• The Korean Journal of Mycology
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• v.21 no.1
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• pp.23-27
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• 1993

Ion effects on $11{\alpha}-hydroxylation$ of progesterone and $5{\alpha}-reduction\;of\;11{\alpha}-hydroxyprogesterone$ by Rhizopus nigricans were investigated. Metal ions such as $Cu^{2+},\;Cd^{2+},\;Co^{2+},\;Mn^{2+},\;Zn^{2+},\;Fe^{2+},\;Mg^{2+},\;Fe^{3+}\;and\;Na^+$ reduced the $11{\alpha}-hydroxylation$ activity, while $K^+$ stimulated the same reaction. Enzyme activity for the $5{\alpha}-reduction$ of $11{\alpha}-hydroxyprogesterone$ was increased in the presence of $Fe^{2+},\;Mn^{2+},\;Mg^{2+},\;Co^{2+},\;Zn^{2+},\;Fe^{3+},\;K^+\;and\;Na^+$, whereas it was decreased in the presence of $Cd^{2+}\;and\;Cu^{2+}$. Potassium ion of $10^{-3}\;M\;$ of concentration was found to be effective for the promotion of $11{\alpha}-hydroxylation$. On the other hand, cadmium ion of $10^{-4}\;M$ was proved to suppress the $5{\alpha}-reduction$ reaction. Progesterone is reported to be transformed into $11{\alpha}-hydroxyprogesterone$ which, in turn, is converted further into $11{\alpha}-hydroxy-allopregnane-3$, 20-dione by R. nigricans. From this point of view, the highest yield of $11{\alpha}-hydroxyprogesterone$ could be obtained when potassium ion of $10^{-3}\;M$ was given initially followed by addition of cadmium ion of $10^{-4}\;M$ to limit conversion of 11{\alpha}-hydroxyprogesterone into $11{\alpha}-hydroxy-allopregnane-\;3$, 20-dione.

• Korean Journal of Microbiology
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• v.31 no.4
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• pp.306-311
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• 1993

The effects of methanol. used as a solvent for the hydrophobic substrate progesterone. on the morphology of Rhizopus nigricans and 11$\alpha$-hydroxylation of progesterone was investigated. The methanol tolerance of the 11$\alpha$-hydroxylase system in polyacrylamide immobilized R. nigricans mycelia as well as in free mycelia has been induced by adding various unsaturated fatty acids. biotin and ions into the cultivation medium. Immobilization of the cell seemed to protect the cells from denaturation by methanol. It gave higher reaction rate of progesterone than the free mycelia in the presence of methanol.500 $\mu$g/l of biotin was found to be the most effective induction agent for the methanol tolerance among tested chemicals. R. nixricans cells sustained its enzymatic activity at higher methanol concentrations as a result of accumulation of unsaturated fatty acids. especially oleic acid. in the membrane phospholipid.

• Korean Journal of Microbiology
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• v.29 no.4
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• pp.258-262
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• 1991

Rate of 11.alpha.-hydroxylation of progesterone with Rhizopus nigricans was accelerated by glucose. Glucose seemed to play an important role in the formation of cofactor because its effects on the reaction were almost same as those of electron acceptors such as NADPH and $NaIO_{4}$. Rate of glucose consumption appeared to increase in proportion as the rate of hydroxylation reaction, which enhanced with increase in the glucose concentration to level off at 0.5 g/l for mycelia and at 20 g/l for spores. However, for mycelia immobilized in polyacrylamide gel, externally added glucose did not affect the reaction rate at all because of the glucose accumulated in the gel during the cultivation period. 5.alpha.-Reduction of 11.alpha.-hydroxyprogesternoe required much higher concentration of glucose than 11.alpha.-hydroxylation of progesternoe so that high yield of 11.alpha.-hydroxyprogesternoe can be obtained by repressing the activity of 5.alpha.-reductase at low concentration of glucose.

• The Korean Journal of Mycology
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• v.15 no.1
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• pp.23-28
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• 1987

Physicochemical factors influencing the $11{\alpha}$, hydroxylation of progesterone by Rhizopus nigricans Ehrenberg were studied. The higher is the concentration of progesterone. the shorter time is need to attain maximum yield in the $11{\alpha}-hydroxyprogesterone$. High concentration in progesterone was supposed to inhibit the hydroxylation reaction of the $11{\alpha}-hydroxyprogesterone$. The range of optimum pH and temperature appeared to be rather broad and these conditions were similar to those of optimum growth of the mycelia. Agitation at a higher speed than a certain limit, and the presence of sugars accelerated the $11{\alpha}-hydroxyprogesterone$ reaction. Glucose was found to be more effective for the reaction using spores rather than mycelia as enzyme sources. Organic solvents were proved to supress the enzyme activity, which decreased with increase in the concentration of the organic solvents and in the time for pretreatment with the solvents.

• The Korean Journal of Mycology
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• v.18 no.2
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• pp.84-88
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• 1990

Hydroxylation in the $11{\alpha}$-position of progesterone molecules was carried out using Rhizopus nigricans spores immobilized within various gel matrices, among which polyacrylamide and agar gel were proved to be the most effective. Reactions with the immobilized cells and in­tact free cells showed almost identical conversion rate of progesterone, optimal pH and reaction time for attaining maximal yield, from which were confirmed absence of any decay and modification of enzyme activities.

• Mycobiology
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• v.47 no.3
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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.

• Korean Journal of Microbiology
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• v.23 no.4
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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.

• Korean Journal of Microbiology
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• v.29 no.2
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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.

• Microbiology and Biotechnology Letters
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• v.18 no.3
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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.

• Korean Journal of Microbiology
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• v.27 no.1
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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.