Biotransformation of aromatic compounds Monitoring fluorinated analogues by NMR

The results presented here illustrate the power of NMR in the non-invasive analysis of microbial transformations. Whilst the definitive identification of products requires purification and full structural elucidation, NMR can provide rapid insights into the nature of these reactions and their regulation in vivo. In addition, once the products have been identified NMR methods allow rapid assessment of the effects of genetic and physiological manipulation, and on competing metabolic fluxes with mixed substrates and branched pathways.     

Biotransformation of N-substituted aromatic compounds in mammalian spermatozoa. Nonoxidative formation of N-hydroxy-N-arylacetamides from nitroso aromatic compounds

The metabolism of N-substituted aromatic compounds, i.e. aniline, acetanilide, N-hydroxyacetanilide, nitrosobenzene, and nitrobenzene in mammalian spermatozoa was investigated. In boar spermatozoa fortified with glucose, no acetylation, deacetylation, and monooxygenation of these compounds were found. Nitrobenzene was reduced slowly, but nitrosobenzene was a good substrate for this reductive activity. In the latter reaction, the products were N-hydroxyacetanilide, azoxybenzene, and an organic phase-nonextractable metabolite(s). Pyruvate was found to be involved in the formation of N-hydroxyacetanilide from nitrosobenzene, and the reaction occurred through a ping-pong mechanism. This enzymatic activity, located in the mid-piece fraction of spermatozoa, was enhanced by thiamine pyrophosphate and Mg2+ and inhibited by thiamine thiazolone pyrophosphate. N-Hydroxyacetanilide formed from [3(-13)C]pyruvate showed complete retention of the isotope at the methyl carbon of the molecule. 2-Nitrosofluorene and 4-nitrosobiphenyl were also transformed into the corresponding N-hydroxy-N-arylacetamides. N-Hydroxyacetanilide was also formed in rat and human spermatozoa. These facts suggest that the formation of N-hydroxy-N-arylacetamides from the nitroso aromatic compounds and pyruvate is mediated by a pyruvate dehydrogenase complex located in the mitochondria of spermatozoa. The formation of both azoxybenzene and the organic phase-nonextractable metabolite(s) was found to be a pyruvate-independent nonenzymatic reaction.     

Synthesis of regioselectively substituted cellulose derivatives and applications in chiral chromatography

Various cellulose-2,3-bis-arylcarbamate-6-O-arylesters and cellulose-2,3-bis-arylester-6-O-arylcarbamates, designed to test the possible combined effects of the known tris-arylcarbamate and tris-arylester classes, were synthesized with high regioselectivity at O-C(6), and their use as CSP s in liquid chromatography for enantiomeric separations was investigated. The separations obtained with the synthesized CSP s were compared to the separations achieved on a self-packed reference column, consisting of cellulose-tris-(3,5-dimethylphenyl-carbamate) as CSP standard. Among the synthesized, regioselectively substituted cellulose derivatives, 2,3-bis-O-(3,5-dimethylphenylcarbamate)-6-O-benzoate-cellulose and 2,3-bis-O-(benzoate)-6-O-(3,5-dichlorophenylcarbamate)-cellulose gave the best CSP s for the separation of the test racemates. CSP s from regioselectively substituted cellulose derivatives seem to exhibit higher selectivities than cellulose-tris-(3,5-dimethylphenylcarbamate) for certain classes of racemic compounds. Chirality 10:294–306, 1998. © 1998 Wiley-Liss, Inc.     

Preparation and evaluation of regioselectively substituted amylose derivatives for chiral separations

Six novel regioselectively substituted amylose derivatives with a benzoate at 2‐position and two different phenylcarbamates at 3‐ and 6‐positions were synthesized and their structures were characterized by ¹H nuclear magnetic resonance (NMR) spectroscopy. Their enantioseparation abilities were then examined as chiral stationary phases (CSPs) for high‐performance liquid chromatography (HPLC) after they were coated on 3‐aminopropyl silica gels. Investigations indicated that the substituents at the 3‐ and 6‐positions played an important role in chiral recognition of these amylose 2‐benzoate serial derivatives. The derivatives demonstrated characteristic enantioseparation and some racemates were better resolved on these derivatives than on Chiralpak AD, which is one of the most efficient CSPs, utilizing coated amylose tris(3,5‐dimethylphenylcarbamate) as the chiral selector. Among the derivatives prepared, amylose 2‐benzoate‐3‐(phenylcarbamate/4‐methylphenylcarbamate)‐6‐(3,5‐dimethylphenylcarbamate) exhibited chiral recognition abilities comparable to that of Chiralpak AD and may be useful CSPs in the future. The effect of mobile phase on chiral recognition was also studied. In general, with the decreased concentration of 2‐propanol, better resolutions were obtained with longer retention times. Moreover, when ethanol was used instead of 2‐propanol, poorer resolutions were often achieved. However, in some cases, improved enantioselectivity was achieved with ethanol rather than 2‐propanol as the mobile phase modifier.     

