• KSBB Journal
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• 제26권3호
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• pp.243-247
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• 2011

Using Bacillus subtilis spore display system, with cotG as an anchoring motif, levansucrase from Zymomonas mobilis, was displayed on the outer surface of Bacillus subtilis spore. Flow cytometry of DB104 (pSDJH-cotG-levU) spore, proved the surface localization of CotG-LevU fusion protein on the spore compared to that of DB104. Enzymatic activity of DB104 (pSDJH-cotG-levU) spore showed more than 1.5 times higher levansucrase specific activity compared to that of the host spore, which is a remarkable increase of enzymatic activity considering the existence of sacA (sucrase) and sacB (levansucrase) in the Bacillus subtilis chromosome. The spore integrity, revealed by sporulation frequency test after heat and lysozyme treatment of spore, did not changed at all in spite of the CotG-LevU fusion protein incorporation into the spore coat layer during spore formation process. These data prove again that Bacillus subtilis spore could be considered as good live immobilization vehicle for efficient bioconversion process.

• KSBB Journal
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• 제26권3호
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• pp.199-205
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• 2011

Using tetrameric ${\beta}$-galactosidase as a model protein, anchoring motives were screened in Bacillus subtilis spore display system. Eleven spore coat proteins were selected considering their expression levels and the location in the spore coat layer. After chromosomal single-copy homologous integration in the amyE site of Bacillus subtilis chromosome, cotE and cotG were chosen as possible spore surface anchoring motives with their higher whole cell ${\beta}$-galactosidase activity. PAGE and Wester blot of extracted fraction of outer layer of purified spore, which express CotE-LacZ or CotG-LacZ fusion verified the existence of exact size of fusion protein and its location in outer coat layer of purified spore. ${\beta}$-galactosidase activity of spore with CotE-LacZ or CotG-LacZ fusion reached its highest value around 16~20 h of culture time in terms of whole cell and purified spore. After intensive spore purification with lysozyme treatment and renografin treatment, spore of BJH135, which expresses CotE-LacZ, retained only 1~2% of its whole cell ${\beta}$-galactosidase activity. Whereas spore of BJH136, which has cotG-lacZ cassette in the chromosome, retained 10~15% of its whole cell ${\beta}$-galactosidase activity, proving minor perturbation of CotG-LacZ, when incorporated in the spore coat layer of Bacillus subtilis compared to CotE-LacZ. Usage of Bacillus subtilis WB700, of which 7 proteases are knocked-out and thereby resulting in 99.7% decrease in protease activity of the host, did not prevent the proteolytic degradation of spore surface expressed CotG-LacZ fusion protein.

• KSBB Journal
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• 제26권4호
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• pp.305-310
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• 2011

This study was carried out to establish the optimum condition of cell disruption with a sonicator for the detection of the spore, Bacillus anthracis ${\Delta}$-sterne for the purpose of developing automatic fluorometer. The efficiency of sonication on the ${\Delta}$-sterne spore disruption was very weak. The ${\Delta}$-sterne spore with zirconia bead showed greater disruption than the ${\Delta}$-sterne spore alone when sonificated. The volumn of the zirconia bead added in the spore solution has little effect on the disruption efficiency. The detection limit of the ${\Delta}$-sterne spore with zirconia bead and the ${\Delta}$-sterne spore alone was $10^6$ CFU/mL and $5{\times}10^7$ CFU/mL respectively, when sample was sonicated for 20 seconds with a sonicator probe of 13 mm diameter.

