Applied Biochemistry and Microbiology, Vol. 41, No. 5, 2005, pp. 450456.

Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 5, 2005, pp. 514520. Original Russian Text Copyright 2005 by Donova, Nikolaeva, Egorova.

Enzymes Involved in Modification of the Steroid Nucleus of Industrial Mycobacterial Strains: Isolation, Functions, and Properties
M. V. Donova, V. M. Nikolaeva, and O. V. Egorova
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, pr. Nauki 5, Pushchino, Moscow oblast, 142290 Russia e-mail: donova@ ibpm.pushchino.ru
Received January 19, 2005

AbstractThe key enzymes involved in modication of the steroid nucleus of sterol-transforming mycobacteria3-hydroxysteroid oxidase (3-OH-SO, EC 1.13.1.2) and 17-hydroxysteroid dehydrogenase (17-OHSDH, EC 1.1.1.51)were isolated and characterized. It is shown that 3-OH-SO is a multifunctional enzyme catalyzing oxidation of the 3- group, 5 4 isomerization, and 6-hydroxylation. Two forms of intracellular 17-OH-SDH that catalyze redox reactions at C17 were found, and their properties were determined. The presence of an extracellular 17-OH-SDH in Mycobacterium spp. (VKM Ac-1815 D and Et1) was demonstrated for the rst time.

Microbial transformation of sterols is the major method of industrial production of the key synthetic precursors of various pharmaceutical preparations 17-ketosteroidsandrostenedione (AD) and androstadienedione (ADD). Saprophytic mycobacteria, in which the enzymes that cleave the steroid nucleus are blocked, are commonly used as biocatalysts of this process [1]. Oxidation of the side chain of sterols is a complex multienzyme process including at least 14 chemical reactions catalyzed by nine enzymatic systems. Elimination of the aliphatic chain of sterols is accompanied by reactions of steroid-nucleus modication: oxidation of the 3-OH group, isomerization of the double bond from position 5(6) to position 4(5), redox reactions at 17, and 1(2) dehydration [2]. Modication of the 3-hydroxy-5(6)-ene conguration into the 3-keto-4(5)-ene structure is the initial stage of microbial catabolism of sterols. This process includes dehydration at position 3 yielding 3-keto-5(6)ene steroids; subsequent isomerization of the double bond (5 4) leads to the formation of 3-keto-4(5)ene steroids. This modication of sterol at the A ring can either precede the oxidation of the side chain at 17 or take place in parallel to it [3]. In some microorganisms, dehydrogenase and isomerase, which catalyze the above-mentioned reactions, are representative of different enzymes. In others, this process is catalyzed by cholesterol oxidase (CO, EC 1.1.3.6), which is able to catalyze hydroxylation of steroids at position 6, along with dehydration and isomerization [46]. However, similar data for mycobacteria have thus far been absent in the literature.

The presence of CO oxidizing the 3-hydroxy group in sterol-transforming mycobacteria was rst demonstrated by Stadtman et al. [7]. This enzyme is responsible for the formation of 4-sitostene-3-one and was later classied by different authors with either COs or glucose-methanol-choline reductases [3, 8]. It was discovered that CO of Mycobacterium fortiutum and M. vaccae are located in the cytoplasm [9]. We did not nd published data on the presence of extracellular steroid-transforming enzymes and their properties in this group of microorganisms. One of the terminal stages of oxidation of the side chain of sterols is 17-reduction of 3,17-ketosteroids. This process is catalyzed by 17-hydroxysteroid dehydrogenase (17-OH-SDH, EC 1.1.1.51). 17-OH-SDHs of some fungal and bacterial cultures have been studied. Enzymes isolated from different organisms differed in molecular weight, structure, and functions [1013]. In some bacteria (e.g., Comamonas testosteroni), redox transformations of steroids at positions 3 and 17 are catalyzed by the same enzyme [14]. However, the relationship between such reactions in mycobacteria remained obscure. The goal of this work was to isolate the enzymes modifying the steroid nucleus (3-OH-SO and 17-OHSDH) in mycobacteria degrading the side chain of sterols and to study their properties. MATERIALS AND METHODS Microorganisms. In this study, we used Mycobacterium sp. VKM Ac-1815 D from the All-Russia Collection of Microorganisms, Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian

