hydroxylation of steroids by curvularia lunata mycelium in the presence of methyl-β-cyclodextrine

ISSN 0003�6838, Applied Biochemistry and Microbiology, 2011, Vol. 47, No. 1, pp. 42–48. © Pleiades Publishing, Inc., 2011.Original Russian Text © V.A. Andrushina, A.V. Druzhinina, V.V. Yaderets, T.S. Stitsenko, N.E. Voishvillo, 2011, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2011,Vol. 47, No. 1, pp. 50–57.

42

The modification of steroid molecule using micro�organisms (hydroxylation, 1,2�dehydration, degrada�tion of side chain of sterols, etc.) are required stages ofindustry synthesis of medical drugs of a steroid nature[1–4]. Their advantage over chemical reactions lies inhigh regio� and stereospecificity and ecological com�patibility. However, there is a problem of increase ofavailability of steroid substrates for microbial cell andincrease of their concentration in reaction mediumduring mentioned biotransformations due to difficultsolubility of steroids in aqueous environment (severalmg/l) [1–3].

The dispersion method of steroids in cultural liquidusing organic solvents miscible with water partiallysolves a problem but only for transformation of so�called “hydrophilic” steroids. In comparison withthat, the method of application of steroids as the aque�ous suspensions with size of particles less than 10 μmprovides us with an opportunity to increase the con�centration of steroid substrates until 50 g/l [1, 2]. This

method is not applicable for the processes where thesteroid substrate or the products of its transformationare toxic for a microorganism. For instance, androsta�dienedione (ADD) created as the result of removal ofa side chain of sterols is toxic for bacteria in a concen�tration higher 0.8 g/l [5].

The higher concentrations of steroid substrates canbe reached by using the method suggested by a scien�tist of Gedeon Richter firm in 1981 (Gedeon Richter,Hungary) which includes the transformation of ste�roids solubilized in aqueous mediums using macrocy�clic α�, β�, and γ�cyclodextrines [6, 7]. These cyclo�dextrines, in particular β�cyclodextrine (CD),together with steroids create water soluble complexesthat allow increase of concentration of steroids intransformation medium until 10 g/l [8]. The presenceof CD in reactions of hydroxylation, 1,2�dehydroge�nation, reduction of 17�ketogroup of steroids and oxi�dation of Δ5�3β�hydroxysteroids to Δ4�3�ketosteroidscatalyzed by free or immobilized bacterial cells andfungi has decreased the time of transformation by twotimes and promoted the increase of yield of reactionproducts and, in some cases, changed their ratio [9–15].

As a result of CD substitution by its chemical mod�ified derivatives like methyl�CD (MCD), hydroxypro�pyl�CD (HPCD), etc., the concentration of steroidsin aqueous medium was more than 20 g/l. This chargeand usage of actinobacteria allowed the 1,2�dehydro�genation of hydrocortisone and androstenedione and

Hydroxylation of Steroids by Curvularia lunata Mycelium in the Presence of Methyl�β�cyclodextrine

V. A. Andrushina, A. V. Druzhinina, V. V. Yaderets, T. S. Stitsenko, and N. E. VoishvilloBioengineering Center, Russian Academy of Sciences, Moscow, 117312 Russia

Received September 18, 2009

Abstract—Transformation of 16 Δ5�3β�hydroxy� and Δ4�3�ketosteroids of androstane and pregnane classeswas carried out using Curvularia lunata mycelium suspended in phosphate buffer with methyl�β�cyclodex�trine (MCD). As the result, 20 monohydroxy� and dihydroxy�metabolites, whose structure was determinedusing spectra of proton magnetic resonance and mass�spectra, have been isolated. Hydroxylation of Δ5�3β�hydroxy�steroids occurred mostly in the C�7α position whereas hydroxylation of Δ4�3�ketosteroids was in theC�11β position. Only androst�4�en�3,17�dione, 9α�hydroxy�androstenedione, and androsta�1,4�diene�3,17�dione were hydroxylated at C�14α position. Besides main 11β�derivatives, the 6β� and 7β�hydroxy�derivatives with yield 10 and 30%, respectively, were isolated during transformation of progesterone andhydroxymethyl pregnadienone. The ratio of MCD to transforming steroid was 1 : 1 (mol/mol). Hydrocorti�sone and 7α�hydroxyandrostenolone with the yield 55 and 77%, respectively, were obtained at the maximalconcentrations of cortexolone 20 g/l and androstenolone acetate 10 g/l in the presence of MCD. Absorptionof steroids on mycelium, lower speed of their transformation, low concentrations of modifying substrates, andlow yield of hydroxyderivatives have been observed in the absence of MCD.

