Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria <Emphasis Type="Italic">Mycobacterium neoaurum,Pimelobacter simplex</Emphasis>, and <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5–15 μm) or the transformation in the presence of randomly methylated β-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The crude product ADD was obtained in 75% yield, based on phytosterol. It contained as by-products 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment—the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD—no by-products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5% AD was obtained. The yield of 9-OH-AD (m.p. 218–220°C) based on transformed phytosterol was 56%.     

Biocatalytic synthesis of pharmacology perspective stigmast-4-en-3-one using rhodococci cells

Conditions for a directed biocatalytic oxidation of β-sitosterol to pharmacologically valuable stigmast-4-en-3-one using Rhodococcus actinobacteria were determined. It was shown that palmitic acid induced the cholesterol oxidase reaction and allowed for the decrease in the bioconversion process duration from 7 to 5 days. The maximum level of stigmast-4-ene-3-one formation was achieved using an additional growth substrate n-hexadecane. With increased concentrations of β-sitosterol (up to 2 g/l) an effective target product formation (80%) was achieved in the presence of Tween-80 and β-cyclodextrin. R. erythropolis strains were 1.5–2 times more active than R. ruber strains in catalyzing the β-sitosterol biotransformation process.     

In vitro fermentation of prebiotics by <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> CFR 2194: selectivity,viability and effect of metabolites on β-glucuronidase activity

Prebiotic Fructooligosaccharides (FOS) escape metabolism in upper GI tract undergo microbial metabolism in colon and thereby influence the nature, type and number of intestinal microbiota to improve host’s health. The present study focuses on the ability of Lactobacillus plantarum CFR 2194 to utilize FOS as a selective carbon and energy source. The effect of fermentative metabolites of L. plantarum on the β-glucuronidase was also investigated. A total of 16 strains of lactobacilli were assessed for their ability to ferment oligosaccharides. L. plantarum CFR 2194, an isolate from kanjika was found to utilize FOS effectively. Lactic acid was the main metabolic end product, followed by acetic acid, butyric acid, formic acid and ethanol. The inhibitory effects of these metabolites have been confirmed through the reduction of β-glucuronidase activity. L. plantarum when co-cultured with β-glucuronidase producing E. coli, in a basal media containing FOS as an energy source, could inhibit the growth of the pathogen during the course of fermentation. The results showed that L. plantarum CFR 2194 has the ability to utilize the prebiotic FOS as a selective carbon and energy source. The organism could inhibit the growth of the pathogen which produces β-glucuronidase and lowered its activity by the metabolites of FOS which indicates the probable use of L. plantarum through dietary intervention in combating colon carcinogenesis.     

Biotransformation of β-myrcene to geraniol by a strain of Rhodococcus erythropolis isolated by selective enrichment from hop plants

The biocatalytic generation of high-value chemicals from abundant, cheap and renewable feedstocks is an area of great contemporary interest. A strain of Rhodococcus erythropolis designated MLT1 was isolated by selective enrichment from the soil surrounding hop plants, using the abundant triene β-myrcene from hops as a sole carbon source for growth. Resting cells of the organism were challenged with β-myrcene, and the major product of biotransformation was determined by mass spectrometric analysis to be the monoterpene alcohol geraniol. Controls demonstrated that the product was biogenic and that an aerobic environment was required. The ability to transform β-myrcene was shown to be restricted to cells that had been grown on this substrate as sole carbon source. Pre-incubation of cells with the cytochrome P450 inhibitors metyrapone or 1-aminobenzotriazole reduced geraniol production by 23% and 73% respectively, but reduction in activity was found not to correlate with the inhibitor concentration. A comparative analysis of insoluble and soluble cell extracts derived from cells of MLT1 grown on either β-myrcene or glucose revealed at least four proteins that were clearly overproduced in response to growth on β-myrcene. Mass spectrometric analysis of tryptic digests of three of these protein bands suggested their identities as an aldehyde dehydrogenase, an acyl-CoA dehydrogenase and a chaperone-like protein, each of which has a precedented role in hydrocarbon metabolism clusters in Rhodococcus sp. and which may therefore participate in a β-myrcene degradation pathway in this organism.     