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Academy of Science. The mutant strain Et1 of Mycobacterium sp. was obtained earlier from Mycobacterium sp. VKM Ac-1815 D using chemical mutagenesis [15]. Cultures were maintained as described in [16]. Mycobacterium sp. Et1 was grown in an ANKUM 2M fermenter on mineralorganic medium under inducing conditions [15]. Cells were separated by centrifugation at 4 at 6000 g for 10 min and washed twice with 0.01 M cooled sterile potassiumphosphate buffer (pH 7.2). Mycobacterium sp. strain VKM Ac-1815 D was cultured in a BIOR fermenter (Russia) in a mineralorganic medium supplemented with 3 mM -sitosterol [16]. Cells were separated by centrifugation at 20000 g for 2 h. To isolate 3-OH-SO, cell-free cultivation broth was used. To isolate enzymes associated with the cell wall, cells (100 g) were treated with 0.1% Triton X-100 (100 ml) in 10 mM potassium-phosphate buffer (pH 6.8). Reagents. -Sitosterol was from Ultra Kaukas (Finland); AD, ADD, dehydroepiandrosterone (DHEA), and other steroids were from Sigma (United States); yeast extract and agar from Difco (United States); reagents for electrophoresis, phenazine methosulfate, Nitro Blue Tetrazolium, and EDTA were from Serva (Sweden); and NAD and NADH were from DiaM (Russia). Solvents and other reagents were of reagent and special purity grade. Enzymes were isolated using columns and carriers from Pharmacia, BioRad, Serva (Sweden), Toyo Soda (Japan), and Sigma (United States). Isolation and purication of 3-OH-SO. All manipulations were performed at 4C. (1) Cell-free cultivation broth (50 l) was concentrated to 2 l using hollow-ber ultraltration membranes. The concentrate was subjected to 85% saturation with ammonium sulfate. The precipitate was dialyzed against 2 l of 10 mM potassium-phosphate buffer (pH 6.8; buffer K); buffer solution was changed twice. (2) The preparation was loaded onto a column (0.9 40 cm) packed with DEAE Toyopearl, which was equilibrated with buffer K. Proteins were eluted with a gradient of NaCl concentrations (01 M, 10 ml/h, 200 ml). (3) Desalted preparation was loaded onto a column (1.5 20 cm) packed with hydroxylapatite, and 3-OHSO was eluted with a gradient of potassium-phosphate buffer (0.011.0 M, pH 6.8, 11 ml/h, 200 ml). Fractions with 3-OH-SO activity were pooled and concentrated to 1.8 ml; the concentrates were designated as HA-I and HA-II. (4) The preparation HA-I was loaded onto a column (0.8 55 cm) with Biogel A-0.5 M. Proteins were eluted with buffer K. Fractions eluted in free volume were concentrated to 1.8 ml and gel-ltered again. The preparation HA-II was gel-ltered under the conditions described above.
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The molecular weight of enzymes was estimated using the following protein markers: catalase (240 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and chymotrypsin (25 kDa). Isolation and purication of 17-OH-SDH. All manipulations were performed at 4C. (1) Cells were disintegrated by solid-phase extrusion on a press (Skryabin Institute of Biochemistry and Physiology of Microorganisms) and resuspended in ve volumes of 10 mM TrisHCl buffer containing 1 mM EDTA and 2 mM MgCl2 (pH 8.0; buffer A). Cellfree extract was treated with 0.1% sodium deoxycholate. Cell debris was removed by centrifugation at 30000 g for 1 h. Nucleic acids were precipitated with 0.6% streptomycin sulfate. (2) Cell-free extract was fractionated with ammonium sulfate (3055%) and centrifuged at 30000 g for 40 min. The pellet obtained was resuspended in 10 25 ml of buffer A containing 5 mM MgCl2 and dialyzed using Centricon Plus 20 concentrating units (United States). (3) After desalting, the preparation was loaded onto a column (1.6 40 cm) with DEAE Trisacryl Plus M, which was equilibrated in buffer A. The enzyme was eluted with a KCl gradient (01.0 M, 20 ml/h, 300 ml). (4) After desalting, the preparation was loaded onto a column (1 15 cm) with hydroxyapatite, which was equilibrated in 20 mM potassium-phosphate buffer containing 1 mM EDTA and 2 mM gCl2 (pH 7.4). 17-OH-SDH eluted with a gradient of K24 concentrations (0.011.0 M, 20 ml/h, 150 ml). (5) Ammonium sulfate was added to a concentration of 1.5 M. After centrifugation (30000 g, 20 min), the preparation was loaded onto a column (1.8 20 cm) with phenylsepharose CL 4B, which was equilibrated in buffer A containing 1.5 M (NH4)2SO4. The enzyme was eluted with a gradient of (NH4)2SO4 concentrations (1.50 M, 11 ml/h, 150 ml). (6) After concentration, the above preparation was loaded onto a column (1 80 cm) with Sephacryl S-220 equilibrated in buffer A. The enzyme was eluted with buffer A (ow rate, 4.5 ml/h). Fractions with 3-OH-SO or 17-OH-SDH activity were pooled and dialyzed for 12 h against buffer K or A, respectively, containing 50% glycerol. Preparations were stored at 15C. Enzymatic activities. The amount of 3-OH-SO that oxidized 1 mol steroid in 1 min was taken as one unit of activity of 3-OH-SO. The reaction mixture (2 ml) contained 20 mol of TrisHCl (pH 6.8), 0.1 mol DHEA in dimethyl formamide (DMFA), 0.2 mol of NADPH, and 100500 l of enzyme. The reaction mixture was air-saturated at 37 for 30 min. Steroids were analyzed by HPLC as described below. The activity of 17-OH-SDH was determined spectrophotometrically at 340 nm. The amount of the
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Table 1. Scheme of purification of 3-hydroxysteroid oxidase from Mycobacterium sp. VKM Ac-1815D Purification step Degree Vol- Total proSpecific activity, Total activity, U Yield, % of purifiume, ml tein, mg U/mg cation 300.0 250.0 17.6 1.1 1.2 0.7 0.2 0.15 6.9 5.9 3.2 3.4 2.9 2.5 1.4 1.3 0.02 0.02 0.18 3.17 2.40 3.51 6.90 8.67 100 86 46 49 42 36 20 19 1 1 8 138 104 153 300 378