DOI: 10.1134/S0003683811010029

Abbreviations: AD—androstenedione, ADD—androstadienedione,DEA—dehydroepiandrosterone (androstenolone), ADEA—DEAacetate, DHA—3β,17β�dihydroxyandrost�5�en, THA—3β,7α,17β�trihydroxyandrost�5�en, Δ5�PG—pregnenolone, Δ4�PG—progesterone, 17�OH�Δ4�PG—17β�hydroxyprogesterone,16,17�epoxy�Δ4�PG—16α,17α�epoxyprogesterone, comp. “S”—cortexolone (Compound “S” Reichstein), comp. “F”—hydrocorti�sone (compound F Kendall), MAR and TAR—mono� and triace�tate of compound “R” Reichstein, HMPD—21�hydroxy�20�meth�ylpregna�1,4�diene�3�on, CD—β�cyclodextrine, MCD—methyl�β�cyclodextrine, HPCD—hydroxypropyl�β�cyclodextrine

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 47 No. 1 2011

HYDROXYLATION OF STEROIDS BY Curvularia lunata MYCELIUM 43

removal of a side chain of sterols [16–18]. There is alack of literature data on hydroxylation of steroids byfungi in the presence of chemically modified cyclo�dextrines [1–4, 6, 19].

The aim of this study was hydroxylation of Δ5�3β�hydroxy� and Δ4�3�ketosteroids of androstane andpregnane classes as complexes with methyl�β�cyclo�dextrine using the strains of fungi Curvularia lunata.

METHODS

Transformations of Δ5�3β�hydroxysteroids werecarried out using fungus Curvularia lunata F�981 fromthe All�Russia Collection of Industrial Microorgan�isms (VKPM) [20] and transformations of Δ4�3�keto�steroids were carried out using Curvularia lunataVKPM F�988 [21]. Randomly methylated β�cyclo�dextrine (MCD) and hydroxypropyl�β�cyclodextrine(HPCD) (China) were used for solubilization of trans�forming steroids. The ratio of cyclodextrines to trans�forming steroid was 1 : 1 (mol/mol).

Cultivation and transformation of steroids was car�ried out in 750 ml conic flasks at 28°C on a rotor�shaker 220 rpm in particular cultivation in 100 ml ofnutrient medium and transformation in 75 ml 1/15 Mphosphate buffer. The strain F�981 was grown onmedium containing 30.0 g/l sucrose, 2.5 g/l yeastautolysate, 2.0 g/l NaNO3, 1.0 g/l K2HPO4, 3.0 g/l(NH4)H2PO4, 0.55 g/l KCl, 0.5 g/l MgSO4 ⋅ H2O;pH 6.0–6.3 (medium I). The strain F�988 was culti�vated on the medium containing 20.0 g/l glucose, 5.0 g/lpeptone, 5.0 g/l yeast extract, 10.0 g/l soya meal,4.0 g/l KH2PO4; pH 6.0–6.3 (medium II).

The mediums were inoculated with suspension offungal spores grown for 7–10 days on agar slants pre�pared on the basis of the medium for strain F�981.Cultivation of the first generation was carried out for3 days and used as the inoculum which was added tothe fresh medium (I or II). Mycelium was separatedfrom the medium after 32–34 h of growth and 9.5–19.0 g/l (calculated per dry weight of biomass) weresuspended in buffer solution preliminary added withcyclodextrine and microcrystalline steroid. The sus�pension further was divided into several flasks, andafter some time intervals, probes not less than 5 mlwere taken which were further extracted by ethylacetate.

