during sporulation. In this report, the $\delta$-endotoxin produced by Bacillus thuringiensis BT-1 and BT-2 were characterized by Scanning Electron Microscope(SEM), Transmission Electron Microscope(TEM), SDS-PACE, and solubilization activity by alkaline solution. BT-1, BT-2 were cultured in the GBY medium, and the $\delta$-endotoxin of them was purified with discontinuous sucrose density gradient centrifugation. Their $\delta$-endotoxin was observed by SEM and TEM. Morphologically, the $\delta$-endotoxin of BT-1 was a square and flat type, whose size was $1.73{\mu}m{\times}0.7{\mu}m$, and the $\delta$-endotoxin of the BT-2 was spherical form whose size was $1.1{\mu}m{\times}0.9{\mu}m$ determined by SEM and TEM. The $\delta$-endotoxin of the BT-1 was composed of 28 kDa and 21 kDa, however, it of the BT-2 was composed of 50 kDa, 35 kDa, and 22 kDa bands determined by SDS-PACE. The purified crystals of BT-1 and BT-2 were dissolved gradually in alkaline solution as time goes by, and it was perfectly dissolved after 3 hours. It is supposed that the $\delta$-endotoxin of crystal was converted to a state of activation in the course of time in the intestines of insect.

• Journal of Microbiology and Biotechnology
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• v.5 no.3
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• pp.138-142
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• 1995

Bacillus thuringiensis strain BT-209 was isolated from a soybean grain dust sample in Korea. The strain BT-209 produced two different sizes of cuboidal crystals and one spore in the cell. In the biochemical characterization, the strain BT-209 showed negative reactions on the production of urease, and the utilization of citrate and sucrose. Examination of its antibiotic resistance revealed that while the strain BT-209 showed higher sensitivity than B. thuringiensis subsp. kurstaki HD-1 to ampicillin, bacitracin, chlortetracycline, gentamycin, neomycin, penicillin G, tetracycline and tobramycin, it was more resistant to methicillin than B. thuringiensis subsp. kurstaki HD-1. The $\delta$-endotoxin crystal of strain BT-209 consisted of three proteins with apparent molecular weights of appoximately 148, 135 and 62 kDa on a 10% SDS-PAGE. The strain BT-209 had at least eight different plasmids with sizes of 4.1, 5.2, 6.3, 8.6, 14.6, 24.5, 67.6 and 77.6 Kb. The strain BT-209 showed strong lethalities of 70% and 87% against Bombyx mori and Hyphantria cunea larvae. at 72 h, respectively.

• Korean journal of applied entomology
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• v.33 no.3
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• pp.178-183
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• 1994

The prolein components of each crystal toxin of Bacillus thuringiensis kurstaki and alzawai were separated by SDS-PAGE. The major clqistal proteins from both shins were composed of paiypeptides having molecular weights of 130 kd and 64 kd. Digestive mixture of both. toxins with t~ypsin and gut juices shared 62 kd polypeptide which may be major actwe toxh. However, aizawal produced much less amount of 62 kd polypeptide than kurstaki did. On ingestion with Bt &-endotoxin, larvae of Hyphantria cunea developed 45 kd stress protein in the several tissues including fat body, which was induced by heat and cold shock.

• Journal of Microbiology and Biotechnology
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• v.4 no.4
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• pp.322-326
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• 1994

Bacillus thuringiensis strain BT-14 was isolated from alfalfa dust in Korea. The strain BT-14 produced one bipyramidal crystal and one spore in the cell. The biochemical characteristics of the strain BT-14 were similar to those of Bacillus thuringiensis subsp. kurstaki HD-l. Examination of its antibiotic resistance revealed that while the strain BT-14 was less resistant than BTK HD-l to ampicillin, gentamycin, neomycin and tobramycin, it was more resistant to amikacin than BTK HD-l. The $\delta$-endotoxin crystal of strain BT-14 consisted of a single protein with a high molecular weight of ca 135 KD on a 10% SDS-PACE. The strain BT-14 contained at least nine different plasmids with sizes of 2.9, 5.3, 5.8, 6.2, 9.4, 15.1, 18.1, 23.1 and 79 Kb. In insect bioassay, the isolated strain BT-14 showed lethality of 67% against Plutella xylostella larvae at dilution of 5$\times$$l0^{-4}$ (5$\times$l0 to 3$\times$$l0^2$ spores/ml), which is, almost equivalent to that of BTK HD-l.

• Microbiology and Biotechnology Letters
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• v.13 no.3
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• pp.315-319
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• 1985

Delta-endotoxin crystals of B. thuringiensis var. kurstari and B. thuringiensis var. israelensis were purified by NaBr density gradient centrifigation and the wet weight of the BTK endotoxin was approximately 23.79％ of the cell wet weight and that of BTI was 25％. The shape of BTK crystal was bipyramidal, whose size was 1.7${\mu}{\textrm}{m}$ $\times$ 0.9${\mu}{\textrm}{m}$ and that of BTI was a spheroid, whose size was about 1.6$\times$0.45${\mu}{\textrm}{m}$. The molecular weight of BTK crystal protein was approximately 134,000 daltons and that of BTI was about 128,000 daltons.

