• 한국균학회지
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• 제21권1호
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• pp.23-27
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• 1993

Rhizopus nigricans에 의한 progesterone의 $11{\alpha}-hydroxylation$과 ${5\alpha}-reduction$반응에 미치는 이온의 효과를 조사하였다. $Cu^{2+},\;Cd^{2+},\;Co^{2+},\;Mn^{2+},\;Zn^{2+},\;Fe^{2+},\;Mg^{2+},\;Fe^{3+}$ 및 $Na^+$은 $11{\alpha}-hydroxylation$ 반응의 활성을 저하시켰으나 $K^+$은 이 반응을 촉진시켰다. $11{\alpha}-hydroxyprogesterone$을 $5{\alpha}-reduction$시키는 효소는 $Fe^{2+},\;Mn^{2+},\;Mg^{2+},\;Co^{2+},\;Zn^{2+},\;Fe^{2+},\;K^+,\;Na^+$에 의하여 그 활성이 증가되었고 $Cd^{2+}$과 $Cu^{2+}$은 이 반응을 억제하였다. $11{\alpha}-hydroxylation$ 반응을 촉진하는데는 $10^{-3}\;M$의 $K^+$이 효과적이었으며 $5{\alpha}-reduction$반응을 엑제하기 위하여는 $10^{-4}\;M$의 $Cd^{2+}$이 적합하였다. R. nigricans는 세포내의 다효소체계에 의하여 progesterone이 $11\{alpha}-hydroxyprogesterone$으로 전환된 후 $11{\alpha}-hydroxy-allopregnane-3, 20-dione으로 $5{\alpha}-reduction$되므로 $10^{-3}\;M$의 $K^+$을 먼저 첨가하여 2시간 동안 progesterone 전환 반응을 실시한 후에 $10^{-4}\;M$의 $Cd^{2+}$을 첨가하였을 때 가장 높은 $11\{alpha}-hydroxyprogesterone$의 수득률을 얻을 수 있었다.

• 미생물학회지
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• 제31권4호
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• pp.306-311
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• 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.

• 미생물학회지
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• 제29권4호
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• pp.258-262
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• 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.

• 한국균학회지
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• 제15권1호
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• pp.23-28
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• 1987

R. nigricans Ehrenberg 에 의한 progesterone 의 $11{\alpha}-hydroxyprogesterone$에 영향을 미치는 여러 물러 화학적 요인에 대하여 조사하였다. Progesterone의 농도가 낮을수록 $11{\alpha}-hydroxyprogesterone$이 최고의 수득율에 도달하는데 소요되는 시간이 짧았으며, 높은 기질 농도에서의 Progesterone은 $11{\alpha}-hydroxyprogesterone$의 hydroxylation을 저해하였다. 이 반응에 대한 최적 pH와 온도 범위는 비교적 넓은 범위로 나타났으며, 균사제의 성장 죄적 조건과 유사하였다. 일정 속도 이상의 진탕 속도와 당류들은 $11{\alpha}-hydroxyprogesterone$ 반응을 촉진시키는 효과를 보였으매, 균사체에서 보다 포자를 효소원으로 사용하였을 때 glucose의 효과는 더 크게 나타났다. 유기 용매는 효소 활성 저해제로 작용하었으며, 저해 정도는 유기 통매 농도가 높을수록, 용매에 처리한 시간이 길어질수록 높게 나타났다.

• 한국균학회지
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• 18권2호
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• pp.84-88
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• 1990

Rhizopus nigricans 생존포자체를 여러 종류의 고정용 담체에 고정화시켜 progesterone의 $11{\alpha}-hydroxylation$ 효소 활성을 조사한 결과 Polyacrylamide와 agar gel이 가장 우수한 것으로 나타났다. 이때 고정화는 free cell이 나타내는 progesterone의 전환율과 최적 pH, 생성물이 최고 수득률에 도달하는 반응시간에 변화를 유발하지 않았으므로 고정화 과정에서 호소 활성의 감소와 변성이 없었음을 확인할 수 있었다.

• Mycobiology
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• 제47권3호
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• pp.301-307
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• 2019

The $11{\alpha}$-hydroxylation of $16{\alpha}$, 17-epoxyprogesterone (EP) catalyzed by Rhizopus nigricans is crucial for the steroid industry. However, lower conversion rate of the biohydroxylation restricts its potential industrial application. The $11{\alpha}$-steroid hydroxylase CYP509C12 from R. oryzae were reported to play a crucial role in the $11{\alpha}$-hydroxylation in recombinant fission yeast. In the present study, the CYP509C12 of R. oryzae (RoCYP) was introduced into R. nigricans using the liposome-mediated mycelial transformation. Heterologous expression of RoCYP resulted in increased fungal growth and improved intracellular reactive oxygen species content in R. nigricans. The $H_2O_2$ levels in RoCYP transformants were approximately 2-folder that of the R. nigricans wild type (RnWT) strain, with the superoxide dismutase activities increased approximately 45% and catalase activities decreased approximately 68%. Furthermore, the $11{\alpha}$-hydroxylation rates of EP in RoCYP transformants (C4, C6 and C9) were 39.7%, 38.3% and 38.7%, which were 12.1%, 8.2% and 9.4% higher than the rate of the RnWT strain, respectively. This paper investigated the effect of heterologous expression of RoCYP in R. nigricans, providing an effective genetic method to construct the engineered strains for steroid industry.