Analysis of transformation products. The quantita�tive analysis of the products of transformation was car�ried out using TLC and Silufol UV 254 (Kavalier,Czech Republic) and Sorbfil plates (Imid Ltd, Rus�sia). The separation of steroid compounds was carriedout in the system of solvents acetone�chloroform (7 : 3).After separation of transformation products of Δ5�3β�hydroxysteroids, the chromatograms were visualizedby 1% vanillin solution in 10% solution of water chlo�

ric acid. The transformation products of Δ4�3�keto�steroids were visually evaluated in UV�light at the wavelength 254 nm.

In order to determine the concentration of initialΔ5�3β�hydroxysteroids and the products of their trans�formation, ethyl acetate extract was evaporated untildry; they were weighed and the percentage concentra�tion of steroids in probes using specter of proton mag�netic resonance was determined. The concentration ofΔ4�3�ketosteroid substrates and the products of theirtransformation were evaluated using HPLC [20].

Isolation of hydroxylation products. After transfor�mation, the mycelium was separated and tested forsteroid compounds. In case of their absence, the waterfraction was extracted by ethyl acetate until totalextraction of steroids. The extract was evaporated. Ifmycelium contained the transformation products itwas additionally extracted by ethyl acetate. Extractswere mixed and evaporated in a vacuum. The rest waswashed by ether and crystalline products were filteredwhose yield (B) was calculated according to the for�mula: B = 100 × Mp/Ms × (Mpr/Msb), where Mp ismass of isolated crystalline product, Ms is amount ofsubstrate used for reaction, Mpr is molecular weight ofthe product, and Msb is molecular weight of the sub�strate.

RESULTS AND DISCUSSION

Hydroxylation of Δ5�3β�hydroxysteroids. The schemein Fig. 1 shows that all Δ5�3β�hydroxysteroids (dehy�droepiandrosterone (DEA)), acetate DEA (ADEA),3β,17β�dihydroxyandrost�5�en (DHA) and its 3β�ace�tate, pregnenolone (Δ5�PG), 16α,17α�epoxyprogester�one (16,17�epoxy�Δ4�PG), and monoacetate and triace�tate of compound “R” Reichstein (MAR and TAR) con�tained 7α�hydroxyl group after transformation usingmycelium of Curvularia lunata VKPM F�981.

The ability of different strains of C. lunata to 11β�hydroxylation and 14α�hydroxylation of some Δ4�3�ketosteroids [1–4, 22] is well known, but the capabilityof this species to selective 7α�hydroxylation of Δ5�3β�hydroxysteroids is shown for a first time by us [20].

Transformation of dehydroepiandrosterone (DEA).Full conversion into 7α�hydroxy�DEA occurred using10 g/l of mycelium (calculated per weight of dry biomass)for 20 and 24 h, respectively, at the charge of DEA orADEA in the concentration 2 g/l added to buffer solu�tion as the cryptocrystalline suspension (Fig. 2);7α�hydroxylation occurred with creation of an insuffi�cient amount of reduction product of 17�ketogroup—3β,7α,17β�trihydroxyandrost�5�en (THA). THA isinteresting as the perspective compound for synthesisof steroid preparations but it is the side product for thisreaction [23]. The increase of concentration of micro�crystals of ADEA in reaction mixture until 5 g/l

44


ANDRUSHINA et al.

CH3

CH3

O

H3C CH2OH

HOCH3

CH3

O

O

HO

CH3

CH3

O

H3C CH2OH

CH3

CH3

O OH

H3C CH2OH

CH3

CH3

O

O

HO

CH3

CH3

O

O

CH3

CH3

CH2OH

O

O

OH

CH3

CH3

CH2OH

O

O

HO OH

CH3

CH3

HO

CH2OH

O

OH

OH

CH3

CH3

OR1

CH2OR2

O

OR3

CH3

CH3

O

CH3

OCH3

CH3

O

CH3

O

HO

CH3

CH3

HO

CH3

O

OH

HOCH3

CH3

HO

CH3

O

CH3

CH3

O

OHCH3

CH3

O

OH

HOCH3

CH3

O

O

OH

CH3

CH3

O

O

CH3

CH3

HO

OHCH3

CH3

HO OH

OHCH3

CH3

HO OH

OCH3

CH3

HO OH

OCH3

CH3

RO

O

R = H�DEAR = Ас�АDEA

7�ОН�DEA

АD 14�OH�АD 11�OH�ТS ТS

Pregnenolone 7,11�OH�pregnenolone 11�OH�progesterone Progesterone

R1 = R2 = R3 = H�comp. “R”

