• Microbiology and Biotechnology Letters
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• v.23 no.4
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• pp.390-396
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• 1995

Bacillus sphaericus 1593 is a larvicidal toxin-producing mosquitocidal bacterium. The toxin contains a parasporal crystalline inclusion which is composed of a protein that is activated under alkaline condition. To enhance alkaline environment around toxin protein, cryptic plasmid cured, B. sphaericus 1593 was transformed by the Bacillus pasteurii urease gene which generate ammonia from urea. Transformant produced urease at about 80% more than wild type strain. B. sphaericus 1593, and the urease gene was stably maintained. It also produced crystalline toxin protein at the same level as the wild type strain B. sphaericus 1593.

• The Korean Journal of Food And Nutrition
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• v.14 no.1
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• pp.77-81
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• 2001

Regulation of inulinase biosynthesis was studied in Bacillus sphaericus 188-1 Biosynthesis of inulinase was effectively induced in the presence of 0.5% inulin for 8 hrs. Fructose (0.5%) repressed the inulinase induction by inulin and as late as addition time of fructose, inulinase formation was decreased. Catabolite repression was not reduced by the addition of CAMP for 8 hrs of induction.

• Microbiology and Biotechnology Letters
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• v.16 no.5
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• pp.389-392
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• 1988

A clone pSL 2-1, which is a recombinant plasmid believed to contain the mosquitocidal crystal-line protein gene of the Bacillus sphaericus 1593, was expressed in Escherichia coli JM83 and the product of the clone was purified and identified. The unsolubilized mosquitocidal crystal proteins from the B. sphaericus had formed 43, 58, 64, 100, 113, and 130 Kd bands in the SDS-polyacrylamide gel, but the NaOH-solublized proteins at pH 12 formed 2 protein bands of 43- and 64Kd in the gel because the larger protein (precursor) bands were cleaved. The products of the pSL 2-1 clone was purified by Sephadex G-200 and only the fractions having lethal activity to the 3rd in-star larvae of mosquito Culex pipiens were analyzed by the gel. The only single protein band of 42 Kd toxic to the larvae was formed. The major toxic protein being produced from the B. sphaericus 1593 and the pSL 2-1 clone was found to be the 42 Kd.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.156-163
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• 1995

Bacillus sphaericus 1593 is pathogenic to the larvae of a number of mosquito species that are known as important vectors for the transmission of certain human and animal diseases. As a preliminary experiment for developing a multfunctional B. sphaericus 1593 as a potent antagonist, we investigated the conditions for the protoplast transformation system of B. sphaericus 1593 using the plasmid pGB215-110$\Delta$B. The protoplast of B. sphaericus 1593 were obtained most efficiency by treating the cells with 500 $\mu$g/ml of lysozyme in the SMM buffer containing 0.5 M sucrose at pH 8.0 and 40$\circ$C for 60 minutes. The cell wall was regenerated on the plate containing 1.2% agar and 0.8 M mannitol. Under the best condition for protoplast formation and regeneration established in the work the highest frequency of transformation was achieved with the 40% PEG (M.W 4,000) treatment for 15 minutes of incubation at 4$\circ$C, and subsequently for 120 minutes incubation at 30$\circ$C for phenotypic expression. The highest transformation efficiency were observed at 1.0 $\mu$g/ml of the final concentration of the plasmid DNA and the plasmids were found to be fairly stable since about 70% of the plasmids were maintained after 8 successive daily transfers onto the fresh medium.

• The Korean Journal of Food And Nutrition
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• v.14 no.3
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• pp.193-198
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• 2001

고정화 inulinase를 이용하여 inulin으로부터 fruc-tose 시럽을 연속적으로 생산하기 위하여 Bacillus sphaericus 188-1이 생성하는 inulinase를 부분 정제한후 5시간 NaIO$_4$로 산화시킨 cellulose에 고정화시킨 다음 이들의 성질을 조사하였다. 산화 cellulose에 고정화시킨 inulinase의 활성은 g당 47 Unit 이었고 고정화수율과 활성수율은 각각 41%와 39%이었다. 고정화시킨 inulinase의 작용 최적온도와 pH는 각각 5$0^{\circ}C$, pH 9.0이었고 5$0^{\circ}C$, pH 8.0~10.0에서 비교적 안정 하였다 고정화시킨 inulinas의 활성은 K+, Ca2+, Mn2+과 Hg2+에 의하여 활성화 되었으며 EDTA(10mM)에 의하여 심하게 저해되었다.

• International Journal of Industrial Entomology and Biomaterials
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• v.2 no.2
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• pp.185-189
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• 2001

Wild type Bacilus thuringiensis subsp. israelensis and B. sphaericus toxins have been used separately as active in ingredients for bacterial insecticides to control mosquito larvae due to their comparable toxicity to chemical insecticides. Cry11B, recently cloned from B. thuringiensis subsp. jegathesan, shows higher toxicity against three major species of mosquito larvae than Cry11A, one of the major component of B. thuringiensis subsp. israelensis inclusion body. To determine whether the combination of cry11B and B. sphaericus binary toxins is as toxic as B. thuringiensis subsp. israelensis parental strain, cry11B and B. sphaericus binary toxins genes were co-expressed as an operon using cytlA promoters/STAB-SD hybrid expression system in B. thuringiensis subsp. israelensis acrystalliferous strain 4Q7. However, unexpectedly, B. sphaericus binary toxins were barely produced, whereas relatively large amount of Cry11B was produced. When this strain was grown in four different media, NB+G and Peptonized Milk produced more toxin proteins and spores per unit of media than GYS and G-Tris. Toxicity of this strain against fourth instar Culex quinquefasciatus was ranged from of 8.3 to 45.7 ng/ml, with NB+G culture being the highest, and GYS culture was the lowest.

