11α-hydroxylation Of Progesterone Research Articles

Metabolism of steroids by cytochrome P450 2C9 variants.

CYP2C9 is a human microsomal cytochrome P450c (CYP). Much variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and ten mutants were co-expressed with NADPH-cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward steroids were examined. CYP2C9.2, CYP2C9.3, CYP2C9.4, CYP2C9.16, CYP2C9.28, CYP2C9.48 and CYP2C9.52 had higher testosterone 6β-hydroxylation than CYP2C9.1. CYP2C9.4 showed higher progesterone 6β-hydroxylation activity than CYP2C9.1. CYP2C9.28 and CYP2C9.48 showed higher progesterone 11α-hydroxylation activity than CYP2C9.1. CYP2C9.48 showed higher progesterone 16α-hydroxylation activity than CYP2C9.1. CYP2C9.2, CYP2C9.3, CYP2C9.16 and CYP2C9.30 had higher estrone 16α-hydroxylation activity than CYP2C9.1. CYP2C9.3 had higher estrone 11α-hydroxylation activity than CYP2C9.1. CYP2C9.39 and CYP2C9.57 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.39 and CYP2C9.57 was not changed, but CYP2C9.2, CYP2C9.3, CYP2C9.4, CYP2C9.16, CYP2C9.28, CYP2C9.30, CYP2C9.48 and CYP2C9.52 showed different hydroxylation activities toward steroids compared with CYP2C9.1.

Development of a continuous steroid biotransformation process and product extraction within microchannel system

Results of an integrated approach for the improvement of progesterone 11α-hydroxylation by Rhizopus nigricans, which is one of the key steps in the production of corticosteroid drugs and hormones, are presented. Several issues have been addressed in order to increase process productivity, including biocatalyst immobilization, improvement in the solubility of the lipophilic substrate, setting-up of continuous reaction process and easing downstream processing. After preliminary studies on the effects on Rhizopus nigricans morphology, fungal pellets of a desired size and structure could be obtained. The mycelia grown in spherical agglomerates further enabled its continuous use in the process of biotransformation. The problem of low steroid solubility was addressed by the addition of organic solvents or β-cyclodextrin. In order to develop an integrated biotransformation process, a continuous two-liquid phase extraction within microchannel device has been set up, where very efficient steroid extraction was achieved within a few seconds.

Role of the biomass and pelleted forms of Aspergillus ochraceus ATCC 3150 on the 11α-hydroxylation of progesterone

Role of the biomass and pelleted forms of Aspergillus ochraceus ATCC 3150 on the 11α-hydroxylation of progesterone

Batch and continuous transformation of progesterone by Rhizopus nigricans pellets in the presence of β-cyclodextrin

A pelleted morphological form of Rhizopus nigricans, which might be regarded as a naturally immobilized biomass, enabled development of a continuous process of progesterone 11α-hydroxylation in a laboratory-scale stirred tank bioreactor. β-Cyclodextrin was used to enhance steroid solubility in aqueous transformation media and consequently to increase the rate of steroid hydroxylation. Batch and continuous processes were performed with pellets of different average diameters, ranging from less than 1 mm to 7.5 mm. Mathematical model simulations of both operational modes confirmed the previously defined Michaelis–Menten kinetics for the biotransformation. A decrease in overall hydroxylation rate was noticed when pellets larger than 5 mm were used, which correlated with the lower area of outer surface of agglomerates where, presumably, most of the enzyme induction had taken place.

The influence of β-cyclodextrin on the kinetics of progesterone transformation by Rhizopus nigricans

The process of progesterone 11α-hydroxylation by the pelleted growth form of the filamentous fungus Rhizopus nigricans has been described with a mathematical model, based on Michaelis-Menten enzyme kinetics and the rate of substrate dissolution. It was confirmed that the low water solubility of steroids is the limiting step of this process at high steroid concentrations. In order to overcome this problem, β-cyclodextrin, which is known to form inclusion complexes with these organic compounds, was added to the production medium. The phase solubility of the steroid-β-cyclodextrin system was investigated and the effect of β-cyclodextrin addition on progesterone biotransformation evaluated. Enhancement of steroid solubility was demonstrated and nearly two-fold increase in reaction rate was found in the presence of β-cyclodextrin.