R1 = R3 = H, R2 = Ac�MARR1 = R2 = R3 = Ac�TAR

7�OH�comp. “R” comp. “S”

11�OH�ADD 11�OH�HMPD

14�OH�АDD

DHАТHА

7�OH�HMPD

comp. “F”

ADD

Fig. 1. Scheme of hydroxylation of steroids by Curvularia lunata strains.

resulted in inhibition of transformation and amount of7α�hydroxy�DEA was not higher than 1 g/l. Anincrease of the mass of biocatalyst to 18.5 g/l (Fig. 2)did not influence its yield.

The presence of MCD in combination with a dou�ble amount of mycelium (Fig. 2) considerably influ�enced the speed of conversion of ADEA and yield of7α�hydroxy�DEA. The concentration of 7α�hydroxy�



DEA at the transformation of 5 g/l of ADEA as thecomplex with MCD over 24 h was 85%.

The results of hydroxylation of 10 g/l of ADEA incomplex with CD, MCD, and HPCD at the molarration ADEA�cyclodextrine 1 : 1 are shown in Table 1.Transformation continued for 44 h. During thisperiod, the maximal level of ADEA conversion to 7α�hydroxy�DEA isolated with yield 77% was reached inthe presence of MCD. High concentration of 7α�hydroxy�DEA in reaction mixture (90%) was observedusing HPCD. However, HPCD in comparison withMCD formed a more stable complex with 7α�hydroxy�DEA that made the product extraction diffi�cult and resulted in the obtaining of a smaller yield—65% (Table 1). The minimal synthesis of side THA(15% in control without solubilizator; 3% in experi�ment with MCD) was observed in the presence of bothMCD and HPCD. The concentration of 7α�hydroxy�DEA in reaction mass did not exceed 50% at solubili�zation of ADEA using CD. Besides that, the latter cre�ated a more stable complex with CD than withHPCD, which resulted in a yield of final product of20.5% (Table 1).

Transformation of 3α,17β�dihydrooxyandrost�5�en(DHA). The results of DHA transformation and itsacetate in the concentration 2–5 g/l are similar toresults of hydroxylation of DEA and ADEA. Thetransformation of DHA in the concentration 2 g/l(either microcrystals or complex with MCD), resultedto the total conversion to THA (the concentration inreaction mixture 70 and 75%, respectively) over 20–24 hof incubation. The speed of hydroxylation as the com�plex with MCD insufficiently increased in comparisonwith control (Fig. 3). Transformation of 5 g/l of DHAas the complex with MCD terminated after 30 h, andconcentration of THA in reaction mixture was 80%(Table 2), while the conversion of 5 g/l of DHA asthe microcrystals even over 48 h was not completedand the amount of 7α�hydroxy product did notexceed 20%.

Transformation of pregnenolone (Δ5�PG),16α,17α�epoxyprogesterone (16,17�epoxy�Δ4�PG),

monoacetate and triacetate of compound R Reichstein(MAR and TAR). Mycelium of C. lunata introducedhydroxyl group into these substrates not only in theC�7α�position but also in the C�11β�position; trans�formation of Δ5�PG occurred with the creation of 7α,11β�dihydroxyproduct but not with monohydroxy�compunds (Table 2). In contrast to DEA and DHAand their acetates, the production of sufficient amount ofhydroxyderivatives of mentioned Δ5�steroids (excluding16,17�epoxy�Δ5�PG) even at a low load 2 g/l was possi�ble only in the presence of MCD (Table 2). The totalyield of isolated steroids was not higher than 10–25%in the absence of solubilizator over 27 h transforma�tion of Δ5�PG and 23 h transformation of MAR andTAR due to intensive destruction of both initial sub�strate and reaction products.

