Victoria V. Fokina

The influence of β-cyclodextrin on the kinetics of 1-en-dehydrogenation of 6α-methylhydrocortisone by Arthrobacter globiformis cells

Abstractβ-Cyclodextrin changes the kinetic peculiarities of microbial 1-en-dehydrogenation of 6α-methylhydrocortisone: the reaction rate, degree of conversion and respiratory-chain activity are increased. Respiratory-chain activity in the presence of β-cyclodextrin is increased both in the presence and in the absence of 6α-methylhydrocortisone. A mathematical model is proposed to describe the kinetics of the process. This model suggests the formation of different multi-component complexes consisting of inclusion complexes and the functional unit involving the enzyme and the respiratory chain. All the parameters of the model were estimated by data fitting. In the model framework the formation of multi-component complexes leads to an increase of the maximal reaction rate and to a decrease of substrate inhibition. An approach is suggested for optimisation of 6α-methylhydrocortisone 1-en-dehydrogenation in the presence of β-cyclodextrin. The optimal concentrations of 6α-methylhydrocortisone and β-cyclodextrin have been calculated.

Kinetic characteristics of 1-en-dehydrogenation of 6α-methylhydrocortisone by cells of Arthrobacter globiformis 193

Abstract Biochemical characteristics of 6α-methylhydrocortisone 1-en-dehydrogenation by bacterial cells of Arthrobacter globiformis 193 have been studied. The reaction follows the kinetics of substrate inhibition; the inhibition reveals itself at the level of the respiratory chain. A mathematical model describing multiple substrate inhibition during the process of 6α-methylhydrocortisone 1-en-dehydrogenation with whole cells was proposed. The solution of the constructed model agrees well with experimental data. There is little or no inhibitory effect at low substrate concentrations, and the reaction rate is determined by the enzyme-substrate interaction rather than the respiratory chain activity.

Genome-wide bioinformatics analysis of steroid metabolism-associated genes in Nocardioides simplex VKM Ac-2033D

Actinobacteria comprise diverse groups of bacteria capable of full degradation, or modification of different steroid compounds. Steroid catabolism has been characterized best for the representatives of suborder Corynebacterineae, such as Mycobacteria, Rhodococcus and Gordonia, with high content of mycolic acids in the cell envelope, while it is poorly understood for other steroid-transforming actinobacteria, such as representatives of Nocardioides genus belonging to suborder Propionibacterineae. Nocardioides simplex VKM Ac-2033D is an important biotechnological strain which is known for its ability to introduce ∆1-double bond in various 1(2)-saturated 3-ketosteroids, and perform convertion of 3β-hydroxy-5-ene steroids to 3-oxo-4-ene steroids, hydrolysis of acetylated steroids, reduction of carbonyl groups at C-17 and C-20 of androstanes and pregnanes, respectively. The strain is also capable of utilizing cholesterol and phytosterol as carbon and energy sources. In this study, a comprehensive bioinformatics genome-wide screening was carried out to predict genes related to steroid metabolism in this organism, their clustering and possible regulation. The predicted operon structure and number of candidate gene copies paralogs have been estimated. Binding sites of steroid catabolism regulators KstR and KstR2 specified for N. simplex VKM Ac-2033D have been calculated de novo. Most of the candidate genes grouped within three main clusters, one of the predicted clusters having no analogs in other actinobacteria studied so far. The results offer a base for further functional studies, expand the understanding of steroid catabolism by actinobacteria, and will contribute to modifying of metabolic pathways in order to generate effective biocatalysts capable of producing valuable bioactive steroids.

Bioconversion of C19- and C21-steroids with parent and mutant strains of Curvularia lunata

Regio- and stereospecificity of microbial hydroxylation was studied at the transformation of 3-keto-4-ene steroids of androstane and pregnane series by the filamentous fungus of Curvularia lunata VKM F-644. The products of the transformations were isolated by column chromatography and identified using HPLC, massspectrometry (MS) and proton nuclear magnetic resonance (1H NMR) analyses. Androst-4-ene-3,17-dione (AD) and its 1(2)-dehydro- and 9α-hydroxylated (9-OH-AD) derivatives were hydroxylated by the fungus mainly in position 14α, while 6α-, 6β- and 7α-hydroxylated products were revealed in minor amounts. At the transformation of C21-steroids (cortexolone and its acetylated derivatives) the presence of 17-acetyl group was shown to facilitate further selectivity of 11β-hydroxylation. Original procedures for protoplasts obtaining, mutagenesis and mutant strain selection have been developed. A stable mutant (M4) of C. lunata with high 11β-hydroxylase activity towards 21-acetate and 17α,21-diacetate of cortexolone was obtained. Yield of 11β-hydroxylated products reached about 90% at the transformation of 17α, 21-diacetate of cortexolone (1 g/l) using mutant strain M4.

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.

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.

21-Acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione conversion by Nocardioides simplex VKM Ac-2033D.

The conversion of 21-acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (I) by Nocardioides simplex VKM Ac-2033D was studied purposed selective production of its 1(2)-dehydroanalogues-value precursors in the synthesis of modern glucocorticoids starting from 9alpha-hydroxyandrostenes. 21-Acetoxy-pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (II), pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (III) and pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (IV) were revealed as metabolites, and the structures were confirmed by mass spectrometry and (1)H nuclear magnetic resonance (NMR) spectroscopy. The metabolic pathways of I by N. simplex included 1(2)-dehydrogenation and deacetylation. The sequence of the reactions was shown to depend on the transformation conditions. The presence of both soluble and membrane associated steroid esterases in N. simplex was demonstrated using cell fractionation. Unlike inducible 1(2)-dehydrogenase, steroid esterase was shown to be constitutive. The conditions providing selective accumulation of II from I by whole N. simplex cells were determined.

Complete Genome Sequence of Steroid-Transforming Nocardioides simplex VKM Ac-2033D

ABSTRACT Nocardioides simplex VKM Ac-2033D is an effective microbial catalyst for 3-ketosteroid 1(2)-dehydrogenation, and it is capable of effective reduction of carbonyl groups at C-17 and C-20, hydrolysis of acetylated steroids, and utilization of natural sterols. Here, the complete genome sequence is reported. An array of genes related to steroid metabolic pathways have been identified.

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.