• Journal of Microbiology and Biotechnology
• /
• 제15권1호
• /
• pp.188-191
• /
• 2005

Abstract Cyclosporins are a family of clinically-important immunosuppressive cyclic peptides produced by Tolypocladium inflatum. The structural modification of cyclosporins via hydroxylation at various positions of N-methyl leucines in cyclosporin A leads to a dramatic change of their bioactive spectra. Among over 100 soil actinomycetes screened, two actinomycetes species, Sebekia benihana and Pseudonocardia autotrophica, were identified to contain superior cyclosporin A hydroxylation activities. A HPLC-based cyclosporin A hydroxylation assay revealed that each strain possesses distinctive hydroxylation specificity and regiospecificity; mono-hydroxylation at the 4th N-methyl leucine of cyclosporin A by S. benihana, and di-hydroxylations at both 4th and 9th N-methyl leucines of cyclosporin A by P. autotrophica. The conversion yields for cyclosporin A hydroxylation by both S. benihana and P. autotrophica were significantly improved from less than 10% and 18% up to 58% and 45%, respectively, in the optimized culture containing molybdenum with 0.05 g/l of cyclosporin A concentration. An ancymidol-specific inhibition of cyclosporin hydroxylation also suggested that the regiospecific cyclosporin hydroxylation might be catalyzed by a putative cytochrome P450 mono-oxygenase enzyme.

• BMB Reports
• /
• 제32권3호
• /
• pp.226-231
• /
• 1999

In vitro effects of acetone on cytochrome P450 (P450)-dependent benzo(a)pyrene (B(a)P) hydroxylation supported by cumene hydroperoxide (CuOOH) or NADPH/$O_2 $ systems were studied using 3-methylcholanthrene-pretreated rat liver microsomes. The maximal rate of B(a)P hydroxylation at constant concentration ($80\;{\mu}M)$ of the substrate was observed in the presence of $30\;{\mu}M$ CuOOH. However, at concentrations higher than $30\;{\mu}M$ CuOOH the hydroxylation rates were rapidly decreased. In contrast to CuOOH, at a concentration of $200\;{\mu}M$ NADPH, B(a)P hydroxylation rate reached a plateau. At concentrations higher than $200\;{\mu}M$ NADPH, the rates of substrate hydroxylation were maintained at the maximal rate with no inhibition. Acetone at 1% (v/v) enhanced both CuOOH- and NADPH/$O_2$-supported B(a)P hydroxylation at the optimal concentrations of the cofactors. At concentrations higher than 1% (v/v) acetone, substrate hydroxylation was sterero specific under the support of these two cofactors; it was strongly enhanced with $30\;{\mu}M$ CuOOH, but rather inhibited in the $200\;{\mu}M$> NADPH/$0_2 $ system. The lipid peroxidation rate induced during CuOOH-supported P450-dependent B(a)P hydroxylation was increased as CuOOH concentrations were increased. Acetone in the concentration range of 2.5~7.5%(v/v) inhibited lipid peroxidation during CuOOH supported B(a)P hydroxylation. The finding that CuOOH-supported B(a)P hydroxylation is greatly enhanced by acetone suggests that acetone may contribute more to the activation of oxygen (for the insertion of oxygen into the substrate) in the presence of CuOOH than with NADPH/$O_2$. Acetone may also contribute to the partial inhibition of destruction of microsomal membranes by lipid peroxidation.

• Archives of Pharmacal Research
• /
• 제15권1호
• /
• pp.41-47
• /
• 1992

In order to obtain acetaminophen, a popular analgesic-antipyretic, though microbial p-hydroxylation and N-acetylation of aniline, various Streptomyces strains were screened. Aniline N-acetylation activity was rather ubiquitous but-hydroxylation activity was selective. Microbial conversion pathway of aniline to acetaminophen was considered to be through N-acetylation and p-hydroxylation or vice versa. However, depending on species used, o-hydroxylation and its degradation activity (S. fradiae) and acetaminophen degradation activity (S. coelicolar) were also detected. Among the screened Streptomyces strains, S fradiae NRRL 2702 showed the highest acetanilide p-hydroxylation activity (203% conversion rate). Furthermore, in S. fradiae carbon source and its concentration, phosphate ion concentration and pH of growth medium were found to play the crucial roles in p-hydroxylation activity. Through the proper combination of factors mentioned above, the ten times more activity (26-30% conversion rate) was attained.

• Archives of Pharmacal Research
• /
• 제15권1호
• /
• pp.30-34
• /
• 1992

In order to obtain acetaminophen, a popular analgesic-antipyretic, through microbial p-hydroxylation and N-acetylation of aniline, various fungi and bacteria were secreened. Among them, Streptomyces species were chosen for strain improvement by the use of interspecific protoplast fusion technique. Two interspecific fused strains were developed between S. rimosus (N-cetylation function) and S. aureofaciens (p-hydroxylation function) and also between S. lividans and S. globisporus. For efficient protoplast fusion and cell wall regeneration, various conditions were examined. In a typical experiment of mixed S rimosus ($pro^- \;his^-$) and S. aureofaciens ($ilv^-$) protoplasts with 40% (w/v) polythylene glycol 3350 (PEG) for 3 min gave $8.3\times10^{-7}$ of fusion frequency. Treatment of mixed S. lividans (pant-) and S. globisporus (leu-) protoplasts with 50% (w/v) PEG for 3 min at $30^\circ{C}$ gave $1.2\times10^{-6}$ of frequency. Among the fused strains, up to 40-50% increase in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation of acetanilide, plasmid curing was attempted. We found that cells treated with acriflavine (at the frequency of 100%) and cells regenerated from protoplsts of S. auroefaciens (2% frequency) lost their p-hydroxylation function.