• Korean Journal of Soil Science and Fertilizer
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• v.40 no.5
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• pp.405-411
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• 2007

This study was carried out to measure insoluble phosphorus fractions content fixed in different soil type and isolate a superior phosphate solublizing bacteria(PSB) producing free phosphate in citrus orchard soil. Distribution of insoluble phosphate fraction ordered Al-P>Ca-P>Fe-P in the investigated citrus orchards. Insoluble phosphate fraction such as Al-P, Ca-P, Fe-P were higher in volcanic ash than in non-volcanic ash soil. A PSB with high holo zone in PDA-P medium isolated from citrus orchard soil. This strain identificated by MIDI system as Bacillus sphaericus. The optimum growth of pH and temperature were at 4~5, $30^{\circ}C$, respectively. When Bacillus sphaericus cultured at $25^{\circ}C$, 150 rpm condition in LB broth medium included different phosphate. Bacillus sphaericus produced free phosphate in the culture broth medium from tricalcium-phosphate(207.0 ppm), aluminium phosphate(324.5 ppm) and hydroxyapatite(334.8 ppm) and Phosphatase activity of Bacillus sphaericus was higher at $35^{\circ}C$ culture condition than that of $25^{\circ}C$. Two type preparation inoculated Bacillus sphaericus made with carrier materials such as Bentonite, $CaCO_3$, Sodium alginate. Density of PSB in this preparation conserved at $10^5c.f.u.\;g^{-1}$ level during storage in different temperature condition for 7 month. It also showed that free phosphate produced at PDA-P medium.

• Microbiology and Biotechnology Letters
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• v.13 no.2
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• pp.157-162
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• 1985

The cell cultures and crude extracts of Bacillus sphaericus 1593 K-5 and its mutant Spo-Dl216 were respectively bioassayed against Culex pipiens var. pollens mosquito larvae. The B. sphaeriucs 1593 K-5 showed toxic activity against the larvae. LC$_{50}$ values (cells/$m\ell$) was 2.6$\times$10$^2$. Also the LC$_{50}$ ($\mu\textrm{g}$ Protein/$m\ell$) of the crude extract was 10.26. However, B. sphaericus Spo-Dl216 didn't show toxic activity against the larvae. The soluble cytoplasmic toxin in broken B. sphaeriucs 1593k-5 cells was partially purified by gel permeation chromatography and ion exchange chromatography. Among the fractions of the gel permeation chromatography only a single fraction was found to be toxic. LC$_{50}$ values ($\mu\textrm{g}$ protein/$m\ell$) of the active fraction was 0.182. The active fraction of the gel permeation was subjected to ion exchange chromatography. Only a single fraction showed toxic activity and its LC$_{50}$ values ($\mu\textrm{g}$ protein/$m\ell$) was 0.02..02.

• Microbiology and Biotechnology Letters
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• v.32 no.3
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• pp.224-229
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• 2004

Several microorganisms (esterase-producing group) were isolated by the solid selective media containing-naphtylacetate. Among them, strain 366M-9 having a high activity of cephalosporin-C deacetylase (CAH; EC 3.1.1.41) was selected. The strain 366M-9 was identified as Bacillus sphaericus on the basis of morphological, physiological, and biochemical characteristics. The production of CAH reached at maximum value after 32 hrs, when cultivated in the optimal medium containing dextrin 2.5%, peptone 2.5%, sodium chloride 0.5%, dipotassium phosphate 0.25%, ferrous sulfate 0.02%, and 7-ACA 0.1% at $30^{\circ}C$ with initial pH 6.0. The CAH was purified by 3 steps with ammonium sulfate precipitation, adsorption chromatography on hydroxyapatite column, and Sephadex G-200 gel chromatography. The final enzyme preparation was homogeneous as judged by the analysis of SDS-PAGE and HPLC. Optimum temperature and pH for CAH activity were $50{\circ}C$ and around 7.0, respectively. And the enzyme was stable at pH 6.0～8.0, up to $50^{\circ}C$. The Michaelis-Menten constants ($K_{m}$ ), $V_{max}$ were 0.87 mM and 1.22 unit/ml, respectively.

• Microbiology and Biotechnology Letters
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• v.21 no.5
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• pp.446-452
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• 1993

The cholesterol oxidase produced from Bacillus sphaericus was purified and characterized. Through a series of purification procedures including DEAE-Toyoperal 650C, Sephadex G-200 and DEAE-Sephadex A-50 column chromatography, the purified enzyme was shown to have a specific activity of 0.179 units/mg protein having 31.8 fold purification and final yield of 12%. The molecular weight of the enzyme was estimated to be 47kDa and 47.tkDa by Sephadex G-200 chromatography and SDS-PAGE. The optimum temperature and pH for the enzyme were 30C and 6.0, respectively. The activity of the purified cholesterol oxidase was inhibited by Fe2+ and Hg+.