• KSBB Journal
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• 제13권6호
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• pp.644-648
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• 1998

In the production of a low cost bacterial insecticide, it is important to produce a high spore concentration using low price substrates. Experiments were carried out to investigate the effects of the addition of mineral salts and glucose, and of dissolved oxygen concentration on the cell growth and spore formation of Bacillus thuringiensis var aizawai using a cheap wheat and soybean meal in the batch culture. The maximum viable cell number was 1.2${\times}$109 CFU/mL at 12 hr culture and spore yield was 54.2% at 74 hr culture using an industrial medium containing 20 g/L wheat meal and 30 g/L soybean meal under 1.0 vvm aeration and 200 rpm agitation. The cell growth and the spore formation were not enhanced by the addition of mineral salts in industrial medium, whereas th addition of 10g/L glucose decreased the cell growth and spore formation. We could obtain a maximum viable cell number of 2.2${\times}$109 CFU/mL and spore number of 1.9${\times}$109 CFU/mL at the dissolved oxygen concentration of 60% of saturation. The spore concentration was enhanced approximately by 2 times as compared to the dissolved oxygen concentration of 50%. In the bench-scale culture, the maximum viable cell and spore number were 2.5${\times}$109 CFU/mL, and 2.2${\times}$109 CFU/mL, respectively under 1.0 vvm aeration and 400 rpm agitation. The spore yield was 88% based on the maximum viable cell number. As a result, it was confirmed that the production of high spore concentration could be obtained by a bench-scale culture using an industrial medium.

• International Journal of Industrial Entomology and Biomaterials
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• 제18권2호
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• pp.83-89
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• 2009

Multiplication and spore production of three microsporidia(Nosema bombycis, Nosema sp. 1 and Nosema sp. 2) in selected tissues of pupa and adult of silkworm, Bombyx mori L. were studied in two seasons (SI, SII) with distinct temperature (SI: $20.1{\pm}0.8^{\circ}C$ and SII: $25.1{\pm}0.7^{\circ}C$) regimes. Multiplication of the microsporidia followed a logistic pattern with a lag phase, an exponential phase and a stationary phase. In SII, spore production was significantly (P<0.01) higher in various tissues. Highest spore production was observed 30 days post inoculation (p.i.) in SI and in SII, it was $21{\sim}23$ days p.i. Spore production was significantly (P<0.01) higher in the gut tissues than other tissues. Nosema sp. 2 registered significantly (P<0.01) higher spore production in both the seasons compared to Nosema bombycis and Nosema sp. 1. Results indicate that the multiplication and spore production of microsporidia are tissue specific and extremely sensitive to the temperature at which the host is reared. Through this study, the precise day that the spore numbers of the microsporidia are maximized can be predicted in both pupa and adult in case the infection is initiated in the first instar.

• International Journal of Industrial Entomology and Biomaterials
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• 제12권2호
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• pp.87-93
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• 2006

Multiplication and spore production of three microsporidia viz., Nosema bombycis, Nosema sp. 1 and Nosema sp. 2 in fifth instar larval tissues of silkworm, Bombyx mori L. in two seasons with distinct temperature regimes were studied. Nosema sp. 2 produced significantly (P < 0.01) higher number of spores in various tissues. Among the tissues, spore production was highest in silk gland, followed by fat body and gut. Spore production was significantly (P < 0.01) higher in season-II (Average temperature $29.4{\pm}1.1^{\circ}C$). Maximum spore production was observed 25 days post inoculation (p. i.) in season-I (Average temperature $18.9{\pm}1.1^{\circ}C$), whereas in season-II, it was 14 days p. i. In season-I, spore production was low up to 21 days p. i., then increased sharply. In season-II, there was a steady increase in spore production. The results indicate that the microsporidian multiplication is tissue specific and extremely sensitive to temperature at which the host is reared. It also reveals that, silk gland, fat body and gut are the most appropriate tissues for microscopic identification of microsporidia in the larval stage.

• 한국식물병리학회지
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• 제3권2호
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• pp.120-123
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• 1987

자연상태하에서 잎도열병 병반으로부터 포자의 량을 측정하기 위하여 4종의 포자탐집기(금의 원형, 금의 개량형, 길야의 원형, 금$\cdot$길야의 개량형)를 검토한 결과, 원골한 포자형성 및 이탈, 포자채집, 검경에 있어서 한 두가지의 결점이 발견되었다. 따라서 위의 결점을 보완할 수 있는 새로운 포자채집기를 고찰, 성능을 검토하여 상기 4종의 채집기의 결점을 보충할 수 있는 결과를 얻었으므로 금후 자연상태하에서 포자이탈량 조사에 유용할 것으로 생각된다.