Effects of β-lactam antibiotics, auxins, and cytokinins on shoot regeneration from callus cultures of two hybrid aspens, Populustremuloides?×?P. tremula and P. xcanescens?×?P. gradidentata

The effects of β-lactam antibiotics (penicillin, carbenicillin and cefotaxime), cytokinins, and auxins including phenylacetic acid, a β-lactam breakdown product, were evaluated during in vitro shoot morphogenesis in two hybrid aspens; P. tremuloides × P. tremula (XTTa) and P. x canescens × P. grandidentata (XCaG). Although different callus and shoot induction media were used for both hybrids, the β-lactams often engendered similar responses. At concentrations of 1,000 mg l⁻¹, carbenicillin adversely impacted shoot elongation and, to a lesser degree, shoot regeneration. Cefotaxime enhanced caulogenesis for all of the concentrations evaluated (125–500 mg l⁻¹) especially when the cytokinin thidiazuron was used for shoot induction. The shoots formed faster and in greater numbers; and the improvements were significant (α = 0.05) for both hybrids. However, hyperhydricity was more problematic when cefotaxime was included in the media. The incidence of shoot hyperhydricity for the XCaG hybrid was more than twice as great for the highest cefotaxime concentration evaluated (500 mg l⁻¹) than for the control (>90% vs. ~40%). Penicillin had an opposite effect. Hyperhydricity frequencies for the XCaG hybrid were lower when the media were supplemented with penicllin and the reductions were statistically significant at concentrations of 500–1,000 mg l⁻¹. The effects of the antibiotics were generally not reproduced by the auxins (0.1–100 μM), including phenylacetic acid, or the other potential β-lactam degradation products evaluated (e.g. phenylmalonic acid, aminopenicillanic acid). The antibiotics may have affected shoot hyperhydicity indirectly via changes in the time course of shoot regeneration.     

Anaerobic Degradation and Transformation of p-Toluidine by the Sulfate-Reducing Bacterium Desulfobacula toluolica

The ability of the strictly anaerobic sulfate-reducing bacterium Desulfobacula toluolica (strain Tol2) to cometabolically degrade p-toluidine (p-methylaniline) while using toluene as the primary source of carbon and energy has been studied. This organism has been shown to modify and degrade toluidine in dense cell suspensions when no other source of carbon and energy is added. The metabolism led to the formation of a variety of metabolites. From these metabolites a biphenyl-like compound as well as phenylacetic acid have been identified by means of HPLC/MS techniques. The probable conversion of p-toluidine to p-aminophenylacetic acid and phenylacetic acid as dead end products suggested that this organism initiates p-toluidine degradation by the carboxylation of the methyl group. If this could be validated in further experiments, it would be the first time that a toluidine was carboxylated at the methyl moiety by an anaerobic, sulfate-reducing bacterium. Received: 6 March 1998 / Accepted: 3 April 1998     

Effect on the Medium Salinity on Oil Degradation by Nocardioform Bacteria

Oil degradation by cultures of Rhodococcus erythropolisand Dietzia mariswas found to depend on the NaCl concentration in the medium. Optimal utilization of turbine oil by R. erythropolisand D. mariswas observed at 0.5 and 2 to 5% NaCl concentration, respectively. Mineral oil and a mixture of paraffins (C₁₄–C₁₈) were utilized within a broader range of the medium salinity. As shown by fluorescent microscopy, D. mariscolonies formed on the oil drop surface, whereas R. erythropoliscells penetrated the drops. The strains studied may populate various ecological niches in oil-containing ecosystems. They are promising for the development of microbial preparations for cleaning the environment from oil pollution.     

Simultaneous Biodetoxification of S, N, and O Pollutants by Engineering of a Carbazole-Degrading Gene Cassette in a Recombinant Biocatalyst

The gene cassette encoding enzymes responsible for degrading carbazole to anthranilic acid was introduced into a dibenzothiophene degrader. The resultant strain, Rhodococcus erythropolis XPDN, could simultaneously transform the model pollutants dibenzothiophene, carbazole, and dibenzofuran to nontoxic metabolites and may have an application potential for bioremediation.     

A gene <Emphasis Type="Italic">linB2</Emphasis> responsible for the conversion of β-HCH and 2,3,4,5,6-pentachlorocyclohexanol in <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. BHC-A

Commercial formulations of hexachlorocyclohexane (HCH) consist of a mixture of four isomers: α, β, γ, and δ. All four isomers are toxic and recalcitrant pollutants. β-HCH is more problematic due to its longer persistence in the environment. Sphingomonas sp. BHC-A was able to degrade not only α-, γ-, and δ-HCH but also β-HCH. To clone a gene responsible for the degradation of β-HCH, a Tn5 mutation was introduced into BHC-A, and one mutant BHC-A45 defective in β-HCH degradation was selected. Sequencing analysis showed this mutant had a Tn5 insertion at the site of one haloalkane dehalogenase gene, designated linB2. linB2 was overexpressed in Escherichia coli and the 32-kDa product LinB2 showed the conversion activity of not only β-HCH to β-2,3,4,5,6-pentachlorocyclohexanol (β-PCHL) but also β-PCHL to β-2,3,5,6-tetrachloro-1,4-cyclohexanediol.     