Induction of steroidal 11α-hydroxylase activity in the filamentous fungus Rhizopus nigricans by tomatidine and Primula veris root extract

The inducement of commercially important steroid 11α-hydroxylation in the filamentous fungusRhizopus nigricans by several plant saponins and saponin aglycones was tested. The α-tomatine derived aglycone tomatidine and a saponin rich Primula veris root extract were found to be effective inducers of progesterone 11α-hydroxylation in this saprophytic fungus. Furthermore, the transformation of progesterone to 5α-pregnane-3,6,20-trione was detected after pre-incubation with these plant constituents. However, the progesterone 11α-hydroxylase activity remained near the constitutive level after treatment of R. nigricans with solanidine, oat-leaf extract and its β-glucosidase treated derivative, while the presence of β-escin and especially α-tomatine lowered the transformation capability of mycelia.

Improved adsorption of Aspergillus niger 589 spores on high-density polyethylene for progesterone biotransformation

Aspergillus niger NCIM 589 spores were immobilized on high density polyethylene (HDPE), using polyethyleneimine (PEI) as the surface coating with as high as 50% adsorption compared with 30% reported earlier. The adsorption time was reduced to 2 h from the earlier 24 h, and pretreatment with a solvent was eliminated. The biocatalyst could be used for 11α-hydroxylation of Progesterone to give better conversions than alginate-entrapped or free mycelia.

11α-hydroxylation of progesterone using modified alginate-immobilized cells

The major disadvantage of using alginate-immobilized Aspergillus ochraceus for 11α-hydroxylation of progesterone is its instability in phosphate solutions. In this study, different gel-hardening methods were used to improve the stability of alginate gels. Addition of Fe 3+ and Fe 2+ ions increased the activity of hydroxylation, but Fe 2+ was not a good gelling agent. Replacement of CaCl 2 by FeCl 3 as the gelling agent increased the activity of the immobilized cells by about 33%. Hardening the alginate-immobilized cells with polyacrylamide (PAA) increased their stability in a 0.1 m phosphate buffer from 0.35 h to over 36 h. The productivity was increased by a factor of 1.8 compared to those without hardening, and the maximum yield was also increased from 84.3% to 90.8%. The physical properties and biocatalytic characteristics of PAA-hardened, immobilized cells were found to be feasible for application in a steroid bioconversion system.

11α-Hydroxylation of progesterone in biphasic media using alginate-entrapped Aspergillus ochraceus gel beads coated with polyurea

A novel cell-immobilization technique was developed in this study for increasing substrate partition to the gel matrix by coating a polyurea thin layer on the surface of Ca-alginate beads. The proposed method was simple and could be performed under mild conditions. The bioconversion of progesterone to 11α;hydroxyprogesterone with these polyurea-coating alginate-entrapped Aspergillus ochraceus cells was investigated using different organic solvents in biphasic media. The reaction medium of ethyl acetate could markedly enhance the bioconversion rate with the existence of a hydrophobic layer, most likely resulting from the increasing partition of substrate to gel matrix. Bioconversion with higher substrate concentration was possible using an ethyl acetate-water medium. The conversion rate increased almost linearly with increasing substrate concentration from 10 to 80 g l −1. The rate with 80 g l −1 progesterone increased up to six times greater than the rate with the immobilized cells without coating, and also exhibited a much higher rate than that reported in the literature.

Characterization of progesterone 11α-hydroxylase of Aspergillus ochraceus TS: A cytochrome P-450 linked monooxygenase

The monooxygenase of Aspergillus ochraceus TS capable of 11α-hydroxylation of progesterone has been resolved into three components and characterized as (i) cytochrome P450, (ii) NADPH-cytochrome P450-reductase and (iii) phosphatidyl choline. The 11α-hydroxylase was observed to be NADPH dependent, and hydroxylation was enhanced by a NADPH regenerating system. This fungal monooxygenase has many features in common with that of mammalian liver microsomes. The role of mammalian cytochrome P450 inducers were tested for induction of 11α-hydroxylase in Aspergillus ochraceus TS. The reductase has been partially purified.

