Isolation and identification of a new metabolite of microbial conversion of upgraded neutral fraction of Polish tall oil by means of four strains of Mycobacterium sp.

Summary A new metabolite, 5-alpha-androstane-3,6,17-trione, was isolated as a product of microbial conversion of upgraded neutral fraction of the Polish tall oil byMycobacterium NRRL B-3683, NRRL B-3805, MB 3683, and MB 3805.     

Microbial conversion of solasodine to 1-androstene-3,17-dione (AD), a key intermediate for androgen synthesis

The spirosolane side chain of solasodine has been cleaved by cholesterol preinduced Mycobacterium sp. NRRL B-3805 to yield 1-androstene-3,17-dione (AD), a key intermediate for the synthesis of androgenic drugs. Conversion up to 34% has been recorded in shake flask culture after 192 h incubation period using dimethyl-formamide as carrier for solasodine addition.     

Dependence of steroid 1(2)-dehydrogenation on the C-17 side chain during cholesterol metabolism by immobilized Mycobacterium fortuitum

1(2)-Dehydrogenation of 4-cholestene-3-one by immobilized Mycobacterium fortuitum NRRL B-8153 and free growing Mycobacterium sp. NRRL B-3805 and Micromonospora chlacea MTCC 329 depended on the nature of the C-17 side chain. The 1(2)-dehydrogenation did not appear to occur before C-17-side-chain cleavage of the 4-cholestene-3-one.S. Patil is and A. Srivastava was with the School of Life Sciences, Devi Ahilya Vishwavidyalaya. Vigyan Bhawan, Khandwa Road Campus, Indore-452 001, India. A. Srivastava is now with the Shripati Singhania R&D Centre. J. K. Pharmaceuticals. 13th Mile Stone. Mathura Road, Faridabad (Haryana) 121 003, India.     

1-Ene-steroid reductase of Mycobacterium sp. NRRL B-3805

The microbial enzymatic reduction of 1,4-androstadiene-3,17-dione (ADD) to 4-androstene-3,17-dione (AD), testosterone and 1-dehydrotestosterone (DHT) is described. Two reducing activities observed in washed cell suspensions and cell free extracts of Mycobacterium sp. NRRL B-3805 were found to account for these bioconversions. One was a 1-ene-steroid reductase and the other a 17-keto steroid reductase. The first reducing activity was found to appear in the soluble cell fraction whereas the latter could be precipitated by centrifugation. Maximum 1-ene-steroid reductase specific activity was achieved during the exponential growth phase of the organism and significantly increased upon induction with ADD. The 1-ene-steroid reductase was partially purified (30-fold) by ammonium sulfate fractionation, gel-filtration and ion-exchange chromatography, and was eluted from a Sephacryl S-300 column with an Mr = 115,000. The 1-ene-steroid reductase activity was NADPH-dependent and had specificity towards steroid compounds containing C-1,2 double bond with an apparent Km for ADD of 2.2 X 10(-5) M. The reverse reaction catalyzing C-1,2 dehydrogenation could not be detected in our preparations. The results suggest that in Mycobacterium sp NRRL B-3805 and B-3683 the steroid C-1,2 dehydrogenation and 1-ene reduction are two separable activities.     

Microbial Degradation of Sterols

A process is described for the microbial degradation of cholesterol and plant sterols, to produce androsta-1, 4-diene-3, 17-dione and androst-4-ene-3, 17-dione, by two newly isolated bacteria designated Mycobacterium sp. NRRL B-3683 and Mycobacterium sp. NRRL B-3805. These myocbacteria produce substantial amounts of 17-ketonic compounds without appreciable degradation of the steroid nucleus. No ring degradation inhibitory agents are necessary. The first microbiological production of 20alpha-hydroxymethylpregna-1, 4-dien-3-one is also reported.     