• 미생물학회지
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• 제23권4호
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• pp.297-301
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• 1985

The dependence of activities of Aspergillus phoenicis on the culture conditions in the progesterone transformation reaction was investigated. In the beginning of the reaction, $6{\beta},\;11{\alpha}-dihydroxyprogesterone$ was not produced even at high concentration of $11{\alpha}-hydroxyprogesterone$. However, large amount of the product was obtained after the complete exhaustion of progesterone. When spores of A.phoenicis replaced mycelia as enzyme source, $11{\alpha}-hydroxyprogesterone$ was produced after a considerably long indyction period, and its maximum production rate followed the exponential growth phase. The $6{\beta}-hydroxylation\;of\;11{\alpha}-hydroxyprogesterone$ continued, even after the stationary growth phase. A. phoenicis showed high enzyme activity for these reactions when the phosphate buffer solutions were used in place of the ordinary culture medium. The buffer solutions of low pH gave more yield of $11{\alpha}-hydroxyprogesterone$ than those of high pH. However, the addition of flucose to the buffer solutions did not activate the transformation reaction. The presence of progesterone seems to be necessary for the induction of enzymes for the $6{\beta}-hydroxylation\;of\;11{\alpha}-hydroxyprogesterone\;since\;6{\beta},\;11{\alpha}-dihydroxyprogesterone$ is not produced in the reaction medium containing only $11{\alpha}-hydroxyprogesterone$ as a substrate.

• 미생물학회지
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• 제29권2호
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• pp.111-116
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• 1991

Rhizopus nigricans produces 11.alpha.-hydroxyprogesternoe with a unidentified byproduct, which is hardly separated. Results of chromatography, IR and NMR spectroscopy identified the byproduct to be 11.alpha.-hydroxy-allopregnane-3,20-dione. R. nigricans was found to transform progesternoe into a monoform intermediate, 11.alpha.-hydroxyprogesterone, from which 11.alpha.-hydroxy-allopregnane-3,20-dione and 6.betha., 11.alpha. - dihydroxyprogesterone were formed respectively by 5.alpha.-reduction and 6.betha.-hydroxylation.

• 한국미생물·생명공학회지
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• 제18권3호
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• pp.280-285
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• 1990

Biotransformation of progesterone to 11 $\alpha$ -hydroxyprogesterone by growing cells of Rhizopus nigricans was investigated. As the concentration of progesterone increased, the specific growth rate of R. nigricans decreased linearly, and consequently the conversion yield lowered. The hyphae of the microorganism were observed to become thicker, shorter, and more densely branched at high concentrations of progesterone. In order to improve the process productivity, biotransformation was conducted with continuous addition of progesterone. When the substrate was added continuously at a rate of 0.86 g/hr for 30 hrs, overall conversion yield reached upto 56% while a single addition of the same amount of progesterone yielded about 40% eonversion. When additional feeding of glucose was carried out upon its depletion, an improved br'oconversion yield upto 68% was obtained.

• 미생물학회지
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• 제27권1호
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• pp.70-76
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• 1989

Progestrone bioconversion by immobilized Aspergillus phoenicis was studied. Progesterone was converted into 11$\alpha$-hydroxyprogesterone and 3-minor byproducts. Whole cells of A. phoenicis were immobillized by enreappment with calcium-alginate, K-carrageenan, or polyacrylamide. Of these materials tested, cell immobilized in $Ca^{2+}$ -alginate gels showed the highest activity for 11$\alpha$-hydroxylation of progesterone. In the case of mycelia immobilized in $Ca^{2+}$-alginate, futher progressing hydroxylation of 11$\alpha$-hydroxyprogesterone was greatly reduced. Spores of A. phoenicis which were immobillized with $Ca^{2+}$-alginate and germinatedin situ for 25 hours showed higher 11$\alpha$-hydroxylase activity than those of entrapped whole mycelia and maintained initial enzyme activity for all 8 times of repeated use. After 16 times of reuse, the activity was declined 30% or more. When culture media and $Zn^{2+}$ were introduced into the reaction media, the activity of the immobilized mycelia which had been lowered due to many times of reuse was effectively reactivated.