• 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%.

• Journal of Sericultural and Entomological Science
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• v.35 no.2
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• pp.120-128
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• 1993

The objective of this study is to identify plasmids of Bacillus thuringiensis var. kurstaki HD-1(B. t k HD-1) toxic to lepidopteran larvae. The results from agarose gel electrophoresis indicated that the bacterium contained 9 plasmids with approximate sizes of 1.4, 4.9, 5.4, 9.3, 10, 29, 44, 52, and 150 megadaltons(Md). By treating the wild type of B. t k HD-1 with either SDS or EtBr as curing agent, 26 cured mutants of the bacterium were obtained, 9 of them were crystallifereous(cry+) and the others acrystallifereous(cry-). Plasmids from B. t k HD-1 were transferred to B. cereus 569 strR cry- recipients(Bc569 M1). Among 13 isolates of Bc569 M1 transcipient, 11 of them were capable of producing the crystal toxic proteins. The plasmid patterns of Bc569 M1 transcipients and partially curved mutants of B. t k HD-1 on agarose gel electrophoresis suggested that the 29 and 44Md plasmids should be involved in the production of crystalline toxic proteins.

• Journal of Microbiology and Biotechnology
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• v.23 no.1
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• pp.88-91
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• 2013

The Cyt1Aa protein of Bacillus thuringiensis subsp. israelensis is known to synergize mosquitocidal proteins of B. thuringiensis and Bacillus sphaericus strains. Cyt1Aa is highly lipophilic, and after binding in vivo to the midgut microvillar membrane serves as a "receptor" for mosquitocidal Cry proteins, which subsequently form cation channels that kill mosquito larvae. Here we report that Cyt1Aa can serve a similar function for lepidopteran-specific Cry proteins of B. thuringiensis in certain mosquito larvae. Engineering Cyt1Aa into the HD-1 isolate of B. thuringiensis subsp. kurstaki enhanced toxicity against $4^{th}$ instars of Aedes aegypti, but not against $4^{th}$ instars of Culex quinquefasciatus.

• The Korean Journal of Pesticide Science
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• v.14 no.4
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• pp.446-455
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• 2010

The characteristics of parasporal inclusion body from Bacillus thuringiensis subsp. kurstaki KB099 isolate which is high bioactive to the tobacco cutworm, Spodoptera litura, were examined. Parasporal inclusion of B. thuringiensis subsp. kurstaki KB099 isolate showed only 1 band at 130 kDa compared with B. thuringiensis subsp. kurstaki HD-l isolate producing 2 protein bands at 130 kDa and 60 kDa from by SDS-PAGE analysis without any enzyme treatment. Also, we confirmed that gut extract of sensitive S. litura KB099 isolate had digested only 60 kDa ${\delta}$-endotoxin protein. When the digestive enzyme of sensitive insect responsible for parasporal inclusion from KB099 and HD-l isolate was treated to each of them, protein band 60 KDa of KB099 was maintained up to 12 hours but all bands of HD-l were disappeared within 6 hours. In KB099 isolate, 6 genes (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry1D and Cry1I) were identified by PCR analysis. Also, $Cry^-$ mutant of KB099 isolate was investigated by phase- contrast microscope, SDS-PAGE and PCR.

• Korean journal of applied entomology
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• v.28 no.2
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• pp.69-75
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• 1989

The extracellular amylase production by Bacillus thuringiensis subsp. kurstaki HD-l in amylase production media and its characteristics were investigated. The amylase production was highest in the medium composed of 0.2% soluble starch, 1.0% Bacto-peptone, 0.3% beef extract, 0.3% yeast extract, 0.5% NaCl, 0.3% $K_2HPO_4$, 0.1% $KH_2PO_4$, 0.012% $CaCl_2$.$2H_2O$, 0.005% $MnSO_4$.$H_2O$, and 0.03% $MgSO_4$.$7H_2O$. The amylase activity was inhibited by 50mM EDT A. The enzyme was optimally active from pH 6.5 to 7.0 at $55^{\circ}C$, The specific activity of the enzyme in the ethanol precipitate was 2.01 units/mg, and the Km value was approxi-mately 0.8 mg/ml.