Hydroxylation of Δ4�3�ketosteroids. The condi�tions of transformation of Δ4�3�ketosteroids that wasfulfilled using Curvularia lunata VKPM F�988 and dif�fered from the previous strain by the presence ofmarker of resistance to antibiotic geneticin G�418were similar to examples described previously, butmycelium for transformation was grown in themedium where the mineral source of nitrogen was sub�

4

3

2

1

0 4836247h

g/l1

2

34

Fig. 2. The speed of 7α�OH�DEA synthesis dependent onamount of mycelium and presence of MCD at the ADEAconcentration 5 g/l.

Table 1. Influence of cyclodextrines on transformation ofADEA with the load 10 g/l

Polymer

Concentration of steroids in reaction mixture, %

Yield 7α�OH�DEA,

%ADEA THA 7α�OH�DEA

CD 45 5 50 20.5

MCD 5 3 92 77

HPCD 10 traces 90 65

Control* 70 15 15 9

* Transformation without polymer.

2

1.5

1

0.5

0 18 246h

g/l

1

2

3

4

Fig. 3. Transformation of 3β,17β�dihydroxyandrost�5�enin the concentration 2 g/l (biomass 9.5 g/l): 1 is substrateDHA in complex with MCD, 2 is microcrystals of DHA,3 is product of THA from microcrystalline substrate, and4 is product of THA in the presence of MCD.

46


ANDRUSHINA et al.

stituted by organic. Androstanes like testosterone (TS),androstenedione (AD), 9α�hydroxy�AD (9�OH�AD),and androstadienedione (ADD) and pregnanes likeprogesterone (Δ4�PG), 17�hydroxy�Δ4�PG (17�OH�Δ4�PG), cortexolone (comp. “S”), and its acetate, 21�hydroxy�20�methylpregna�1,4�diene�3�on (HMPD)(Fig. 1) were selected for evaluation of hydroxylaseactivity of fungus in regard to solubilized Δ4�3�keto�steroids using MCD. The selection of these steroids isstipulated that TS, AD, Δ4�PG, 16,17�epoxy�Δ4�PG,and acetate of compound “S” are Δ4�3�ketoanaloguesof DHA, DEA, Δ5�PG, 16,17�epoxy�Δ5�PG, andMAR, respectively. In addition, AD, 9�OH�AD, and

ADD are interesting as substrates for hydroxylation inthe C�14α�position in the presence of MCD.

Transformation of testosterone (TS). It is deter�mined that C. lunata inserts hydroxyl group into TS asin steroids of pregnane class in C�11β�position.Dynamics of 11β�hydroxylation of TS at load 4 g/l asthe complex with MCD and in its absence is shown inFig. 4. The conversion of TS to 11β�hydroxy�TSoccurred in the presence of MCD without accumula�tion of side products and finished after 24 h. Duringthis time, 75% of 11β�hydroxy�TS accumulated inreaction solution, while without solubilizator only

Table 2. Products of hydroxylation of steroids of (1–7) androstane class and (8–16) pregnane by mycelium of Curvularia lunatacreated in the presence of MCD and absence of solubilizator (control)