Solvent damage during free cell catalysis and its avoidance: studies of 11α-hydroxylation of progesterone by Aspergillus ochraceus

The solvent tolerance of the progesterone 11α-hydroxylase system of Aspergillus ochraceus has been defined and, given its limited extent for conventional organic solvents, a number of natural oils have been examined. They have been found superior and represent an interesting solvent class for organic reactants of partial polarity. The study emphasizes that solvents for the products of biocatalytic action on organic reactants must often be partially polar and must not interact strongly with cellular lipids.

Resolution and reconstitution of cytochrome P-450 containing steroid hydroxylation system of Rhizopus nigricans

11α-hydroxylation of progesterone in the eucaryotic filamentous fungus Rhizopus nigricans is catalyzed by a monooxygenase. Three components of this multienzyme system, cytochrome P-450, rhizoporedoxin and a FAD containing rhizoporedoxin reductase have been separated from the postmitochondrial fraction on DEAE cellulose. Using NADPH as electron donor we showed that the presence of all three components was necessary for the reconstitution of the active electron transport chain.

Effects of sterol biosynthesis-inhibiting (SBI) fungicides on cytochrome P-450 oxygenations in fungi

The fungicides miconazole, fenarimol, and etaconazole block ergosterol biosynthesis in fungi by inhibiting sterol 14α-demethylation, which is mediated by a cytochrome P-450 enzyme. The sensitivity of cytochrome P-450-dependent hydroxylation or demethylation of several substrates to these fungicides and similar compounds was compared to that of fungal growth and sterol 14α-demethylation. Demethylation of p-chloro- N-methylaniline (PCMA) by sporidia of Ustilago maydis and 11α-hydroxylation of progesterone by Aspergillus nidulans were relatively insensitive to these compounds and to metyrapone. The ability of a sterol 14α-demethylation-deficient mutant to demethylate PCMA indicates that this substrate is not demethylated by the sterol 14α-demethylation system of U. maydis. The 14α-hydroxylation of progesterone by cells of Curvularia lunata was quite sensitive to the three fungicides, and also to metyrapone and isopropylphenylimidazole. This system was less sensitive to the three fungicides than sterol 14α-demethylation, but was appreciably more sensitive than PCMA demethylation. A study of progesterone 14α-hydroxylation in cell-free preparations of C. lunata showed the reaction to be inhibited by CO, and to be competitively inhibited by low concentrations of miconazole. These data suggest that the primary action of sterol biosynthesis-inhibiting (SBI) fungicides is competitive inhibition of sterol/steroid-type cytochrome P-450 enzymes rather than interference with the function of sterol carrier proteins or enzyme-modulating phospholipids.

The uptake of Aspergillus ochraceus spores on diatomaceous particles and their use in the 11?-hydroxylation of progesterone

The entrapment of Aspergillus ochraceus spores on to diatomaceous earth particles occurs rapidly, the number of spores entrapped at equilibrium being dependent upon the initial spore:particle ratio. The rate of agitation during spore uptake markedly affected entrapment. Immobilized spores carried out the 11α-hydroxylation of progesterone as effectively as free spores.

Studies on the microsomal 11α-hydroxylation of progesterone in Aspergillus ochraceus: isolation, characterization, and solubilization of the hydroxylase system

A suitable procedure for the preparation of cell-free extract with high 11α-hydroxylase activity from the induced vegetative cell cultures of Aspergillus ochraceus was developed. The presence of EDTA (10 mM), glycerol (10%), and dithiothreitol (5 mM), in the grinding medium was found to be necessary for optimal hydroxylase activity. Although the pH optimum of the hydroxylase was found to be 7.7, the ideal pH for preparing the cell-free extract from mycelium was 8.3. Microsomes (2.0 mg) isolated from the cell-free extract hydroxylated progresterone in very high yields (85–90% in 30 min) in the presence of NADPH and O2. Cytosolic involvement in the hydroxylation reaction was not noticed. The apparent Km for NADPH and progesterone were 0.052 and 0.625 mM, respectively. The hydroxylase activity was inhibited by metyrapone, carbon monoxide, SKF-525A, and p-chloromercuribenzoate indicating the involvement of cytochrome P-450 system in the reaction. Inhibition of the hydroxylase activity by cytochrome c and the presence of significant levels of NADPH–cytochrome c reductase in the microsomal fraction suggested that the reductase could be one of the components of the hydroxylase system. The membrane-bound hydroxylase was solubilized using sodium cholate. Solubilized membrane fraction contained considerable levels of cytochrome P-450 and NADPH–cytochrome c reductase, besides hydroxylase activity.

