Tetrameric β를 이용한 고초균 포자에서의 미생물 표면 발현 모체 선별

김준형,반재구,김병기,Kim, June-Hyung,Pan, Jae-Gu,Kim, Byung-Gee 한국생물공학회 2011 KSBB Journal Vol.26 No.3

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.

Tetrameric β-galactosidase를 이용한 고초균 포자에서의 미생물 표면 발현 모체 선별

김준형(June-Hyung Kim),반재구(Jae-Gu Pan),김병기(Byung-Gee Kim) 한국생물공학회 2011 KSBB Journal Vol.26 No.3

Using tetrameric β-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 β-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. β-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 β-galactosidase activity. Whereas spore of BJH136, which has cotG-lacZ cassette in the chromosome, retained 10~15% of its whole cell β-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 ctivity of the host, did not prevent the proteolytic degradation of spore surface expressed CotG-LacZ fusion protein.

고초균 포자를 이용한 Zymomonas mobilis 유래의 levansucrase 표면 발현

김준형(June-Hyung Kim),최수근(Soo-Keun Choi),정흥채(Heung-Chae Jung),반재구(Jae-Gu Pan),김병기(Byung-Gee Kim) 한국생물공학회 2011 KSBB Journal Vol.26 No.3

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.

Ashbya gossypii로부터 riboflavin 대량생산을 위한 배지 최적화와 유가식 배양

남수완,장형욱,반재구,김익환,민태익 한국산업미생물학회 1993 한국미생물·생명공학회지 Vol.21 No.6

본 연구에서는 Ashbya gossypii를 이용한 비타민 B2(riboflavin) 생산에 있어서 발효배지의 최적화를 위해 탄소원(포도당과 대두유), 유기 질소원(CSL외 8종), 전구체(glycine 및 glutamate)의 영향을 검토하였다. 또한 생성균주의 고농도 배양을 통한 riboflavin 생산성 향상을 위해 유가식 배양을 수행하였다. 최적 발효배지의 탄소원은 기질 저해를 피할 수 있는 3%의 포도당과 0.5%의 대두유를 사용함으로써 riboflavin 생산수율을 증가시킬 수 있었다. 유기 질소원으로는 corn steep liquor(CSL)가 가장 좋았으며, 최적 CSL의 농도는 1%였다. Riboflavin 생합성의 전구체인 glycine과 glutamate는 배양 초기에 0.5%로 첨가하는 것이 riboflavin 생산에 가장 좋았다. 세포의 riboflavin 생합성 활성을 장기간 유지시키고 균체의 균체의 고농도 배양을 위해 포도당과 yeast extract로 구성된 복합배지를 연속적으로 공급한 유가식 발효 결과, 플라스크 배양(3.5g/ℓ)보다 약 100%, 발효조를 이용한 회분식발효(5.4 g/ℓ)보다 약 20% 향상된 6.8 g/ℓ의 최종 riboflavine 생산농도를 얻었다. 이상의 결과로부터 탄소원, 전구체 및 필수 영양소(특히 비타민) 공급원인 CSL의 최적화된 공급 전략을 통한 유가식 발효와 고농도 균체 배양 기술의 확립으로 riboflavin 생산수율을 더욱 향상시킬 수 있을 것으로 생각된다. In order to maximize the riboflavin production by a mutant strain Ashbya gosspyii, the optimization of medium and fed-batch fermentation were performed. As carbon sources, glucose and soybean oil were necessary for the riboflavin overproduction. Optimal concentrations of glucose and soybean oil in the flask cultures were found to be 3.0% and 0.5%, respectively, in a complex medium containing corn steep liquor (CSL) 1%. Among the various organic nitrogen sources tested, CSL as the most effective one both for the cell growth and riboflavin overproduction. More than 1% concentration of CSL, however, was inhibitory to the cell growth and riboflavin production. Glycine and glutamate at each 0.5% concentration stimulated the riboflavin production. The riboflavin concentration was reached to 5.4 g/ℓ in the batch fermentation with the optimized medium. The riboflavin production could be enhanced upto 6.8 g/ℓ through the fed-batch fermentation employing continuous feeding of glucose and yeast extract.