No. Steroid substrate Concentra�tion, g/l Transformation products

Concentration of hydroxys�teroids in reaction mixture, %

MCD control *

C19�Steroids

1 TS 4.0 11β�Hydroxy�TS 75 35

2 AD 4.0 14α�Hydroxy�AD 70 25

11β,14α�Dihydroxy�AD 15 10

3 9�OH�AD 2.0 9α,14α�Dihydroxy�AD 55 50

4 ADD 3.0 14α�hydroxy�ADD 70 5

5 DHA 5.0 7α�hydroxy�DHA 80 20

6 DEA 4.0 7α�hydroxy�DEA 85 10

7 ADEA 10.0 7α�hydroxy�DEA 95 15

C21�Steroids

8 Δ5�PG 2.0 7α,11β�dihydroxy�Δ5�PG 70 10

9 Δ4�PG 2.0 11β�hydroxy�Δ4PG 40 10

7α�hydroxy�Δ4PG 35 15

6β�hydroxy�Δ4PG 10 –

10 17�OH�Δ4�PG 2.0 11β,17α�dihydroxy�Δ4�PG 85 70

11 16,17�epoxy�Δ5�PG 2.0 7α�hydroxy�16,17α�epoxy�Δ5�PG 80 75

12 16,17�epoxy�Δ4�PG 1.0 11β,14α�dihydroxy�16,17α�epoxy� Δ4�PG 50 10

13 MAR 2.0 Comp. “R” 30 –

7α�hydroxy�"R" 30 15

11β�hydroxy�"R" 25 10

14 TAR 1.0 Comp. “R” 60 –

7α�hydroxy�comp. “R” 20 10

11b�hydroxy�comp. “R” 15 5

15 Sub. S 10.0 Comp. “F” 65 55

14α�hydroxy�comp. “F” 30 40

C22�Steroids

16 HMPD 4.0 11β�hydroxy�HMPD 60 –

7β�hydroxy�HMPD 30 –

7β,11β�dihydroxy�HMPD – 15



35% of the product accumulated (Table 2), after whichits destruction began.

Transformation of androstens AD, 9�OH�AD, andADD. The main products of transformation of indi�cated 17�ketosteroids were 14α�hydroxyderivatives. Inthe absence of MCD, 14α�hydroxy�AD (14�OH�AD)was obtained with the yield 55–60% over 24–26 h onlyif concentration of AD was less than 2 g/l. During thistime, the total conversion of 4 g/l AD to 14�OH�AD,whose concentration in reaction mixture was 70%(Table 2), was finished in the presence of MCD. Sixty�two percent of 14�OH�AD and, as the side compound,15% of 11β,14α�dihydroxy�AD were extracted.

Since further increase of single dose of AD even asthe complex with MCD was not desired due toincrease of amount of side products, the two repeatedcycles of transformations in aqueous phase obtainedafter separation of mycelium and extraction of trans�formation products were carried out. Both cycles wereconducted with 4 g/l of AD using fresh mycelium (10–11 g/l) but without regeneration and addition of newportion of MCD. At the total conversion of AD over24 h, the concentration of 14�OH�AD in aqueousphase of second and third cycles of transformation was60%. Thus, 12 g of AD were converted to 14�OH�ADwith the use of MCD in the concentration calculatedper 4 g/l of AD and as the result of repeated using of asimilar portion of MCD. However, hydroxylase activ�ity of mycelium was low in the fourth cycle.

Transformation of 2 g/l of 9�OH�AD in 9α,14α�dihydroxy�AD and 1 g/l ADD to 14α�hydroxy�ADDresulted in high yield at the application of both micro�crystalline and substrates solubilized with MCD, but itwas two times faster in the latter case. However,hydroxylation of ADD at the concentration 3 g/l in theabsence of MCD entailed the reduction of 17�keto�group and destruction of the reduction product—1,2�dehydro�TS.

The prevalence of 14α�hydroxylase activity asregards to Δ4�3,17�diketo�androstens is a peculiarityof C. lunata against other fungi species. For instance,mycelium of Congronella butleri showed 7α�hydroxy�lase activity not only during transformation of DEA

but also at AD creating 7α�hydroxy�AD (27%) and14�OH�AD (22%) as the main products and 6β� and7β�hydroxy�AD as the side compounds [24].

Transformation of pregnans Δ4�PG, 17�OH�Δ4�PG,HMPD, comp. “S”, and 21�acetate of comp. “S”. Themain process of transformation of the mentionedcompounds was 11β�hydroxylation. The maximalyield of 11β�hydroxyderivatives was possible only inthe presence of MCD (Table 2) even at the minimalconcentration of other steroids 1–2 g/l, excluding 17�OH�Δ4�PG; 11β�hydroxy�HMPD with yield 50% wasobtained from solubilized HMPD.