Culture-liquid concentrate 2000 Dialysate of the fraction of 85% saturation 100 with (NH4)2SO4 Chromatography on DEAE Toyopearl 70 (0.4250.5 M KCl) Chromatography on hydroxylapatite, 20 HA-I (0.1 M KH2PO4) Chromatography on hydroxylapatite, HA-II 25 (0.30.345 M KH2PO4) Gel filtration of HA-I on Biogel A-0.5 M (first) 2.8 Gel filtration of HA-I on Biogel A-0.5 M 0.8 (second; 3-OH-SO II) Gel filtration of HA-II on Biogel A-0.5 M (3-OH-SO II)

enzyme that changed the optical density at 340 nm by 0.01 unit in 1 min at 25 was taken as one unit of activity of 17-OH-SDH. The molar extinction coefcient for NAD is 6.22 1 cm1 [17]. Determination of KM. The Michaelis constant was determined as described earlier [5]. DHEA concentration varied from 1.99 105 to 22 105 M. Protein concentration was determined by the Lowry method. Electrophoresis. The purity of enzymatic preparations and the molecular weight of proteins were determined by SDS-PAGE (9% polyacrylamide) by the Laemmli method. The kit of molecular weight markers (Sigma) contained carbonic anhydrase (29 kDa), glyceraldehydes-3-phosphate dehydrogenase (36 kDa), ovalbumin (45 kDa), bovine serum albumin (66 kDa). PAGE (under nondenaturing conditions) was performed by the Davis method. Specic staining for 17-OH-SDH was performed as described in [10]. Transformation of 17-keto and 17-hydroxysteroids by the preparations of 17-OH-SDH. Steroids were transformed in a medium containing either 100 M TrisHCl buffer (pH 8.0) and 2.26 mM NAD (in the case of 17-OH-steroids) or 100 mM potassiumphosphate buffer (pH 7.0) and 2 mM NADH (in the case of 17-ketosteroids), 1.73 mM steroid substrate in DMFA, and 0.51.0 ml of enzyme. Aliquots of the reaction mixture were taken every eight to twelve hours. Metabolites were identied as described below. Analysis of steroids. Steroids were analyzed by TLC and HPLC. TLC was performed on Silufol UV254 plates (Kavalier, Czech Republic) and Kieselgel 60 F254 (Merck, Germany). Steroids were separated in the benzeneacetone system (3 : 1, v/v) and visualized in UV light at 254 nm.

