• 한국식품영양과학회지
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• 제28권1호
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• pp.87-93
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• 1999

This study was carried out to investigate the effects of nucleic acid related compounds and metal ions on activities of cytidine deaminase from Bacillus subtilis ED 213. The purified cytidine deaminase was weakly inhibited by 1mM GMP, IMP and ATP, but not affected by other nucleic acid related compounds such as CMP and UDP. The apparent Km values for cytidine, deoxycytidine, 5 methylcy tidine, fluorodeoxycytidine, and 5 bromocytidine were calculated to be 6.6$\times$10-4M, 6.0$\times$10-4M, 0.9$\times$10-4M, 0.8$\times$10-4M, and 2.0$\times$10-3M, respectively. The cytidine deaminase was completely inhibited by 1mM Hg2＋, and mildly inhibited over 40% by metal ions such as Na＋ and Fe2＋. However the enzyme activity was activated more than 40% by 1mM Mg2＋.

• 미생물학회지
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• 제35권2호
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• pp.133-138
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• 1999

Bacillus subtilis ED 213의 cytidine deaminase 의 활성부위에 존재하는 필수 아미노산잔기를 화학수식 방법으로 측정하였다. 본 효소는 1mM o-phenanthroline 에 의하여 효소활성이 43% 저해되어 효소활성 발현에 Fe\sup 2+\가 요구된다고 추정되며, 1mM ethylenediaminetetraacetic acid 에 의해서는 효소활성이 오히려 28% 정도 촉진되었다. 본 효소는 1mM N-bromosuccinimide, 1mM chloramine-T 와 1mM $\rho$-chloromercuribenzoic acid에 의하여 100% 저해되었으며, 그의 저해 양상은 경쟁적 저해 양상을 나타내었다. 본 효소의 효소활성은 1mM pyridoxal-5-phosphate 에 의항 36% 저해되었으며, 1mM 1ethyl-3-carbodiamide 와 1mM glycine methylester에 의해 저해된 효소활성이 5mM cysteine에 의해 완전히 회복되었다. 이상의 결과로부터 Bacillus subtilis ED 213 cytidine deaminase의 활성부위에는 tyrosine, methionine, cysteine 과 serine 잔기가 관여할 뿐만 아니라 lysine 과 glycine 도 효소활성에 관여하는 것으로 추정된다.

• 한국미생물·생명공학회지
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• 제14권6호
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• pp.479-485
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• 1986

Bacillus amyloliquefaciens가 생성하는 액화형 $\alpha$-amylase의 구조유전자를 클로닝하기 위하여 이 균주 염색체의 2 - 5kb 획분을 분리하여 Mbo I으로 부분분해한 후 pUB110플라스미드의 BamHI 부위에 삽입하여 hybrid vector pSKS3 를 제작하였다. 이 재조합플라스미드를 세한수식결손 세포인 Bacillus subtilis ED 107에서 subcloning한 후 당화형 $\alpha$-amylase를 생성하는 Bacillus subtilis NA 64의 $\alpha$-amylase결실변이주 Bacills subtilis KM 107 주에서 형질 발현시켰다. 이때 형질전환빈도는 $10^{-5}$ - $10^{-7}$정도였으며 그중 약 50%가 $\alpha$-amylase 분비능이 있었으나 거의 대부분이 쉽게 실활되었다. 최종선별과정에서 $\alpha$-amylase를 가장 강하게 생성하는 형질전환주 Bacillus subtilis KM213으로부터 재 추출한 재조합플라스미드 pSKS3는 5.7kb 삽입된 단편이 BamH I/Mbo I 부위에 결합되어 있었다. pSKS3에 클로닝된 유전자에 의해 발현되는 $\alpha$-amylase 활성은 B. amyloliquefaciens 활성의 약 25%였으며, 이 pSKS3에 의해 발현되는 $\alpha$-amylase 단백은 B.amyloliquefaciens의 $\alpha$-amylase와 동일한 전기영동성을 나타내었음을 볼 때 pSKS3에 클로닝된 유전자는 B. amyloliquefaciens에서 유래함을 알 수 있었다.

• 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.