Anaerobic degradation of phenylacetic acid by mixed and pure cultures

Summary A phenylacetic acid-degrading mixed culture was enriched from effluent of an anaerobic reactor for the treatment of waste water from cellulose bleaching. From this consortium a phenylacetic acid-degrading pure culture, strain DSU3, was isolated and, due to its typical morphology and substrate spectrum, tentatively classified as a Desulfosarcina sp. It could grow on and degrade phenylacetic acid, cyclohexane carboxylate, cyclohexylacetate, benzoate, fumaric acid and several volatile fatty acids, while phenol, o-hydroxybenzoate, p-hydroxybenzoate and glucose were not utilized. Production of mandelic acid from phenylacetic acid by the enrichment culture and utilization of benzoate, an intermediate of the mandelic acid pathway, by strain DSU3 may presumably indicate degradation of phenylacetic acid via the mandelic acid pathway.     

Metabolic engineering for synthesis of aryl carotenoids in Rhodococcus

Rhodococcus erythropolis naturally synthesizes monocyclic carotenoids: 4-keto-γ-carotene and γ-carotene. The genes and the pathway for carotenoid synthesis in R. erythropolis were previously described. We heterologously expressed a β-carotene desaturase gene (crtU) from Brevibacterium in Rhodococcus to produce aryl carotenoids such as chlorobactene. Expression of the crtU downstream of a chloramphenicol resistance gene on pRhBR171 vector showed higher activity than expression downstream of a native 1-deoxyxylulose-5-phosphate synthase gene (dxs) on pDA71 vector. Expression of the crtU in the β-carotene ketolase (crtO) knockout Rhodococcus host produced higher purity chlorobactene than expression in the wild-type Rhodococcus host. Growth of the engineered Rhodococcus strain in eight different media showed that nutrient broth yeast extract medium supplemented with fructose gave the highest total yield of chlorobactene. This medium was used for growing the engineered Rhodococcus strain in a 10-l fermentor, and ∼18 mg of chlorobactene was produced as the almost exclusive carotenoid by fermentation.     

Biodegradation and metabolic pathway of β-chlorinated aliphatic acid in Bacillus sp. CGMCC no. 4196

In this study, a bacterial Bacillus sp. CGMCC no. 4196 was isolated from mud. This strain exhibited the ability to degrade high concentration of 3-chloropropionate (3-CPA, 120 mM) or 3-chlorobutyrate (30 mM), but not chloroacetate or 2-chloropropionate (2-CPA). The growing cells, resting cells, and cell-free extracts from this bacterium had the capability of 3-CPA degradation. The results indicated that the optimum biocatalyst for 3-CPA biodegradation was the resting cells. The 3-CPA biodegradation pathway was further studied through the metabolites and critical enzymes analysis by HPLC, LC-MS, and colorimetric method. The results demonstrated that the metabolites of 3-CPA were 3-hydroxypropionic acid (3-HP) and malonic acid semialdehyde, and the critical enzymes were 3-CPA dehalogenase and 3-HP dehydroxygenase. Thus, the mechanism of the dehalogenase-catalyzed reaction was inferred as hydrolytic dehalogenation which was coenzyme A-independent and oxygen-independent. Finally, the pathway of β-chlorinated aliphatic acid biodegradation could be concluded as follows: the β-chlorinated acid is first hydrolytically dehalogenated to the β-hydroxyl aliphatic acid, and the hydroxyl aliphatic acid is oxidized to β-carbonyl aliphatic acid by β-hydroxy aliphatic acid dehydroxygenase. It is the first report that 3-HP was produced from 3-CPA by β-chlorinated aliphatic acid dehalogenase.     

Chemosystematic investigations on phenolics from flowerheads of Central European taxa of Hieracium sensu lato (Asteraceae)

 HPLC-UV and HPLC-MS investigations of phenolic acids and flavonoids in flowerheads of 84 samples of 76 taxa belonging to 66 species of Hieracium resulted in the identification of three phenolic acids (chlorogenic acid, 3,5-dicaffeoyl quinic acid, 4,5-dicaffeoyl quinic acid) and six flavonoids (apigenin 4^′-O-β-D-glucuronide, isoetin 4^′-O-β-D-glucuronide, luteolin, luteolin 7-O-β-D-glucoside, luteolin 7-O-β-D-glucuronide, luteolin 4^′-O-β-D-glucoside). The contents of these secondary metabolites were quantified by HPLC using quercetin and cynarin as internal standards. In contrast to the previously investigated genera Leontodon and Crepis, cichoric acid and caffeoyl tartaric acid were not found in any of the investigated Hieracium taxa. Results of HPLC analyses revealed only a limited degree of qualitative variation between the different taxa, and luteolin 7-O-β-D-glucuronide and isoetin 4^′-O-β-D-glucuronide were the only compounds, which were not detectable in some of the investigated taxa. Quantitative patterns of phenolics differed markedly between particular taxa and Principal Component Analysis of the quantification results yielded separate clusters for the members of the subgenera Hieracium and Pilosella. Received January 23, 2001 Accepted October 11, 2001     