• Journal of Life Science
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• v.12 no.3
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• pp.369-373
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• 2002

The waste-water from the industry for production of a soybean curd (the soybean curd waste-water) was investigated to use for the substrate to produce the endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1 used as one of well known microbial pesticides. The pH of the soybean curd waste-water was 9.8 and its chemical oxygen demand (COD), total nitrogen (TN) and phosphate (TP) were 276.0, 71.1 and 5.5mg/$\ell$, respectively. The higher was the concentration of the soybean curd waste-water in the medium, the more endotoxin was produced. Maximal sporulation occurred at which concentration of $K_2$HPO$_4$in the medium supplied with the soybean curd waste-water was 1% (w/v). Production of the endotoxin with the optimized medium supplied with the soybean curd waste-water was 1.5 times higher than that without the soybean curd waste-water. The soybean curd waste-water was found to be suitable substrate for production of the endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1.

• Microbiology and Biotechnology Letters
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• v.17 no.2
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• pp.142-147
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• 1989

Six plasmids of B. thuringiensis serovar. kurstaki HD73 were detected, with approximate sizes of 7.4, 7.8, 8.1, 11.3, and 75 Kb, as well as a low copied plasmid of similar length to 75 Kb. Partially cured mutants from B. thuringiensis HD73 were obtained either by the treatment of the curing agent, ethidium bromide(0.02 $\mu\textrm{g}$/$m\ell$) or by spontaneous curing, Acrystalliferous mutants(Cry$^-$) were identified by microscopic observation and immunoblotting with polyclonal antibody against 133 KD deltaendotoxin of HD73. Ten Cry$^-$ mutants were found to be lack of 75 Kb plasmid. These results implicated that this plasmid was associated with delta-endotoxin production, After isolating the mutants, we streaked them on potato dextrose agar, spizizen casamino acid glucose, starch agar, and nutrient agar. Only on starch agar medium did morphologies of Cry$^-$ appear translucent and light greyish. On the other hand, the mutants of B. thuringiensis isolated from Korean soil, designated KBS722, were obtained by the treatment of novobiocin (3 $\mu\textrm{g}$/$m\ell$). Acrystalliferous mutants of KBS722 were less translucent than HD73 mutants' only on nutrient agar medium. Compared the plasmid profile of the mutants with delta-endotoxin production, the results seemed to indicate that the insecticidal protein gene of B. thuringiensis isolates KBS722 located on about 225 Kb plasmid DNA.

• The Korean Journal of Food And Nutrition
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• v.9 no.4
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• pp.459-465
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• 1996

Bacillus thuringiensis serovar. darmstadiensis produced bipyramidal endo-toxin. The toxin protein was purified by Renografin-76 step gradient centrifugation and investigated by electron microscope. Analysis of total plasmid DNA patterns showed that four different size of plasmids existed in wild type B. thuringiensis serovar. darmstadiensis. Total plasmids DNA was isolated and transformed into pst I site of pBR322 cloning vector. Ten clones containing crystal toxin gene were forst screened colony hybridization by using PUYBT 9044 probe ontained B. thuringiensis kurskaki HD 1 toxin gene. Cloned-DNA was digested with EcoR1 and HindIII and transformed to pIBI30 sequencing vector. Finally, 2.6kb and 3.6kb size fragments contatined toxin-gene were cloned with restriction analysis.

• Journal of Sericultural and Entomological Science
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• v.35 no.1
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• pp.36-42
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• 1993

Expression of insecticidal protein by fusion product of truncated HD-1[CryIA(a)] N-terminal and HD-73[CryIA(c)] C-Terminal fragment of Bacillus thruingiensis subsp. kurstaki was investigate. Immunological analysis of transformants by using polyclonal antisera raised against the whole-crystal protein of HD-1 revealed that SK4 and SK5 were observed cross-reaction with polypeptides of 77-kDa and 105-kDa, respectively. Bioassay of the transformant pSK5 to Plutella maculipennis and Heliothis assulta were 96% and 97%, respectively.

• Microbiology and Biotechnology Letters
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• v.18 no.6
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• pp.647-652
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• 1990

The production of delta-endotoxin crystal and the cloning of endotoxin protein gene in Bscillus thuringiensis subsp. kurstaki HD1 strain were studied. The strain produced bipyramidal crystals ($2.9\times 1.0 \mu m$) in their cells during sporulation. The B. thuringiensis contained about 10 plasmid DNA elements ranging from 2.1 to 80 kilobases. The 73 kb plasmid DNA, the 29 kb BamHI fragment and the 7.9 kb Pstl DNA fragment hybridized to the pHL probe. The 7.9 kb fragment was eluted and cloned in the PstI site of pBR322 vector and transformed into E. coli HB101, which produced insecticidal proteins killing Bornbyx mori larvae.