Immobilized fungal mycelia

Aspergillus niger NCIM 589 spores were immobilized on high density polyethylene (HDPE), using polyethyleneimine (PEI) as the surface coating with as high as 50% adsorption compared with 30% reported earlier. The adsorption time was reduced to 2 h from the earlier 24 h, and pretreatment with a solvent was eliminated. The biocatalyst could be used for 11α-hydroxylation of Progesterone to give better conversions than alginate-entrapped or free mycelia.

Microsomal 11α-hydroxylation of progesterone in [formula omitted]: Part I: Characterization of the hydroxylase system

Microsomes (105,000xg sediment) prepared from induced cells of A.ochraceus was found to hydroxylate progesterone to 11α-hydroxyprogesterone (11α-OHP) in high yields (85–90% in 30 min.) in the presence of NADPH and O 2. The pH optimum for the hydroxylase was found to be 7.7. However, for the isolation of active microsomes grinding of the mycelium should be carried out at pH 8.3. Metyrapone, carbon monoxide, SKF-525A, p-CMB and N-methyl maleimide inhibited the hydroxylase activity indicating the involvement of cytochrome P-450 system. The inhibition of the hydroxylase by cytochrome c and the presence of high levels of NADPH-cytochrome c reductase in induced microsomes suggest that the reductase could be one of the components in the hydroxylase system.

11α-hydroxylation of progesterone by cell free preparation of Aspergillus ochraceus TS

Hydroxylation of progesterone at C 11 by cell-free preparation of A. ochraceus TS has been studied. The hydroxylating enzyme (11α-hydroxylase) is inducible in character and located in post-mitochondrial supernatant. The reaction in vitro is effected in presence of sodium meta-periodate indicating the hemoprotein nature of the enzyme. The in vitro hydroxylation stimulated by cyanide and inhibited by metyrapone suggested that this microbial hydroxylation is possibly mediated by cytochrome P-450.

Inducibility of cytochrome P-450 and of NADPH-cytochrome C reductase in progesterone treated filamenteous fungi Rhizopus nigricans and Rhizopus arrhizus

The inducibility of the enzymes involved in the 11α-hydroxylation of progesterone was studied in filamentous fungi Rhizopus nigricans and Rhizopus arrhizus. We showed that besides the cytochrome P-450, the NADPH cytochrome-C reductase is another component of this steroidal monooxygenase system. The inducibility of cytochrome P-450 and of the reductase was followed by measuring the level of both enzymes during the induction of the hydroxylase in the fungal mycelia with progesterone; neither cytochrome P-450 nor the reductase were present in measurable amounts in the noninduced fungi, whereas the level of both enzymes increased in parallel during the induction. The induction kinetics of the hydroxylase in the intact mycelia is very similar to the induction kinetics of the individual enzymes which was followed in the cell free preparations. It is concluded that the 11α-hydroxylation of progesterone in R. nigricans and R. arrhizus requires the induction, both, of cytochrome P-450 and of NADPH cytochrome-C reductase.

Microbial hydroxylations V. 11α-hydroxylation of progesterone by cell-free preparations of Aspergillus ochraceus

The 11α-hydroxylation of progesterone and 19-nortestosterone by cell-free enzyme systems made from homogenates of vegetative cell cultures of Aspergillus ochraceus NRRL 405 is described. The 11α-hydroxylase is inducible and remains in the supernatant after centrifugation at 24000 × g. The 11α-hydroxylase induced by progesterone or by 19-nortestosterone is free from the associated 6β-hydroxylase or 17β-hydroxysteroid dehydrogenase activities of vegetative cell cultures of A. ochraceus NRRL 405, but induction with 11α-hydroxyprogesterone yields an enzyme preparation with both 6β- and 11β-hydroxylase activities. Although complete conversion of progesterone to 11α-hydroxyprogesterone was accomplished with the cell-free preparations, only 14% conversion of 19-nortestosterone to 11α-hydroxy-19-nortestosterone was possible.