Characterization, Production, and Purification of Leucocin H, a Two-Peptide Bacteriocin from Leuconostoc MF215B

Leuconostoc MF215B was found to produce a two-peptide bacteriocin referred to as leucocin H. The two peptides were termed leucocin Hα and leucocin Hβ. When acting together, they inhibit, among others, Listeria monocytogenes, Bacillus cereus, and Clostridium perfringens. Production of leucocin H in growth medium takes place at temperatures down to 6°C and at pH below 7. The highest activity of leucocin H in growth medium was demonstrated in the late exponential growth phase. The bacteriocin was purified by precipitation with ammonium sulfate, ion-exchange (SP Sepharose) and reverse phase chromatography. Upon purification, specific activity increased 10⁵-fold, and the final specific activity was 2 × 10⁷ BU/OD₂₈₀. Amino acid composition analyses of leucocin Hα and leucocin Hβ indicated that both peptides consisted of around 40 amino acid residues. Their N-termini were blocked for Edman degradation, and the methionin residues of leucocin Hβ did not respond to Cyanogen Bromide (CNBr) cleavage. Absorbance at 280 nm indicated the presence of tryptophan residues and tryptophan-fracturing opened for partial sequencing by Edman degradation. From leucocin Hα, the sequence of 20 amino acids was obtained; from leucocin Hβ the sequence of 28 amino acid residues was obtained. No sequence homology to other known bacteriocins could be demonstrated. It also appeared that the two peptides themselves shared little or no sequence homology. The presence of soy oil did not affect the activity of leucocin H in agar. Received: 10 February 1999 / Accepted: 15 March 1999     

Protein kinase A phosphorylation of the multifunctional protein CAD antagonizes activation by the MAP kinase cascade

Aβ vaccination as a therapeutic intervention of Alzheimer’s has many challenges, key among them is the regulation of inflammatory processes concomitant with excessive generation of free radicals seen during such interventions. Here we report the beneficial effects of melatonin on inflammation associated with Aβ vaccination in the central and peripheral nervous system of mice. Mice were divided into three groups (n = 8 in each): control, inflammation (IA), and melatonin-treated (IAM). The brain, liver, and spleen samples were collected after 5 days for quantitative assessment of plasma lipid peroxides (LPO), an oxidative stress marker, and antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (Gpx). IA group mice have shown the elevated concentration of LPO significantly while there was a reduction at antioxidant enzyme levels. In addition, a significant (P < 0.05) reduction in neurotransmitters like dopamine (DA), 5-hydroxytryptamine (5-HT), and norepinephrine (NE) was also observed in the IA group mice. Nevertheless, their metabolites, such as homovanillic acid (HVA) and 5-hydroxyindole acetic acid (5-HIAA) increased significantly (P < 0.05) as compared to control. Samples were further evaluated at microscopic level to examine the neuropathological changes by immunohistochemical methods. Melatonin treatment effectively reversed these above changes and normalized the LPO and antioxidant enzyme levels (P < 0.05). Furthermore, melatonin salvaged the brain cells from inflammation. Our Immunohistochemical findings in the samples of melatonin-treated animals (IAM group) indicated diminished expression of glial fibrillary acidic protein (GFAP) and nuclear factor kappa B (NfκB) than those observed in the IA group samples. Our results suggest that administration of melatonin protects inflammation associated with Aβ vaccination, through its direct and indirect actions and it can be an effective adjuvant in the development of vaccination in immunotherapy for Alzheimer’s disease (AD).     