On the basis of data of the study of Undisz et al.[25], the hydroxyderivatives of HMPD can have prac�tical interest. This study shows that HMPD and its1,2�dihydroanalogue are the best inductors of 11β�hydroxylase in mycelium of Cochliobolus lunatusamong 24 studied steroids.

In contrast to Δ5�3β�hydroxysteroids, the positionand orientation of side hydroxyl groups (6β�, 7α�, 7β�,and 14α�) introduced into Δ4�3�ketosteroids dependedon the structure of substitutes at C�17. The side prod�uct of Δ4�PG transformation—7α�hydroxy�Δ4�PG—is extracted almost in an equal amount with the main

100

60

40

20

0 18 4012h

1

2

3

4

24

80

%

Fig. 4. Dynamics of transformation (%) of 4 g/l of TS (bio�mass 12 g/l): 1 is 11β�OH�TS in the presence of MCD, 2 ismicrocrystals of TS, 3 is 11β�OH�TS from microcrystal�line TS, and 4 is TS in complex with MCD.

Table 3. Transformation of cortexolone by mycelium of C. lunata VKPM F�988

Charge of cortexolone, g/l

Mass ration steroid/methylcy�

clodextrine

Time of transformation, h

Total amount of transformation products, % Yield of crystalline

hydroxycortisone, %in aqueous phase in mycelium

10.0 – 46 48.8 42.6 43.2

10.0 1/5 22 91.5 traces 58.0

15.0 1/5 24 96.0 traces 56.4

20.0 1/5 48 98.0 traces 55.0

20.0 1/3 50 76.6 10.5 52.0

48


ANDRUSHINA et al.

11β�hydroxy�Δ4�PG both with and without MCD(Table 2). The side products of substance “S” transfor�mation and HMPD were respectively 14α� and 7β�hydroxyderivatives. The study results agree with theliterature data on capability of strains of C. lunata spe�cies to form 6β�, 7α�, 7β�, and 14α�hydroxysteroids asside products [1–4, 26].

High efficiency of MCD as the solubilizator of sub�stance “S” which is the initial compound in the synthe�sis of a great number of medical steroid drugs nature isshown in Table 3. Introduction of 11β�hydroxyl groupinto substance “S” used as the complex with MCD inthe optimal ratio 1 : 1 (mol/mol) was processed at thesubstrate concentration less than 20 g/l during 48 hwithout destruction of steroids. In this case, the prod�ucts of transformation were not represented on myce�lium, which considerably facilitated extraction ofhydrocortisone from aqueous phase.

The analysis of results of hydroxylation of sixteensteroids as the complexes with chemically modifiedcyclodextrines allow for the assumption that the lastare more effective solubilizers in reactions of hydroxy�lation than β�cyclodextrin. In particular, the concen�tration of transforming substrates in reaction mediumwas increased until 10–20 g/l using MCD, their con�version to target hydroxysteroids was enhanced by twotimes and the yield of these products due to theabsence of destruction and decrease of amount of sidecompounds have increased.

It should be noted that the presence of MCDincreased the efficiency of enzymatic hydrolysis. As isseen on the examples of transformation of ADEA,MAR, and TAR, the speed of hydrolysis of aceto�groups in the absence of MCD was low.

REFERENCES

1. Sedlaczek, L., Crit. Rev. Biotechnol., 1988, vol. 7, no. 3,pp. 187–236.

2. Akhrem, A.A. and Titov, Yu.A., Steroidy i mikroorga�nizmy (Steroids and Microorganisms), Moscow:Nauka, 1970.

3. Fernandes, P., Cruz, A., Angelova, B., Pinheiro, H.M.,and Cabral, J.M.S., Enzyme Microb. Technol., 2003,vol. 32, no. 6, pp. 688–705.

