• Journal of Microbiology and Biotechnology
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• 제6권5호
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• pp.326-330
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• 1996

To illustrate whether a hemolysin in $\delta$-endotoxins of Bacillus thuringiensis strain 73E10-2 and subsp. israelensis had immunological identity, a cyt gene of the strain 73E10-2 which encodes a hemolysin was cloned to B. subtilis (transformant 2753). The transformant 2753 containing cyt gene produced the hemolysin which lysed sheep erythrocytes after treatment of proteinase K. The hemolysin was proved also to be toxic against mosquito larvae (Aedes aegypti). The molecular weight of the hemolysin produced from the transformant 2753 was determined to be about 25 kDa by SDS-PAGE and immunoblot. The hemolysin in $\delta$-endotoxin of subsp. israelensis and subsp. kyushensis did not react on immunoblot using polyclonal anti-$\delta$-endotoxin of the strain 73E10-2, but 70-140 kDa mosquitocidal toxins in $\delta$-endotoxin of subsp. kyushuensis reacted.

• 한국미생물·생명공학회지
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• 제11권3호
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• pp.163-168
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• 1983

Pnicillinase를 분필하는 Streptomyces bobili YS-40균주의 plasmid DNA를 phemol방법으로 유출하여 Bocillus subtilis IAM 12118균주에 형질 전환시켜서 penicillin 내성 plasmid DNA가 표현되게 하였다. 형질전환에 미치는 최적 pH와 온도는 각각 7.0, 3$0^{\circ}C$으로 나타났다. DNA와 Competent cell을 20분 이상 접촉시킴으로서 형질전환이 잘 일어났으며 DNA의 농도가 증가할수록 transformant의 수도 증가했다. DNA inhibitor 로 사용되는 lysine 의 첨가로 형질전환의 빈도가 약 6배정도 상승되었으며 chloramphenicol은 형질전환의 빈도에 별 영향을 미치지 않았다.

• Journal of Microbiology and Biotechnology
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• 제1권4호
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• pp.227-231
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• 1991

A gene coding for a $\beta$-galactosidase of Bacillus subtilis HP-4 was cloned in E. coli JM109 by inserting HindIII digested fragment of B. subtilis HP-4 chromosomal DNA into the site of pBR322 and selecting recombinant transformant showing blue color on X-gal plate. The recombinant plasmid, named pBG109, was found to contain the 1.4 Kbp HindIII fragment originated from B. subtilis HP-4 chromosomal DNA by Southern hybridization. The cloned gene was stably maintained and expressed in E. coli JM109 and the pBG109 encoded $\beta$-galactosidase had the same enzymatic properties as those of $\beta$-galactosidase produced by B. subtilis HP-4.

• Journal of Microbiology and Biotechnology
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• 제1권2호
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• pp.116-120
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• 1991

The transformant Bacillus subtilis ED213 carrying the pSO100 which cloned the cdd gene encoding cytidine deaminase (cytidine /2'-deoxycytidine aminohydrolase, EC 3.5.4.5, CDase) originated from wild type B. subtilis was cultivated in Spizizen minimal medium (SMM). To overcome poor expression of the cdd gene in SMM medium, the medium compositions and growth conditions were optimized. The optimized medium compositions and growth conditions were cytidine concentration of 80 mg/l, glycerol of 25 g/l, and $(NH_4)_2SO_4$ of 10 g/l, along with $37^{\circ}C$ and pH 7.0. The intracellular CDase production was increased 3 times from 1,000 unit/ml to 3,200 unit/ml, and extracellular CDase also increased from nearly undetectable amounts to 1,500 unit/ml. The cytidine concentration was signified as the most critical compositional factor for overproduction of CDase by increasing the cell density mainly in culture broth. The plasmids were more stable in cells that were grown in original SMM medium with stability of 90% compared to those grown in optimized SMM medium with stability of 80% after 48 hours cultivation. The most active amplification of plasmid was occurred in the logarithmic phase, which showed a value around four times higher than the initial copy number. In the exponential phase, the CDase production was closely related to the plasmid copy number along with the cell density. However, it was not accorded with cell density at the stationary phase.

• 한국미생물·생명공학회지
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• 제19권4호
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• pp.356-361
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• 1991

식물근부병의 방제균으로 선발된 Bacillus subtilis YBL-7의 식물부균 Fusarium solani에 대한 길항력을 유전공학적 조작에 의해 다목적으로 증강시킬 수 있는지를 타진하기 위해 외부유전자인 Bacillus pasteurii의 urease 유전자를 생물방제균 B.subtilis YBL-7내 도입자하고자 시도하였다. 외부 urease 유전자는 B.pasteurii의 urease gene을 shuttle vector인 pGR71의 HindII site에 삽입하여 E.coli내에서 발현시킨 pGU66을 사용하여 형질전환시켰으며 이때의 최적 형질전환조건과 도입된 urease 유전자의 발현을 조사해 보았다.

