• Microbiology and Biotechnology Letters
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• v.18 no.1
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• pp.66-70
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• 1990

A study on 11 $\alpha$-hydroxylation of progesterone by using Rhizopus nigricans was carried out to produce efficiently 11 $\alpha$-hydroxyprogesterone which is an essential intermediate of corticosteroids synthesis. Firstly, medium was optimized in view of bioconversion yield and cell growth. Glucose and casamino acid were selected as carbon and nitrogen source and the ratio of carbon to nitrogen which maximize bioconversion yield was determined to be 2:1. Secondly, the addition time of progesterone and dispersion method were studied. When progesterone dispersed with 0.01% (v/v) Tween 80 was added at 12-14 hr of cultivation, higher bioconversion yield was obtained. When 20g/$\ell$ of progesterone was added, the yield reached 70% under optimized conditions.

• 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.

• The Korean Journal of Mycology
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• v.11 no.3
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• pp.115-119
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• 1983

The bioconversion of progesterone by Aspergillus phoenicis has been studied. The metabolism in the conditions of experiment gave $11{\alpha}-hydroxyprogesterone$ as main product. The concentration of $11{\alpha}-hydroxyprogesterone$ increased monotonically and leveled off after 40 hours of incubation. Addition of glucose into the medium reduced considerably the time for attaining limiting concentration of $11{\alpha}-hydroxyprogesterone$. The increase in initial progesterone concentration did not affected the percentage of conversion nor the time required for termination of the reaction. But it could not be represented as first order reaction with respect to progesterone concentration. The degree of inhibition of enzymes by organic solvents depended upon the concentration of solvents. At low solvent concentration, acetone proved to yield the highest conversion.

• 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.

• The Korean Journal of Mycology
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• v.13 no.2
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• pp.83-87
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• 1985

The temperature dependency and the stability of enzyme systems for $11{\alpha}-hydroxyla­tion$ of progesterone were investigated using Aspergillus phoenicis. Though A. phoenicis conserves high enzyme activities for lactose hydrolysis even at high temperatures, the bioconversion reaction of progesterone by this strain was found to be very temperature sensitive. The compositions of reaction mediums of inside and outside of cells were analyzed using sonication technique. At early stage of reaction, the concentration of $11{\alpha}-hydroxyprogesterone$ of cell inside was higher than that of outside. But as the reaction proceeded further, the $11{\alpha}-hydroxyprogesterone$ existing inside of cells being converted into another products, its concentration was lower within the cells that in the bulk medium. Even in the reaction mediums containing organic solvents, A. phoenicis was founded to be able to metabolite, so that $11{\alpha}-hydroxyprogesterone$ can be produced continuously from fixed bed reactions packed with immobilized A phoenicis in vivo.

• Journal of Microbiology and Biotechnology
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• v.33 no.3
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• pp.387-397
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• 2023

Cytochrome P450 (CYP) is a heme-containing enzyme that catalyzes hydroxylation reactions with various substrate molecules. Steroid hydroxylases are particularly useful for effectively introducing hydroxyl groups into a wide range of steroids in the pharmaceutical industry. This study reports a newly identified CYP steroid hydroxylase (BaCYP106A6) from the bacterium Bacillus sp. and characterizes it using an in vitro enzyme assay and structural investigation. Bioconversion assays indicated that BaCYP106A1 catalyzes the hydroxylation of progesterone and androstenedione, whereas no or low conversion was observed with 11β-hydroxysteroids such as cortisol, corticosterone, dexamethasone, and prednisolone. In addition, the crystal structure of BaCYP106A6 was determined at a resolution of 2.8 Å to investigate the configuration of the substrate-binding site and understand substrate preference. This structural characterization and comparison with other bacterial steroid hydroxylase CYPs allowed us to identify a unique Arg295 residue that may serve as the key residue for substrate specificity and regioselectivity in BaCYP106A6. This observation provides valuable background for further protein engineering to design commercially useful CYP steroid hydroxylases with different substrate specificities.