• Microbiology and Biotechnology Letters
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• v.13 no.1
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• pp.51-57
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• 1985

Delta-endotoxin in Bacillus thuringiensis var. finitimus, HD-1, HD-9 and HD-73 strains were isolated by NaBr, CsCl and Renografin density gradients. The purity of the toxin was about 98%. The purified o-endotoxin was analyzed by SDS-PAGE, electron microscopic observation and bioassay. According to SDS-PAGE, the molecular weight of subunits of the o-endotoxin were about 66,000 and 130,000 daltons. The shapes of the crystal toxin observed by TEM except finitimus strain were bipyramidal. When the purified endotoxin was bioassayed against tobacco horn worm, the entomocidal activities ($1{\mu}g/ml$) of HD-1 and HD-73 strains were, respectively, 60% and 100% at nine days after treated. The molecular weights of the plasmids isolated from B. thuringiensis were various from 0.5 to 120 Kb. The numbers of plasmids in HD-1, HD-9 and HD-73 strains were 12, 3 and 11, respectively, but B. thuringiensis var. finitimus strain had no plasmid.

• International Journal of Industrial Entomology and Biomaterials
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• v.6 no.1
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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.

• Applied Biological Chemistry
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• v.19 no.4
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• pp.189-201
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• 1976

In an effort to develop a microbial in secticide, B. thdringiensis var. thuringiensis was cultured in the medium composed of cocoon-cooked water from a filature. The results obtained are summarized as followss : (1) Bacillus thuringiensis is a bacterium producing a ${\delta}-endotoxin$ especially toxic to lepidopterous insects and a thermostable exotoxin harmful to dipterous insects. (2) With a view to utilizing the cocoon-cooked water discarded from the filature, as a nutrient source in the B. thuingiensis culture, it was analyzed to contain large amounts of various minerals and protein (7.5 mg/ml) believed to be extracted from the pupae. (3) A large amount of the ${\delta}-endotoxin$ can be obtained most cheeply by using cocoon-cooked water instead of distilled water in preparing GYS and citrate salts media. (4) The largest amount of a mixture of the vegetative cells, spores, and crystals was obtained by addition of 8 gr/l of glucose to the GYS medium. (5) The growth of the bacterium was far better, when leucine, isoleucine, and valine were added all together to the citrate salts medium to the concentration of $1.25{\times}10^{-3}M$. (6) The best growth was observed by addition of Na-glutamate to the citrate salts medium to the concentration of $2.5{\times}10^{-3}M$. (7) The optimal culture time ranged from 9 to 15 days. (8) The highest mortality was shown in Pieris rapae Linne with a pH of the total body extract of 8.4, whereas Dendrolimus spectabilis Butler and Bombyx mori Linne with lower pH's were less susceptible to the ${\delta}-endotoxin$. (9) The presence of the thermo stable exotoxin was confirmed by the fact that the supernatant of the culture was very toxic to the Drosophila melanogaster tested.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.168-173
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• 1988

Immunological analysis between endotoxin proteins produced by Bacillus thuringiensis serovar. kurstaki HD1 and HD73 have been investigated by using polyclonal antibodies. The antisera against the endotoxin proteins were prepared from rabbits injected with the endotoxin protein antigens. When about 2mg/$m\ell$ of the antigens were injected for 7 times, the titers were highest. The stability of the antigens was reduced to about 50% after 9 days incubation at 4$^{\circ}C$. The sensitibity of endotoxin protein from B. thuringiensis HD1 and HD73 by indirect ELISA was 50ng/$m\ell$ and 400ng/$m\ell$, respectively. The cross reaction of antiserum appeared that anti-HD1 partialy reacted with crystal protein but anti-HD73 reacted with HD1 endotoxin about 100%.

• Microbiology and Biotechnology Letters
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• v.23 no.6
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• pp.730-736
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• 1995

The solubilization and proteolytic process of $\delta $-endotoxin was analvsed to compare the biochemical property of the toxin isolated from B. thuringiensis subsp. sotto. The purified crystals were dissolved in 50 mM carbonate buffer containing 10 mM dithiothreitol at pH 10 for various times. The electrophoretic pattern showed that a rapid disappearance of 138 kDa protein band. This disappearance of protein with high molecular weight was accompanied by the appearance of new protein fragment with 104 kDa, 60 kDa, and 25 kDa. For proteolvtic processing, the soluble crystals were digested with trypsin for various times. The soluble crystal protein of 104 kDa was completely disappeared. However, the protein fragment of 60 kDa and 25 kDa still remained after complete proteolysis. The comparative immunoblot analysis showed that the antiserum against intact crystals showed strong immunoreactivity to the homologous inclusion protein of 138 kDa, 104 kDa, and 25 kDa, and to the intact spores of 221 kDa and 138 kDa, but not to the vegetative cell homogenate. The sera against crystals and spores had no immunoreactivity to the vegetative cell homogenate.