Molecular and structural-biological analysis of <Emphasis Type="Italic">Nicotiana plumbaginifolia</Emphasis> mutants for identification of the site on their β-tubulins of interaction with dinitroanilines and phosphorothioamides

The identification of the location of a point mutation on β-tubulin molecules of amiprophosmethyland trifluralin-resistant Nicotiana plumbaginifolia lines are described in this work. It was shown that in the first case, this mutation is related with the substitution of serine residue for proline in position 248; in the second case, with the substitution of phenylalanine for serine in position 317 of the β-tubulin’s amino acid sequence. Three-dimensional models of the β-tubulin molecule from Chlamydomonas with the well-known location of mutations determining dinitroaniline- and phosphorothioamides resistance (the substitution of lysine residue for methionine in position 350), and β-tubulin from Nicotiana plumbagnifolia have been reconstructed. On the basis of the analysis of the interaction site for dinitroanilines and phosphorothioamides located on the Chlamydomonas β-tubulin’s molecule it was concluded that the revealed mutations on Nicotiana plumbaginifolia β-tubulin are affected by the residues of the amino acids, participating in the formation of this site.     

Biotransformation of β-ketosulfides to produce chiral β-hydroxysulfoxides

The biotransformations of a series of substituted phenylthio-2-propanone and benzylthio-2-propanone were carried out using Helminthosporium sp. NRRL 4671, Mortierella isabellina ATCC 42613, or Rhodococcus erythropolis IGTS8. Several products gave microbial oxidation of sulfide to sulfoxide and reduction of carbonyl to secondary alcohol, producing β-hydroxysulfoxides in medium to high enantiomeric and diastereomeric purities. Fungal biotransformations using Helminthosporium sp. and M. isabellina resulted in the opposite sulfoxide configurations of various β-hydroxysulfoxide products.     

Changes in biochemical composition of needles of ornamental dwarf spruce (Picea glauca ‘Conica’) induced by spruce spider mite (Oligonychus ununguis Jacobi, Acari: Tetranychidae) feeding

The impact of spruce spider mite (SSM) (Oligonychus ununguis Jacobi, Acari: Tetranychidae) feeding on needle compounds of young dwarf white spruce (Picea glauca ‘Conica’), important in defence against pests, was determined. It was shown that the direction and intensity of changes in chemical composition of spruce needles was related to the density of spruce spider mite population. Relative to uninfested controls, needles of trees infested by 2–6 mites per 5 cm oftwig during 8 weeks contained markedly higher concentration of soluble proteins, total phenolics and essential oil volatiles (linalool, β-phellandrene, β-myrcene, δ-3-carene, p-cymene, limonene, α-pinene, β-pinene, borneol, methyl salicylate, geranyl acetate). Feeding of a three times larger population of mites (18 specimens per 5 cm of twig) caused either reduction of concentration of those compounds, or no significant difference in comparison to the control. The results of our study show that O. ununguis at relatively low density (not exceeding 2–6 specimens per 5 cm of twig per 8 weeks) stimulates even susceptible host-plant to alter metabolism, however induced responses are suppressed as mite density increases. Findings are discussed in relation to the importance of changes in the concentration of needle primary and secondary metabolites to white spruce defence against O. ununguis.     

Isolation of phenanthrene-degrading bacteria and characterization of phenanthrene metabolites

Three aerobic bacterial consortia GY2, GS3 and GM2 were enriched from polycyclic aromatic hydrocarbon-contaminated soils with water-silicone oil biphasic systems. An aerobic bacterial strain utilizing phenanthrene as the sole carbon and energy source was isolated from bacterial consortium GY2 and identified as Sphingomonas sp. strain GY2B. Within 48 h and at 30°C the strain metabolized 99.1% of phenanthrene (100 mg/l) added to batch culture in mineral salts medium and the cell number increased by about 40-fold. Three metabolites 1-hydroxy-2-naphthoic acid, 1-naphthol and salicylic acid, were identified by gas chromatographic mass spectrometry and UV–visible spectroscopy analysis. A degradation pathway was proposed based on the identified metabolites. In addition to phenanthrene, strain GY2B could use other aromatic compounds such as naphthalene, 2-naphthol, salicylic acid, catechol, phenol, benzene and toluene as a sole source of carbon and energy.     

Efficient expression in <Emphasis Type="Italic">E. coli</Emphasis> of an enantioselective nitrile hydratase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The genes encoding an enantioselective nitrile hydratase (NHase) from Rhodococcus erythropolis AJ270 have been cloned and an active NHase has been produced in Escherichia coli. Maximal activity was found when the genes encoding the α- and β-subunits were transcribed as one unit and the gene encoding the P44k activator protein as a separate ORF on a single replicon. Addition of n-butyric acid and FeSO₄ could improve NHase activity. Coexpression of the GroEL-GroES chaperone proteins increased activity in the absence of P44k protein but had no effect in the presence of P44k. The recombinant enzyme was highly enantioselective in the synthesis of S-(+)-3-benzoyloxy- 4-cyanobutyramide from the prochiral substrate 3-benzoyloxyglutaronitrile.