Application of modern NMR techniques in the structure determination of microbial transformation products from sterols

Microbial transformations of cycloartenol, 24-methylenecycloartanol and lanosterol with Mycobacterium sp. (NRRL B-3805) resulted in the isolation and characterization of five C19 steroids. Details of the tremendous structural modifications of these metabolites from their parent compounds were clarified by 2D nmr techniques. The stereochemistry of these novel metabolites was determined by nOe studies.     

Optimization of androstenedione production in an organic–aqueous two-liquid phase system

A systematic evaluation of the effect of key operational parameters on the selective cleavage of sitosterol to 4-androstene-3,17-dione (AD) with Mycobacterium sp. NRRL B-3805 in a dioctyl phthalate: aqueous buffer two-liquid phase system was performed. Of the parameters assessed, buffer composition, biomedium pH, temperature, and biomass and substrate concentration were those that mostly affected overall bioconversion rate. The optimum pH was 7.5 with Tris buffer. The highest bioconversion rate was observed at 35 °C, although at 40 °C bioconversion activity was virtually lost. Michaelis–Menten type kinetics adequately described the bioconversion system. Increasing biomass concentration from 10 to 70 g_{wet cell weight} l⁻¹ favored AD final yield, although the specific AD yield slightly decreased.     

Cosolvent effect on Δ1-steroid-reductase activity of free and PAAH entrapped Mycobacterium Sp. NRRL B-3805 cells

Summary The effect of water miscible solvents on ¹-steroid reduction by free and polyacrylamide-hydrazide (PAAH) entrapped Mycobacterium sp. NRRL B-3805 cells was investigated. On the basis of retention of reductase activity an order of preference of diols (e.g. ethyleneglycol) > DMSO > DMF and monoalcohols (e.g. ethanol) as cosolvents was recorded. Significant increase in substrate (1,4-androstadiene-3,17-dione) solubility was attained in presence of the cosolvent of choice (ethyleneglycol), accompanied by some inhibition of the ¹-reductase activity. Optimization of ethyleneglycol concentration (10–20% (v/v)) led to specific activity in a homogeneous medium, higher than recorded in the absence of cosolvent. Immobilization in PAAH gel resulted in high retention of immobilized enzymic activity, accompanied by minor diffusional limitations. Moreover, the gel exhibited protective effect of the entrapped cells from cosolvent inhibition. Modification of gel composition (e. g. hydrophobicity) had no significant effect on reaction kinetics.     

Conversion of Unsaturated Fatty Acids by Bacteria Isolated from Compost

A compost mixture amended with soybean oil was enriched in microorganisms that transformed unsaturated fatty acids (UFAs). When oleic acid or 10-ketostearic acid was the selective fatty acid, Sphingobacterium thalpophilum (NRRL B-23206, NRRL B-23208, NRRL B-23209, NRRL B-23210, NRRL B-23211, NRRL B-23212), Acinetobacter spp. (NRRL B-23207, NRRL B-23213), and Enterobacter cloacae (NRRL B-23264, NRRL B-23265, NRRL B-23266) represented isolates that produced either hydroxystearic acid, ketostearic acid, or incomplete decarboxylations. When ricinoleic (12-hydroxy-9-octadecenoic) acid was the selective UFA, Enterobacter cloacae (NRRL B-23257, NRRL B-23267) and Escherichia sp. (NRRL B-23259) produced 12-C and 14-C homologous compounds, and Pseudomonas aeruginosa (NRRL B-23256, NRRL B-23260) converted ricinoleate to a trihydroxyoctadecenoate product. Also, various Enterobacter, Pseudomonas, and Serratia spp. appeared to decarboxylate linoleate substrate incompletely. These saprophytic, compost bacteria were aerobic or facultative anaerobic Gram-negative and decomposed UFAs through decarboxylation, hydroxylation, and hydroperoxidation mechanisms. Received: 3 November 1998 / Accepted: 30 November 1998     

Spectrophotometric estimation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153

A spectrophotometric method for simultaneously estimating 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) in a binary mixture has been developed using sulphuric acid chromogens. The method has been used to estimate both AD and ADD during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153.The authors are with the School of Life Sciences, Devi Ahilya Vishwavidyalaya. Vigyan Bhawan, Khandwa Road, Indore-452 001, India.     