• 한국미생물·생명공학회지
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• 제25권1호
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• pp.30-36
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• 1997

To genetically breed powerful multifunctional antagonistic bacteria, the urease gene of alkalophilic Bacillus pasteurii was transferred into Bacillus subtilis YB-70 which had been selected as a powerful biocontrol agent against root-rotting fungus Fusarium solani. Urease gene was inserted into the HindIII site of pGB215-110 and designated pGU266. The plasmid pGU266 containing urease gene was introduced into the B. subtilis YB-70 by alkali cation transformation system and the urease gene was very stably expressed in the transformant of B. subtilis YB-70(pGU266). The optimal conditions for the transfomation were also evaluated. From the in vitro antibiosis tests against F. solani, the antifungal activity of B. subtilis YB-70 containing urease gene was much efficient than that of the non-transformed strain. Genetic improvement of B. subtilis YB-70 by transfer of urease gene for the efficient control seemed to be responsible for enhanced plant growth and biocontrol efficacy by combining its astibiotic action and ammonia producing ability.

• 미생물학회지
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• 제30권5호
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• pp.403-409
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• 1992

STA1 유전자의 promoter 와 분비신호서열을 이용하여 B. subtilis 의 CMSase 를 분비하는 재조합 효모균주를 육성하였다. STA1A 유전자의 promoter, 분비신호서열, TS region 및 mature glucoamylase N-말단부위의 아미노산 98개와 B. subtilis 의 CMCase 구조유전자가 차례로 연결된 재조합플라스미드 pYESC24 를 제작한후 효모에 형질전환하였으나 CMCase 가 세포외로 분비되지 않았다. 반면에 STA1 의 TS region 및 mature glucoamylase N-말단 아미노산 98 개를 제거하여 CMMase 구조유전자갸 STA1 의 분비신호서열에 바로 연결된 재조합 플라스미드 pYESC11 에 의한 효모형질전환 균주는 CMCase 분비능이 아주 우수하였다. 이 형질전환 균주를 YPD 배지에서 4 일간 배양한 후 세포부위 별 CMCase 역가를 측정한 결과 배양액 1 m/당 총역가 44.7 unit 존재하였으며 이중 93% 이상이 배양상등액에서 관찰되었다.

• 한국식품저장유통학회지
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• 제7권4호
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• pp.414-417
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• 2000

단백질분해효소를 생산하지 않는 균주 B. subtilis MT-2의 염색체 DNA를 추출한 다음, B. subtilis AC819 균주에 상동성 유전자재조합을 이용하여 competent cell 형질전환을 시켰다. 얻어진 형질전환체를 B. subtilis HL-1이라고 명명하였으며, 그 표현형은 histidine 요구성, streptomycin 내성, tetracyclin 내성을 나타내면서 단백질 분해효소를 생산하지 않았다. 플라스미드 pUB110 을 이용한 B. subtilis HL-1의 protoplst 형질전환율은 B. subtilis MT-2의 형질전환율보다 높았다. 따라서 새로운 B. subtilis HL-1균주는 단백질분해효소의 형질전환과 내열성 protease 유전자클로닝에서 숙주로 사용하는데 유용하다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.536-539
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• 2003

A strong constitutive $P_{JH}$ promoter from Bacillus sp. was applied to overexpress the endoxylanase gene in Bacillus subtilis. The expression plasmid, pJHKJ4, was designed to contain the $P_{JH}$ promoter and open reading frame of endoxylanase including its own promoter. The plasmid was introduced into B. subtilis DB431 and the resulting transformant was grown on LB glucose medium. The endoxylanase activity in the culture supernatant reached about 140 unit/ml. The enzyme in the supernatant was efficiently concentrated to 70% by two-step treatments of ammonium sulfate saturation and ultrafiltration. The stabilization of concentrated enzyme solution at different storage temperatures was examined with various stabilizers such as NaCl, $CaCl_2$, sucrose, sorbitol, polyethylene glycol, and Tween-80.

• 한국미생물·생명공학회지
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• 제17권2호
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• pp.160-164
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• 1989

알카리성 Bacillus sp. AL-8의 알카리성 amylase 유전자를 포함하는 5.7Kb의 EcoRI 단편을 pUB 110을 vector로 하여 amylase를 생산하지 못하는 B. subtilis sta-1에서 발현시켰다. 재조합 plasmid pMB802와 pMB809는 숙주세포인 B. subtilis에서 매우 안정하게 유지되었으며 amylase 생산이 공여균 주에서 보다 1.8배 증가하였다. 형질전환주에서 생산된 amylase는 공여균주와 같은 효소적 성질을 나타내었다. 5.7Kb 단편을 E. coli에 subcloning한 결과 3.7Kb의 EcoRI 단편에 알카리성 amylase 유전자가 존재하였다.