HPLC was performed on a column (250 4.6 mm) packed with C18-Octadecyl-Daltodil Si-100 (Serva, Sweden). Steroids were detected at 30 at 240 nm in the acetonitrileH2 system (70 : 30, v/v; ow rate, 1 ml/min). Mass spectrometric analysis was performed on a Finnigan MAT 8430 mass spectrometer (Germany) at an ionization energy of 70 eV. The error in analytical methods did not exceed 5%. Data are presented as an arithmetic mean of three measurements. RESULTS AND DISCUSSION Isolation and purication of extracellular 3-OHSO. The enzyme was isolated from cell-free cultivation broth of Mycobacterium sp. VKM Ac-1815D, which oxidizes the side chain of sterols to AD as a major product. The scheme of isolation of extracellular enzyme is shown in Table 1. Chromatography on hydroxyapatite showed that 3-OH-SO was present in the fractions eluted with 10 mM (3.17 U/mg, HA-I) and 300345 mM (2.4 U/mg, HA-II) potassium-phosphate buffer. Gel chromatography of preparation HA-I on A-0.5 M Biogel revealed two forms of the enzyme exhibiting 3-hydroxysteroid oxidase activity, which were designated 3-OH-SO I (eluted in free volume (20 ml)) and SDH II (eluted in a volume of 36.5 ml). The properties of 3-OH-SO II are summarized in Table 2. 3-OH-SO II (monomer, 64 kDa) was puried to 300 times. The specic activity of the enzyme was 8.67 U/mg, M = 7.9 104 M; NADPH and oxygen were required for the manifestation of activity of this enzyme (Fig. 1).
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ENZYMES INVOLVED IN MODIFICATION OF THE STEROID NUCLEUS Table 2. Properties of steroid nucleus-modifying enzymes of Mycobacterium spp. (VKM Ac-1815 D and Et1) Enzyme Catalyzed reactions 3-OH-SO II 3-oxidation, 4-isomerization, 5 6-hydroxylation 8.67 62 4 Monomer not determined not determined 7.9 104 M, (3.2 105 M/min) yes 17-OH-SDH (1) 17-oxidation 17-OH-SDH (2) 17-oxidation 17-reduction 3-oxidation 23.7 54 2 210 5 Tetramer 8.0 3545 not determined yes

453

Specific activity, U/mg Subunit molecular weight, kDa Total molecular weight, kDa Subunit composition pH optimum Temperature optimum, C KM , (Vmax): Stability*

1.28 68 2 Monomer not determined not determined not determined yes

* Retaining at least 60% of activity for 3 months at 15C.

Analysis of transformation products of sitosterol and DHEA by 3-OH-SO II preparations showed that 6hydroxysitosterol and 6-hydroxyandrostadienedione were present in the reaction medium along with sitosterol and AD (Table 3). This nding indicates that the medium contained not only dehydrogenase and isomerase activities but also 6-hydroxylase activity. The detection of the ability of the enzyme to perform hydroxylation at position 6 is consistent with the known published data. It was found earlier that CO of Pseudomonas sp. also performs 6-hydroxylation of cholesterol [18]. It was shown that CO from Nocardia rhodochrous is an amphipathic protein containing hydrophilic and hydrophobic regions, which allows it to be adsorbed on hydrophobic resins, aggregate in the absence of deter-

gent, and form mixed micelles with detergents and sterols. The presence or absence of the hydrophobic region accounts for the existence of several forms of the enzyme [4]. In light of the data obtained, it can be assumed that 3-OH-SO I possibly represents an aggregated form of sitosterol oxidase, which is bound to the cell wall of Mycobacterium sp. This assumption is corroborated by the detection of the activity of 3-OH-SO in the fraction of enzymes associated with the cell wall (Table 3). Isolation and purication of intracellular 17OH-SDH. The scheme of isolation of intracellular 17OH-SDHs is shown in Table 4. It was shown that Mycobacterium sp. strain Et1 contains two enzymes exhibiting 17-hydroxysteroid dehydrogenase activity, which

a b c d

3 1 2

Fig. 1. SDS-PAGE of 3-OH-SO of Mycobacterium sp. VKM Ac-1815 D. Designations: (1), protein markers (a, 66; b, 45; c, 36; d, 29 kDa); (2), 3-OH-SO II (HA-I preparation, 42 g); (3), 3-OH-SO II after gel ltration on Biogel A-0.5 M (38 g). APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 41