국내 식물시료에서 분리한 Bacillus thuringiensis 균주의 다양성

박승환,구본탁,신병식,최수근,정영미,반재구,김정일 한국산업미생물학회 1997 한국미생물·생명공학회지 Vol.25 No.2

국내 52지역 3,237 지점에서 채집한 식물시료에서 총 1,925주의 B.t.균을 분리하였다. 활엽수, 침엽수와 관목 등 모든 수종에서 B.t.균이 분리되었으며 다소의 빈도 차이는 있었으나 전국 어디에서나 B.t.균이 분리되었다. 토양시료에서 B.t.균을 분리하는 것에 비해 노력이 적게들고 분리되는 빈도도 높아 식물시료가 B.t.균 분리를 위해서는 좋은 분리원임을 알 수 있었다. 분리주의 특성을 조사하여 2단계 연구를 위한 자료를 만들기 위해 독소단백질 결정체의 모양, 독소단백질의 PAGE 패턴, plasmid 패턴, 분리균주의 생화학적 성질, 살충성 검정, serotyping 등 여러 측면에서의 분석을 시도하였다. 독소단백질 결정체의 모양에서는 bipyramid형의 결정체를 생산하는 분리주가 가장 많아 전체의 49.1%(945주)를 차지하였고, 무정형의 inclusion을 만드는 것이 33.1%(638주), 구형의 inclusion을 만드는 것이 7.1%(137주), 네모난 판자형의 inclusion을 만드는 분리주가 1.4%(27주)였고 나머지 9.2%(178주)는 작은 inclusion을 다수 생산하는 것들이었다. 1,644주의 B.t.분리주들에 대해 살충성을 검정한 결과 나비목 해충에 살충효과가 있는 분리주가 전체 분리주의 44.8%인 736주를 차지했고, 그 다음이 모기에 대해 살충성이 있는 분리주로 4.9%(94주), 그리고 딱정벌레목 해충의 하나인 오리나무잎벌레(Alder leaf beetle: Agelastica coerulea)에 대해 살충성을 나타내는 균주가 27주 분리되었다. 독소단백질의 PAGE 패턴, plasmid 패턴 또는 분리균주의 생화학적 성질에 의해서도 분리주들을 분류할 수 있었으며 또한 다양한 부류의 B.t.균들이 분리되었음을 알 수 있었다. 한편 선발된 22주를 대상으로 H-serotyping을 시도한 결과 aizawai(H7), amagiensis(H29), canadensis(H5a, 5c), darmstadiensis(H10a, 10b), galleriae(H5a, 5b), finitimus(H2), kurstaki(H3a, 3b, 3c), morrisoni(H8a, 8b)및 neoleonensis(24a, 24b)와 같은 다양한 아종이 있음을 확인하였고 3 분리주는 새로운 serotype H49(subsp. muju)로 분류되었다.c We collected 3,237 plant samples, mainly leaves of various trees, from many provinces in Korea and a total of 1,925 Bacillus thuringiensis isolates were obtained and characterized. The isolates were characterized in terms of crystal morphology, PAGE pattern of the toxin proteins, plasimids pattern, biochemical characteristics, and bioassay. The microscopic observaton showed that 49.1% of the isolates have bipyramidal shape crystals, 7.1% of spherical shape crystals, 1.4% of rhomboidal shape crystals, and others have small or amorphous inclusions. The insecticidal activities of the spore-crystal mixtures of isolates were tested against Plutella xylostella, Bombyx mori, Culex pipiens, and Agelastica coerulea. Bioassay showed that 51.3% of the isolates were shown to be active; lepidopteran-specific (44.8%), dipteran-specific(4.9%) and coleopteran-specific (1.6%). The remainder(48.8%) did not show any activity against the insects we tested. Interestingly though, some of these non-active isolates were shown to have bipyramidal crystals. By serotyping 22 isolates of our collection, we found that there are various kind of subspecies such as aizawai, amagines, canadensis, dermstadiensis, galleriae, finitimus, kurstaki, morrisoni and neoleonensis, and three isolates have been classified into a new serotype, H49, and one of them, the type strain, named subsp. muju. From his study it was found that phylloplane is a good source for the isolation of Bacillus thuringiensis, and Bacillus thuringiensis is distributed widely in Korea.