Glucose and lactose effect on AD and ADD bioconversion by Mycobacterium sp.

Summary The glucose and lactose effect on the steroid biotransformation reactions by Mycobacterium sp. NRRL B-3683 was studied. The reduction reactions increased at pH 7 and when sugars are added. The oxidation reactions are highers at pH 8 without any sugar added. The dehydrogenation reaction is inhibited when an endogenous carbon source is present in the media.     

Study of key operational parameters for the side-chain cleavage of sitosterol by free mycobacterial cells in Bis-(2-ethylhexyl) phthalate

The selective side-chain cleavage of β-sitosterol by free cells of Mycobacterium sp. NRRL B-3805 is a well-established multi-enzymatic process for the production of the pharmaceutical steroid precursors androstenedione (AD) and androstadienedione (ADD). In this study, bis(2-ethylhexyl) phthalate (BEHP) was used as a reaction medium for carrying out the process with freely suspended cells. The work aimed to show that microbial sitosterol side-chain cleavage is possible in this essentially mono-phasic organic medium, provided that some important parameters are adequately controlled. The effects of the biocatalyst/substrate mass ratio, system aeration rate and minimum buffer addition to the organic medium on the product yield and the reaction rate were thus evaluated.     

Propanol as an end product of threonine fermentation

Clostridium sp. strain 17cr1 was able to ferment l-threonine to propionate and propanol. Electrons arising in the oxidation of 2-oxobutyrate to propionyl-CoA were apparently used in reductive pathway leading to propanol formation. Part of the propionyl-CoA was used to form propionate in an ATP-forming pathway via a propionate kinase, so that the final ATP yield was 0.5 mol per mol of l-threonine metabolised. Other growth substrates were fermented mainly to acetate and butyrate, and the reductive formation of butyrate, from 2 mol of acetyl-CoA or from crotonate or 3-hydroxybutyrate, was the main route for recycling reduced electron carriers arising during oxidative pathways for most substrates.     

A microwell platform for the scale-up of a multistep bioconversion to bench-scale reactors: Sitosterol side-chain cleavage

The microwell-scale approach is widely used for screening purposes and one-pot biotransformations, but it has seldom been applied to complex whole cell multistep bioconversions, requiring prolonged incubation periods. The present study aims to contribute to filling this gap. The side-chain cleavage of sitosterol to androstenedione (AD) with Mycobacterium sp. NRRL B-3805 cells was used as a model system, and focus was given to the screening of suitable bioconversion media with 24-well microwell plates. Results show that to perform this particular bioconversion growing cells are preferred over resting cells due to higher conversion yields obtained in aqueous medium. The use of resting cells may nevertheless present an interesting approach provided catalytic activity is retained throughout successive runs. Maintaining suitable aeration levels (air flow of 1 mL/min) allowed minimizing the decay of catalytic activity typically observed alongside consecutive bioconversion runs with resting cells. Microwell plates with dedicated oxygen and pH monitoring capabilities proved effective in media development for complex multistep bioconversions using relatively slow-growing bacteria. Under constant k_La (0.044/s) similar AD production and dissolved oxygen profiles were observed in microwell plates and in a bench-scale reactor. Selection of a suitable k_La value proved critical, since under lower k_La values scale-up proved unsuccessful. The same pattern was observed when other scale-up criteria were evaluated to perform the scale-up of this particular bioconversion. Results gathered seem to validate the proposed approach “from microwell plate to bench-scale fermentor”.     