Fig. 2. Specic staining of intracellular 17-OH-SDHs. Designations: (1), 17-OH-SDH (2), 198 g; (2), 17-OH-SDH (1), 210 g. No. 5 2005

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Table 3. Composition of the products of steroid transformation by enzymatic preparations Substrate -Sitosterol DHEA Stage of purification Cofactor Product Enzyme

Dialysate of the fraction of 85% saturation with NADPH 4-sitosten-3-one; 3-OH-SO (NH4)2SO4 6-OH-4-sitosten-3-one Dialysate of the fraction of 85% saturation with NADPH AD, ADD, 6-OH-AD 3-OH-SO, 1(2)-SDH (NH4)2SO4 Chromatography on DEAE Toyopearl Chromatography on hydroxylapatite (HA-I) Chromatography on hydroxylapatite (HA-II) NADPH AD, ADD, 6-OH-AD 3-OH-SO, 1(2)-SDH NADPH AD, ADD, T*, DT*, 6-OH-AD 3-OH-SO, 1(2)-SDH, 17-OH-SDH

NADPH AD, ADD, 6-OH-AD 3-OH-SO, 1(2)-SDH 3-OH-SO 3-OH-SO 17-OH-SDH, 1(2)-SDH 17-OH-SDH 17-OH-SDH 1-ene reductase

Gel filtration on Biogel A-0.5 M (3-OH-SO II) NADPH AD, ADD, 6-OH-AD 3-OH-SO, 1(2)-SDH Gel filtration on Biogel A-0.5 M (3-OH-SO II) NADPH AD, 6-OH-AD Fraction of cell wall-associated enzymes Testosterone Chromatography on DEAE Toyopearl Chromatography on DEAE Toyopearl Chromatography on hydroxylapatite (HA-I) Chromatography on hydroxylapatite (HA-II) Fraction of cell wall-associated enzymes ADD Chromatography on hydroxylapatite (HA-I)
* Detected at DHEA concentrations of 3090 g/ml.

NADPH AD, 6-OH-AD NAD NAD AD, ADD, DT AD NADPH not detected NADPH not detected NAD AD AD

Gel filtration on Biogel A-0.5 M (3-OH-SO II) NADPH not detected

Table 4. Purification of extracellular 17-OH-SDHs of Mycobacterium sp. (VKM Ac-1815D and Et-1) Total proTotal Specific acYield, % tein, mg activity, U tivity, U/mg 686.2 87.0 131.8 56.3 0.19 0.65 100 42.7 Degree of purification 1.0 1.3

Purification step

Volume, ml

Cell-free concentrate Fraction of 3055% saturation with (NH4)2SO4 Chromatography of 17-OH-SDH (2) on DEAE Trisacryl (0.440.51 M KCl) Chromatography of 17-OH-SDH (1) on DEAE Trisacryl (0.310.34 M KCl) Chromatography of 17-OH-SDH (2) on hydroxylapatite (0.10.2 M KH2PO4) Chromatography of 17-OH-SDH (2) on phenylsepharose (0.510.48 M (NH4)2SO4) Gel filtration of 17-OH-SDH (2) on Sephacryl S-200 Gel filtration of 17-OH-SDH (1) on Sephacryl S-200

73 10

20

6.7

61.1

9.12

46.4

47.0

8.5

19.7

6.39

0.32

4.9

1.6

10

2.8

45.2

16.08

34.0

84.0

3.5

1.6

33.1

20.37

25.0

106.0

0.8

18.9

23.70

14.3

123.5

2.5

3.2

1.28

2.4

6.8

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Fig. 3. Specic staining of intra- and extracellular 17-OHSDH. Designations: (1), intracellular 17-OH-SDH (2), 178 g; (2), 17-OH-SDH of the fraction of cell wall-associated enzymes, 130 g; (3), extracellular 17-OH-SDH (cultureliquid concentrate, 90 g).

