Galina V. Sukhodolskaya

Screening of mycelial fungi for 7α- and 7β-hydroxylase activity towards dehydroepiandrosterone

In total, 481 fungal strains were screened for the ability to carry out 7(α/β)-hydroxylation of dehydroepiandrosterone (DHEA, 3β-hydroxy-5-androsten-17-one). Representatives of 31 genera of 15 families and nine orders of ascomycetes, 17 genera of nine families and two orders of zygomycetes, two genera of two families and two orders of basidiomycetes, and 14 genera of mitosporic fungi expressed 7(α/β)-hydroxylase activity. The majority of strains were able to introduce a hydroxyl group to position 7α. Active strains selectively producing 3β,7α-dihydroxy-5-androsten-17-one were found among Actinomucor, Backusella, Benjaminiella, Epicoccum, Fusarium, Phycomyces and Trichothecium, with the highest yield of 1.25 and 1.9 g L−1 from 2 and 5 g L−1 DHEA, respectively, reached with F. oxysporum. Representatives of Acremonium, Bipolaris, Conidiobolus and Curvularia formed 3β,7β-dihydroxy-5-androsten-17-one as a major product from DHEA. The structures of the major steroid products were confirmed by TLC, gas chromatography (GC), mass spectra (MS), and 1H-NMR analyses.

Dihydroxylation of dehydroepiandrosterone in positions 7α and 15α by mycelial fungi

The ability of 485 fungal strains is studied for catalysis of the process of 7α, 15α-dihydroxylation of dehydroepiandrosterone (DHEA, 3β-hydroxy-5-androstene-17-one), a key intermediate of the synthesis of physiologically active compounds. The ability for the formation of 3β, 7α, 15α-trihydroxy-5-androstene-17-one (7α, 15α-diOH-DHEA) was found for the first time for representatives of 12 genera, eight families, and six orders of ascomycetes, eight genera, four families, and one order of zygomycetes, one genus, one family, and one order of basidiomycetes, and four genera of mitosporic fungi. The most active strains are found among genera Acremonium, Gibberella, Fusarium, and Nigrospora. In the process of transformation of DHEA (2 g/l) by strains of Fusarium oxysporum VKM F-1600 and Gibberella zeae BKM F-2600, the molar yield was 63 and 68%, respectively. Application of the revealed active strains of microorganisms opens prospects for the efficient production of key intermediates of synthesis of modern medical preparations.

Microbial conversion of pregna-4,9(11)-diene-17α,21-diol-3,20-dione acetates by Nocardioides simplex VKM Ac-2033D

The conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione 21-acetate (I) and 17,21-diacetate (VI) by Nocardioides simplex VKM Ac-2033D was studied. The major metabolites formed from I were identified as pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV). Pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione (III) and pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V) were formed in minorities. Biotransformation products formed from VI were pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17,21-diacetate (VII), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII), pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V). The conversion pathways were proposed including 1(2)-dehydrogenation, deacetylation, 20beta-reduction and non-enzymatic migration of acyl group from position 17 to 21. The conditions providing predominant accumulation of pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) from I and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII) from VI in a short-term biotransformation were determined.

The inhibitory effect of cyclodextrin on the degradation of 9α-hydroxyandrost-4-ene-3,17-dione by Mycobacterium sp. VKM Ac-1817D

The inhibitory effect of methylated β-cyclodextrin (mCD) on steroid degradation was studied using the degradation of 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) by Mycobacterium sp. VKM Ac-1817D as a model process. The formation of the [9-OH-AD–mCD] complex was shown by 1H NMR-spectroscopy. The biodegradation of 9-OH-AD by whole and disrupted cells was carried out at 30°C in aqueous solutions with or without mCD. Enzyme kinetic parameters were calculated by non-linear regression of the Michaelis–Menten plot. The complexation of 9-OH-AD and mCD was evaluated via the stability constant for the [9-OH-AD–mCD] complex. The Vmax and KM values calculated for the free (non-complex) steroid in mCD solutions corresponded to steroid degradation in the absence of mCD. The inclusion complex [9-OH-AD–mCD] was shown to be resistant to enzymatic degradation. The inference is made that the ‘‘guest–host’’ molecular complexation with cyclodextrin can be used for the control of steroid bioconversions.

Poly-N-vinylcaprolactam gel: A novel matrix for entrapment of microorganisms

A new gel-type support poly-N-vinylcaprolactam for microbial cell immobilization is presented. The method allows one to obtain beads of biocatalyst in a single step. The properties of beads obtained using different types of gel stabilizers were compared; the best stabilizer was found to be tannin. The method developed was used for entrapment of viable bacterial cells and fungal spores. The biocatalysts obtained were used for transformations of both hydrophilic (sorbitol, indolyl-3-acetic acid) and lipophilic (cortexolone, hydrocortisone) substrates.