• Mycobiology
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• 제33권4호
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• pp.194-199
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• 2005

Application of fertilizers such as urea, diammonium phosphate (DAP) and muriate of potash in soil adversely affected the spore germination of Arthrobotrys dactyloides. Amendment of soil with urea at the concentrations of 1.0%, 0.5% and 0.1 % completely inhibited spore germination and direct trap formation on the conidium, whereas muriate of potash delayed and reduced the spore germination even at the lowest concentration. DAP also inhibited spore germination at 1.0% concentration, while at lower concentration the percentage of spore germination was reduced. Application of neem cake at the concentration of 0.5% also inhibited spore germination after 24 h of amendment. The inhibitory effect of neem cake was reduced after 15 days of amendment, while after 30 days after amendment the inhibitory effect was completely lost and the spore germinated by direct trap as in unamended soil. Nematodes were not attracted to ungerminated spores after 24 h of amendment. After 15 days of amendment nematodes were attracted to agar blocks containing fewer germinated spores after 24 h of incubation but after 48 h of incubation large number of nematodes were attracted and trapped by the germinated spores with direct traps. After 30 days of amendment, larger number of nematodes were attracted and trapped by direct traps.

• Mycobiology
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• 제29권4호
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• pp.198-204
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• 2001

Analysis of leaf exudates of Vicia faba using paper chromatography to identify individual amino acids and sugars qualitatively was investigated. The results revealed that the number of identified amino acids detected in the leaf exudates of the susceptible plants was more than those of resistant plants. The results also showed an increase in the number of amino acids exuded by infected leaves, but no marked difference in sugars of infected and non infected plants. Lithium chloride application led to decrease in amino acid and sugar contents. The number of amino acids and sugars was also decreased with leaf age. Botrytis fabae and the selected fungal species(Alternaria alternata, Fusarium oxysporum and Aspergillus niger) were used to show the effect of individual amino acid and sugar on their spore germination. It was observed that all amino acids stimulated the fungal spore germination except serine which inhibited its spore germination. In case of A. alternata, spore germination was stimulated by all amino acids except serine, alanine, glutamic acid, arginine and methionine which caused the inhibition. In case of F. oxysporum, aspartic and glutamic acids inhibited spore germination but the other amino acids stimulated its spore germination. Aspartic acid and phenyl alanine inhibited the spore germination of A. niger. All the identified sugars(galactose, glucose, fructose and rhamnose) stimulated spore germination of all tested fungi.

• KSBB Journal
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• 제15권3호
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• pp.219-225
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• 2000

Bacillus thuringiensis를 생물농약으로 이용하기 위해서는 고농도의 포자형성에 의한 높은 살충성의 8 -endotoxin를 생산하는 것이 중요하다. 따라서 본 연구에서는 B. thuringiensis의 고농도 배양에 의한 높은 포자형성 수율을 얻기 위하여 40%의 용존산 소량과 $28^{\circ}C$에서 여랴 가지 방법의 유가식 배양법을 검토하였다. 최종 배양액의 glucose 농도가 50 g/L가 되도록 하는 경우의 유가식 배양에서는 continuos fed-batch culture의 linear gradient f feeding 법에 의하여 $9.37{\times}109$ cell/mL의 최대 생존 세포 수와 8 8.33 X 109 spore/mL의 최대 포자 수를 얻었으며, 이때의 포자 형 성율은 88.9% 이었다 최종 배양액의 glucose 농도가 100 m/L가 되도록 하는 경우의 유가식 배양에서는 intermittent fed-batch culture의 pH-stat 법에 의하여 $1.35{\times}1010$cell/mL의 최대 생존 세포 수와 $1.35{\times}1010$ spore/mL의 최대 포자 수를얻었으며, 이 때의 포자 형성율은 97.8% 이엇다.