were designated as 17-OH-SDH (1) and 17-OH-SDH (2). The properties of these enzymes are summarized in Table 2. 17-OH-SDH (1), a monomer with a molecular weight of approximately 68 kDa, catalyzed 17-oxidation of 17-hydroxysteroids, whereas 17-OH-SDH (2), a tetramer with a molecular weight of approximately 210 kDa, performed both 17-reduction and 17-oxidation. Additionally, 17-OH-SDH (2) also catalyzes the oxidation of the 3-hydroxy group of some androgens, estrogens, and pregnanes [19]. Specic staining for 17-OH-SDH (2) revealed several bands; in the case of 17-OH-SDH (1) only one band was observed (Fig. 2). It is known from the literature that 17-OH-SDH of microorganisms differ in properties, structure, and substrate specicity. For example, the molecular weight of 3(17)-hydroxysteroid oxidase from C. testosteroni (a tetramer) constituted 98.5 kDa [10], whereas 17-OHSDH from Cylindrocarpon radicicola and Alcaligenes sp. represented dimers with molecular weights of 58 and 68 kDa, respectively [11, 12]. The specic activity of high-purity enzymatic preparations varied from 103 to 300 U/mg. It is possible that the differences in 17-OH-SDH properties reect the distinct physiological functions of this enzyme in the organism (e.g., 17-OH-SDH may function in the metabolism of steroids that are used as a source of carbon and energy or attenuate the toxic effect of a substrate by means of its transformation). Extracellular 17-OH-SDH of Mycobacterium sp. When analyzing cell-free cultivation broth of MycobacAPPLIED BIOCHEMISTRY AND MICROBIOLOGY

terium spp. (VKM Ac-1815 D, Et1), we detected activities of not only 3-OH-SO, 3-ketosteroid-1(2) dehydrogenase (1(2)-SDH), and 1-ene-reductase, but also 17-OH-SDH (Table 3). Specic staining using PAGE conrmed the presence of an extracellular 17-OH-SDH in culture-liquid concentrate (one protein band was observed), whereas the intracellular 17-OH-SDH (2) was detected in the form of several stained bands (Fig. 2). The presence of 17-OH-SDH was also detected in the fraction of enzymes associated with the cell wall, which was obtained by treating the cells with 0.1% Triton X-100 (Fig. 3, Table 3). Extracellular 17-OH-SDH catalyzed 17-oxidation of testosterone and 1(2)-dehydrotestosterone, 3-oxidation of DHEA, and 17-reduction of AD(D) in the presence of cofactors. Note that 17-reduction of ADD and 17-oxidation of 1(2)-dehydrotestosterone was coupled to 1(2)-reduction. Analysis of published data [13, 20] showed that the majority of microbial transformations of steroid compounds proceed within the cell. The detection of extracellular 17-OH-SDH, on the one hand, indicates that 17-reduction possibly takes place outside the cell; on the other hand, the detected extracellular enzymes (3-OH-SO, 17-OH-SDH, and 3-ketosteroid-1(2) dehydrogenase) might represent components of the system that transfers steroid substrate into the cell. ACKNOWLEDGMENTS This study was supported by the Russian Foundation for Basic Research, project no. 04-04-49605. REFERENCES
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DONOVA et al. 16. Donova, M.V., Dovbnya, D.V., and Koshcheyenko, K.A., Proc. 8 Int. Symp. on Cyclodextrins, Dordrecht: Kluwer Ac. Publ., 1996, pp. 527530. 17. Hydroxysteroid dehydrogenase, in Enzymes and Related Biochemicals, Worthlington: Bedford, USA, 1979, pp. 105106. 18. Molnar, I., Havayaschi, N., Choi, K., Yamamoto, H., Yamashita, M., and Murooka, Y., Mol. Microbiol., 1993, vol. 7, no. 3, pp. 419428. 19. Egorova, O.V., 17-Reduction of 3,17-Diketosteroids by Sterol-Transforming Mycobacteria, Cand. Sci. (Biol.) Dissertation, Pushchino: IBPM RAN, 2004. 20. Peterson, D., in Biochemistry of Industrial Microorganisms, Rainbow, C. and Rose, A.H., Eds., London: Academic, 1963.

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