• 미생물학회지
• /
• 제27권4호
• /
• pp.366-372
• /
• 1989

Reichstein's substance S의 $11\beta$-hydroxylation 활성이 있는 균주를 선택하고자 21 균주를 실험한 결과 4균주에서 $11\beta$-hydroxylation의 활성이 발견되었으며, 그 중 Pellicularia fillamentosark 가장 높은 효소활성을 보였다. 이 균주의 $11\beta$-hydroxylation의 합성을 강력하게 저해하였다. $11\beta$-hydroxylation의 최적 pH 범위는 2.0-8.0으로 광범위하였다. 성장단계 중 균사체 분지시기와 완전하게 성숙한 균사체 시기에 효소합성의 유도가 가장 활발하였으며, 70시간 배양된 균사체가 가장 좋았다. 반면, 포자에는 기질에 의한 $11\beta$-hydroxylation의 유도현상이 없는 것으로 판정되었다.

• 미생물학회지
• /
• 제31권4호
• /
• pp.306-311
• /
• 1993

소수성 기질인 progesterone 을 수용성 반응액 속에 용해하기 위하여 사용되는 methanol 이 Rhizopus nigricans 의 성장과 progesterone 의 11.alpha.-hydroxylation 반응에 미치는 영향을 조사하고, 세포막 인지질의 불포화 지방산 함량을 증가시켜 유기용매에 대한 내성을 유도하였다. R. nigricans 의 균사체는 methanol 존재하에서 형태적인 변화를 일으켰으며, 세포막 인지질의 지방산 조성에 변화를 가져와 불포화 지방산, 특히 oleic acid 의 함량이 증가되었다. 세포의 고정화는 유기용매로부터 세포를 보호하여 progesterone 의 11-.alpha.-hydroxylation 율을 증가시켰다. 성장배지에 500.$\mu$g/l의 biotin 을 첨가하였을 때 세포막 인지질의 oleic acid 함랴이 증가되었으며 methanol 에 대한 내성이 증진되어 높은 progesterone 의 11 $\alpha$-hydroxylation 율을 얻을 수 있었다.

• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
• /
• pp.334-334
• /
• 1994

Omeprazole의 약동학적 Parameter는 tmax를 제외하고 두 군간에 유의한 (p<0.001) 차이론 보였다 : AUC의 평균치는 5-mephenytoin hydroxylation poor metabol izer에서 extensive metabol izer에 비해 약 6-7배 컸다. Omeprazole sulfone의 parameter는 omeprazole에서 관찰된 두 군간의 차이와 유사하였다. 그러나 5-hydroxyomeprazole의 경우에는 Cmax, AUC 등이 extensive metabolizer에서 더컸다. Omeprazole의 청소율은 S-mephenytoin hydroxylation 대사능(MR)과 유의한 상잔관계(rs=0.79, p<0.01)를 보였으며, omeprazole과 그 대사물(5-hydroxyomeprazole 및 omeprazole sulfone)의 반감기 또한 S-mephenytoin hydroxylation 대사능과 유의한 상관성을 보였다.

• Archives of Pharmacal Research
• /
• 제15권1호
• /
• pp.35-40
• /
• 1992

S. fradiae showed the highest acetanilide p-hydroxylation activity in the tested strains. And S. fradiae was well characterized genetically, especially with respect to tylosin production. Two mutants, which lost hydroxylation, were isolated in 140 regenerated colonies from protoplasts. In restriction enzyme digesion of total DNAs, isolation of giant linear plasmid DNA and determination of antibiotic resistances to chloramphenicol, tylosin, hygromycin B and mitomycin C, any differences among mutants and a wild type strain were not detected. These facts suggest that lesion on 6, 000 Kb chromosomal DNA was responsible for the lack of p-hydroxylation activity induced by protoplast formation and regeneration.

• Bulletin of the Korean Chemical Society
• /
• 제22권1호
• /
• pp.93-96
• /
• 2001

• 미생물학회지
• /
• 제29권4호
• /
• pp.258-262
• /
• 1991

Rhizopus nigricans에 의한 progest$\xi$rone의 Iia-hydroxylation 반응은 포도당에 의하여 촉진되었으며 그 효과가 전자 수용체인 NADPH와 NaJD. 를 투입하였을 때의 효과와 유사한 것으로 부터 표도당은 이 반응에 필요한 보조요소의 생성에 기여하며 hydroxylation 반응을 촉진한다고 생각되었다 포도당의 소모량은 hydroxy lation 반응 속도가 빠을수록 크게 나타났으며 포도당의 농도가 증가함에 따라 반응 속도는 점차 증가하다가 균사체는 0.5 g/l 포자체는 20 g/l에서 거의 일정해지는 경향을 나타내었다. 그러나 polyacrylamide gel에 고정화한 균사체의 경우 배양 기간 동안 gel내에 축적되어 있는 포도당 만으로도 반응이 충분히 촉진되었다. a-Hydroxy progesterone의 Sa-reduction 반응은 p progesterone의 11a-hyroxylation 반응에 버하여 훨씬 더 높은 포도당 농도가 필요하므로 포노당의 농도를 낮게 유지시킴으로써 5a-reducrion 반응을 억제하여 11 a-hydroxyprogesterone의 수득융을 높힐 수 있음 갯으로 판단되었다.