Conversion of β-sitosterol by Mycobacterium sp. NRRL B-3805 cells immobilized on Celite supports

Mycobacterium sp. NRRL B-3805 cells immobilized on Celite were effectively used for the selective side-chain cleavage of sitosterol to androstenedione (AD) in organic media (phthalate derivatives). Kinetic studies were performed with sitosterol concentrations up to 24 mM, with different Celite materials and particle sizes. Higher activity levels were observed when a larger pore size Celite was used as immobilization matrix. Substrate inhibition was observed for sitosterol concentrations above 6 mM. Toxicity effects were not apparently correlated with the high log P solvents (>9) here used as bioconversion media. The use of immobilized cells in repeated batch biotransformations did not prove effective, mainly due to biocatalyst desorption during the periodical, aqueous washing steps used for nutrient delivery to the cells.     

The <Emphasis Type="Italic">Streptomyces violaceusniger</Emphasis> clade: a home for streptomycetes with rugose ornamented spores

The taxonomic status of 16 strains received as Streptomyces hygroscopicus, Streptomyces melanosporofaciens, Streptomyces sparsogenes, Streptomyces sporoclivatus and Streptomyces violaceusniger was evaluated in a polyphasic study. Eleven of the organisms formed a distinct clade in the Streptomyces 16S rRNA gene tree with the type strains of Streptomyces asiaticus, Streptomyces cangkringensis, Streptomyces indonesiensis, Streptomyces javensis, Streptomyces malaysiensis, Streptomyces rhizosphaericus, Streptomyces yatensis and Streptomyces yogyakartensis, the members of this group produced rugose ornamented spores in spiral spore chains. The eleven strains were assigned to three established and four novel species, namely Streptomyces albiflaviniger sp. nov., Streptomyces demainii sp. nov., Streptomyces geldanamycininus sp. nov., Streptomyces griseiniger sp. nov., and Streptomyces hygroscopicus, Streptomyces melanosporofaciens and Streptomyces violaceusniger. It is also proposed that S. sporoclivatus becomes a subjective synonym of S. melanosporofaciens. S. sparsogenes NRRL 2940^T, which produced ridged ornamented spores in spiral spore chains, formed a distinct phyletic line in the Streptomyces 16S rRNA gene tree and was readily distinguished from the other strains using a range of phenotypic properties. S. violaceusniger strains NRRL 8097, NRRL B-5799, NRRL 2834 and ISP 5182 fell outside the S. violaceusniger 16S rRNA gene clade and formed either smooth or ridged ornamented spores in either flexuous or spiral spore chains. These organisms were distinguished from one another and from their closest phylogenetic neighbors and were considered to merit species status as Streptomyces auratus sp. nov., Streptomyces phaeoluteichromatogenes sp. nov., Streptomyces phaeogriseichromatogenes sp. nov., and Streptomyces phaeoluteigriseus sp. nov., respectively. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of the tested strains are S. albiflaviniger NRRL B-1356^T (AJ391812), S. auratus NRRL 8097^T (AJ391816), S. geldanamycininus NRRL 3602^T (DQ334781), S. griseiniger NRRL B-1865^T (AJ391818), S. hygroscopicus NRRL 2387^T (AJ391820), NRRL 2339 (AJ391821) and NRRL B-1477 (AJ391819), S. demainii NRRL B-1478^T (DQ334782), S. melanosporofaciens NRRL B-12234^T (AJ391837), S. phaeogriseichromatogenes NRRL 2834^T (AJ391813), S. phaeoluteichromatogenes NRRL B-5799^T (AJ391814), S. phaeoluteigriseus ISP 5182^T (AJ391815), S. sparsogenes NRRL 2940^T (AJ391817), S. sporoclivatus NRRL B-24330^T (AJ 781369), S. violaceusniger ISP 5563^T (AJ 391823) and NRRL B-1476^T (AJ 391822).     

Testosterone as biotransformation product in steroid conversion byMycobacterium sp.

Summary Testosterone production byMyc. sp. NRRL B-3683 is discussed. The unexpected finding that testosterone is not formed by single reduction of 17-keto group of 4-androstene-3,17-dione (AD) but by a double reduction of both 17-keto group and 1–2 doble bound of 1,4-androstadiene-3,17-dione (ADD) is presented.     