Conversion of 1-benzoylindole by Aspergillus strains

Abstract Biotransformation of 1-benzoylindole (BI) by the strains Aspergillus flavus VKM F-1024 and Aspergillus oryzae VKM F-44 was studied. The major metabolites isolated were identified as 4-hydroxyindole (4-HI), 5-hydroxyindole (5-HI), 4-hydroxy-1-benzoylindole, 4-hydroxy-1-(4′-hydroxy)-benzoylindole and indole. The structure of the metabolites was determined by mass spectrometry and proton nuclear magnetic resonance spectroscopy. The pathways of BI metabolism via initial monohydroxylation at C-4 and C-5 followed by cleavage of the benzoyl substituent to yield 4-HI and 5-HI were proposed. Indole was formed as a by-product, and its role as a potent inhibitor of BI hydroxylation at C-4 and C-5 is discussed.

11β-Hydroxylation of 6α-fluoro-16α-methyl-deoxycorticosterone 21-acetate by filamentous fungi

Selected filamentous fungi—98 strains of 31 genera—were screened for the ability to catalyze 11β-hydroxylation of 6α-fluoro-16α-methyl-deoxycorticosterone 21-acetate (FM-DCA). It was established that representatives of the genera Gongronella, Scopulariopsis, Epicoccum, and Curvularia have the ability to activate 11β-hydroxylase steroids. The strains of Curvularia lunata VKM F-644 and Gongronella butleri VKM F-1033 expressed maximal activity and formed 6α-fluoro-16α-methyl-corticosterone as a major bioconversion product from FM-DCA. The structures of the major products and intermediates of the bioconversion were confirmed by TLC, HPLC, MS and 1H NMR analyses. Different pathways of 6α-fluoro-16α-methylcorticosterone formation by C. lunata and G. butleri strains were proposed based on intermediate identification. The constitutive character and membrane-binding localization were evidence of a 11β-hydroxylating system in G. butleri, while an inducible character and microsomal localization was confirmed for 11β-hydroxylase of C. lunata. Under optimized conditions, the molar yield of 6α-fluoro-16α-methyl-corticosterone reached 65% at a FM-DCA substrate loading of 6 g/L.

Bioconversion of 6-(N-methyl-N-phenyl)aminomethyl androstane steroids by Nocardioides simplex

HIGHLIGHTSNocardioides simplex converts 6‐(N‐methyl‐N‐phenyl)aminomethyl androstanes.Only &bgr;‐stereoisomers undergo 1(2)‐dehydrogenation with N. simplex cells.(N‐methyl‐N‐phenyl)aminomethyl substitution at C6 prevents biodegradation of steroid core. ABSTRACT The newly synthesized (&agr;/&bgr;)‐diastereomers of 6‐(N‐methyl‐N‐phenyl)aminomethylandrost‐4‐ene‐3,17‐dione (5) and 6‐(N‐methyl‐N‐phenyl)aminomethylandrost‐4‐en‐17&bgr;‐ol‐3‐one (6) were firstly investigated as substrates for the whole cells of Nocardioides simplex VKM Ac‐2033D in comparison with their unsubstituted analogs, – androst‐4‐ene‐3,17‐dione (1) and androst‐4‐en‐17&bgr;‐ol‐3‐one (2). 1(2)‐Dehydroderivatives were identified as the major bioconversion products from all the substrates tested. When using the mixtures of (&agr;/&bgr;)‐stereoisomers of 5 and 6 as the substrates, only &bgr;‐stereoisomers of the corresponding 1,4‐diene‐steroids were formed. Along with 1(2)‐dehydrogenation, N. simplex VKM Ac‐2033D promoted oxidation of the hydroxyl group at C‐17 position of 6: both 6(&agr;) and 6(&bgr;) were transformed to the corresponding 17‐keto derivatives. No steroid core destruction was observed during the conversion of the 6‐substituted androstanes 5 and 6, while it was significant when 1 or 2 was used as the substrate. The results suggested high potentials of N. simplex VKM Ac‐2033D for the generation of novel 1(2)‐dehydroanalogs.

Obtaining of a High-Density Culture ofNocardioides sp.

Selection of carbon sources demonstrated ethanol to be the best substrate for a high-density Nocardioides sp. culture. A strategy for control over high-density fed-batch culture production was developed, which permitted maximizing the yield of biomass (21 g/l). The control, based on the ExpoDense algorithm, should be predetermined at the first phase and adaptive in the second phase of the two-phase process of high-density culture production.