4. Mahato, S.B. and Garai, S., Steroids, 1997, vol. 62,no. 4, pp. 332–345.

5. Srivastava, S.K., Srivastava, R.A.K., and Mathur, S.N.,J. Appl. Bacteriol., 1985, vol. 59, no. 5, pp. 399–402.

6. Szejtli, J., Pure Appl. Chem., 2004, vol. 76, no. 10,pp. 1825–1845.

7. Udvardy, E.N., Bartho, I., Hantos, G., Trinn, M.,Vida, Z., Szejtly, J., Stadier, A., Haben, I., andBalazs, M., UK Patent Appl. No. 2108965, 1983.

8. Sukhodol’skaya, G.V., Donova, M.V., Nikolaeva, V.M.,Koshcheenko, K.A., Dovbnya, D.V., and Gulevskaya, S.A.,RF Patent No. 2 156 302, 2000.

9. Alekhina, T.M., Ryzhkova, V.M., Kurakov, V.V., andKlubnichkina, G.A., Khim.�Farm. Zh., 1993, vol. 4,no. 2, pp. 59–62.

10. Chinchokar, S.B., Sukhodol’skaya, G.V., Baklashova, T.G.,and Koshcheenko, K.A., Prikl. Biokhim. Mikrobiol.,1992, vol. 28, no. 6, pp. 685–693.

11. Fokina, V.V., Karpov, A.V., Sidorov, I.A., Andrjushina, V.A.,and Arinbasarova, A.Yu., Appl. Microbiol. Biotechnol.,1997, vol. 47, no. 6, pp. 645–649.

12. Singer, Y., Shity, H., and Bar, R., Appl. Microbiol. Bio�technol., 1991, vol. 35, no. 6, pp. 731–737.

13. Hesselink, P.G.M., Van�Vliet, S., De Vries, H., andWitholt, B., Enzyme Microb. Technol., 1989, vol. 11,no. 7, pp. 398–404.

14. Schlosser, D., Irrgang, S., and Schmauder, H.�P., Appl.Microbiol. Biotechnol., 1993, vol. 39, no. 1, pp. 6–20.

15. Lu, W., Food Bioprod. Proc., 2007, vol. 85, no. 1,pp. 63–72.

16. Donova, M.V., Prikl. Biokhim. Mikrobiol., 2007,vol. 43, no. 1, pp. 1–14.

17. Jadoun, J. and Bar, R., Appl. Microbiol. Biotechnol.,1993, vol. 40, no. 4, pp. 477–482.

18. Alexander, D.L. and Fischer, J.F., Steroids, 1995,vol. 60, no. 3, pp. 290–294.

19. Del Valle, E.M., Process Biochem., 2004, vol. 39, no. 9,pp. 1036–1046.

20. Andryushina, V.A., Druzhinina, A.V., Yaderets, V.V.,Stytsenko, T.S., and Voishvillo, N.E., Prikl. Biokhim.Mikrobiol., 2010, vol. 46, no. 1, pp. 78–83.

21. Andryushina, V.A., Voyshvillo, N.E., Druzhinina, A.V.,Stytsenko, T.S., Yaderets, V.V., and Skryabin, K.G., RFPatent No. 2 377 309 (2008), Byull. Izobret., no. 36,2009.

22. Shuvalova, S.D., Gabinskaya, K.N., Popova, E.V.,Savinova, T.S., and Andryushina, V.A., Khim.�Farm.Zh., 2001, vol. 35, no. 5, pp. 44–46.

23. Chouhary, M.I., Ali, ShahS.A., Musharraf, S.G., andShaheen, F., Atta�Ur�Rahman, Nat. Prod. Res., 2003,vol. 17, no. 3, pp. 215–220.

24. Kollerov, V.V., Shutov, A.A., Fokina, V.V., Suhodol�skaya, G.V., and Donova, M.V., J. Mol. Cat. B: Enz.,2008, vol. 55, no. 1, pp. 61–68.

25. Undisz, K., Groh, H., Stopsack, H., and Horhold�Schubert, C., J. Steroid Biochem., 1992, vol. 43, no. 6,pp. 547–547.

26. Charney, W. and Herzog, H.L., Microbial Transforma�tion of Steroids, New York: Academic Press, 1967.

hydroxylation of steroids by curvularia lunata mycelium in the presence of methyl-β-cyclodextrine

Documents