Synthesis and biological activities of new hydrazide derivatives

The synthesis of a new series of imidazo[1,2-a]pyrazine-2-carboxylic acid arylidene-hydrazides is described. The chemical structures of the compounds were elucidated by IR, ¹H-NMR, FAB⁺-MS spectral data. Their biological activity against various bacteria, fungi species, and Mycobacterium tuberculosis was investigated. Antibacterial activity was measured against Escherichia coli (NRRL B-3704), Staphylococcus aureus (NRRL B-767), Salmonella typhimurium (NRRL B-4420), Proteus vulgaris (NRLL B-123), Enterococcus faecalis (isolated obtained from Faculty of Medicine Osmangazi University, Eskisehir, Turkey), Pseudomonas aeruginosa (NRRL B-23 27853), Klebsiella spp. (isolated obtained from Faculty of Medicine Osmangazi University, Eskisehir, Turkey), while antifungal activity was evaluated against Candida albicans (isolates obtained from Osmangazi Uni. Fac.of Medicine), Candida glabrata (isolates obtained from Osmangazi Uni. Fac.of Medicine). Compounds were also evaluated for antituberculosis activity against Mycobacterium tuberculosis H₃₇Rv using the BACTEC 460 radiometric system and BACTEC 12B medium. The compounds showed moderate inhibitor effects against human pathogenic microorganisms., whereas the preliminary results indicated that all of the tested compounds were inactive against Mycobacterium tuberculosis H₃₇Rv.     

Diversity of Oleic Acid,Ricinoleic Acid and Linoleic Acid Conversions Among <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Strains*

Sixteen Pseudomonas aeruginosa strains, including patent strain NRRL B-18602, three recent isolates from composted materials amended with ricinoleic acid, and 12 randomly selected from the holdings of the ARS Culture Collection, were examined for their fatty acid converting abilities. The study examined the bioconversion of oleic acid to 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) and ricinoleic acid to 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD). A new DOD-like compound from linoleic acid was observed. All strains except NRRL B-247 exhibited varying levels of DOD production. NRRL B-1000, NRRL B-18602 and NRRL B-23258 with yields up to 84% were among the best DOD producers. TOD production generally paralleled DOD production at a relatively lower yield of up to 15%. Strains NRRL B-1000 and NRRL B-23260 were the best TOD producers. A DOD-like product in low yields was obtained from linoleic acid. The fatty acid bioconversion capability was related neither to growth rate nor to variation in the greenish pigmentation of the strains. Production of significant quantities of DOD and TOD from oleic and ricinoleic acids, respectively, appeared to be a characteristic trait of P. aeruginosa strains. A number of highly effective strains for DOD production were identified.     

Principal component analysis applied to bacterial cell behaviour in the presence of organic solvents

The behaviour of cells of Rhodococcus erythropolis DCL14, Xanthobacter Py2, Arthrobacter simplex and Mycobacterium sp. NRRL B-3805, in biphasic systems containing different organic solvents was evaluated and compared. The data, obtained mainly by fluorescence microscopy and image analysis, was interpreted using principal components analysis (PCA). With this technique, the variability of the data could be summarised in 7 components, representing 75.8% of the variance of the data. Over a third of the variance could be explained by the first two principal components which represent solvent toxicity. Apparently this is the major factor influencing cell behaviour in an organic:aqueous system. However, factors such as substrate concentration, cell adaptation ability (resulting in morphological changes and aggregation or separation of cells) and membrane composition (specific to each strain) also play an important role in cell resistance to solvent toxicity. The results regarding cell shape indicate that loss of viability occurs, in the tested bacterial strains, after incorporation of molecules of solvent in the cellular membrane. This should result in an increase in membrane fluidity, and thus, in an alteration of cell shape. The ability to form “self-defence” clusters was observed to be different amongst the four strains. X. Py2 showed, in general, a low tendency to form aggregates under the tested conditions; A. simplex and R. erythropolis aggregated mainly in the presence of low log P solvents; and Mycobacterium. sp. cells showed a high ability to aggregate.