Genetic engineering of Pseudomonas putida KT2442 for biotransformation of aromatic compounds to chiral cis-diols

Toluene dioxygenase (TDO) catalyzes asymmetric cis-dihydroxylations of aromatic compounds. Pseudomonas putida KT2442 (pSPM01) harboring TDO genes could effectively biotransform a wide-range of aromatic substrates into their cis-diols products. In shake-flask culture, approximately 2.7gl(-1) benzene cis-diols, 8.8gl(-1) toluene cis-diols and 6.0gl(-1) chlorobenzene cis-diols were obtained from the biotransformation process. Furthermore, vgb gene encoding Vitreoscilla hemoglobin protein (VHb) which enhances oxygen microbial utilization rate under low dissolved oxygen concentration was integrated into P. putida KT2442 genome. The oxidation ability of the mutant strain P. putida KTOY02 (pSPM01) harboring TDO gene was increased in the presence of VHb protein. As a result, approximately 3.8, 15.1 or 6.8gl(-1) different cis-diols production was achieved in P. putida KTOY02 (pSPM01) grown in shake-flasks when benzene, toluene or chlorobenzene was used as the substrate. The above results indicate that P. putida KT2442 could be used as a cell factory to biotransform aromatic compounds.     

Biotransformation of halogenated compounds

As a result of natural production and contamination of the environment by xenobiotic compounds, halogenated substances are widely distributed in the biosphere. Concern arises as a result of the toxic, carcinogenic, and potential teratogenic nature of these substances. The biotransformations of such halogenated substances are reviewed, with particular emphasis on the biocatalytic cleavage of the carbon-halogen bonds. The physiology, biochemistry, and genetics of the biological system involved in the dehalogenation reactions are discussed for three groups of organohalogens: (1) the haloacids, (2) the haloaromatics, and (3) the haloalkanes. Finally, the biotechnological applications of these microbial transformations are discussed. This includes prospects for their future application in biosynthetic processes for the synthesis of halogenated intermediates or novel compounds and also the use of such systems for the detoxification and degradation of environmental pollutants.     

Mutagenicity of nitro derivatives induced by exposure of aromatic compounds to nitrogen dioxide

Mutagenic nitro derivatives were readily induced when 6 kinds of chemicals were exposed to 10 ppm of nitrogen dioxide (NO2). Single nitro derivatives were formed from pyrene, phenanthrene, fluorene or chrysene. Carbazole and fluoranthene each produced 2 derivatives substituted with nitro groups at different positions. The formation of nitro derivatives was enhanced by exposure of pyrene to NO2 containing nitric acid (HNO3, less than 100-fold enhancement) or sulphur dioxide (SO2, less than 15-fold enhancement). After 24 h of exposure the yields of the nitro derivative were 0.02% with 1 ppm of NO2 in air and 2.85% with NO2 (1 ppm) containing traces of HNO3. The nitro derivatives from all but phenanthrene and carbazole were chemically identified by means of gas chromatography (GC) and mass spectrometry (MS), and the mutagenicity of the 4 kinds of authentic nitro derivatives was tested by using Salmonella strains TA98 and TA1538 with or without the S9 fraction from rat liver treated with Aroclor 1254. The nitro derivative induced from pyrene was determined to be 1-nitropyrene; that of chrysene was 6-nitrochrysene; that of fluorene was 2-nitrofluorene; and those of fluoranthene were 3-nitrofluoranthene, and 8-nitrofluoranthene. Tested with strain TA98 in the absence of the S9 fraction, the first 4 of these derivatives yielded, respectively, 3050, 269, 433 and 13 400 revertants per nmole. Thus, each nitro derivative formed was potentially a direct-acting frameshift-type mutagen. Each compound exposed to NO2 showed a decreased mutagenic activity when tested in the presence of S9 mix. A possible explanation comes from experiments in which 1-nitropyrene was incubated with the S9 mix at 37 degree C for 10 min, and 1-aminopyrene was formed. The mutagenic activity of 1-aminopyrene was appreciable, but only about one-tenth of that of 1-nitropyrene in the Ames test.     

Biotransformation of prednisolone to hydroxy derivatives by Penicillium aurantiacum

Prednisolone, a synthetic adrenal corticosteroid drug, is known to have anti-inflammatory and autoimmune activity. Biotransformation of prednisolone was carried out to obtain more bioactive prednisolone derivatives. Among six different fungi, Penicillium aurantiacum proved to be the best prednisolone hydroxylator. As a result of prednisolone biotransformation by P. aurantiacum, whole cells four different prednisolone derivatives were investigated. 20β-Hydroxyprednisolone (1) and 21,21-dimethoxy-11β-hydroxypregn-1,4-dien-3,20-dione (2) were detected as the main metabolites. These metabolites together with other two metabolites, 11β-hydroxyandrost-1,4-dien-3,17-dione (3) and 11β,17β-dihydroxyandrost-1,4-dien-3- one (4), were purified and assigned by an interpretation of their spectral data using mass spectroscopy (MS), proton nuclear magnetic resonance (¹H-NMR), carbon nuclear magnetic resonance (¹³C-NMR) and infrared spectroscopy (IR) analyses. The best fermentation conditions for production of compounds 1–4 were as follows: medium (3) consisting of (g/l): glucose 20; l-asparagine 0.7; MgSO₄.7H₂O 0.5; KH₂PO₄ 1.52; KCl 0.52; Cu (NO₃)₂ traces; ZnSO₄.7H₂O traces, supplemented with prednisolone concentration of 0.3?mg/ml, inoculated by 10% of microorganism and incubated for 72?h. Under these optimized conditions, ～94.8% of the added prednisolone was converted to aforementioned derivatives, which have the potential to be used in industrial production of important pharmaceutical compounds.     

Binding of aromatic compounds to cornstarch polysaccharides

Sorption of aromatic compounds from aqueous solutions by cryotextures and suspensions of native cornstarches was studied by capillary gas chromatography. Acetophenone and benzyl alcohol were not sorbed by cryotropic-cornstarch gel and native-cornstarch suspension. A linear concentration dependence was found for aldehydes. Phenylethyl alcohol was characterized by a nonlinear concentration dependence. The presence of a benzene ring contributed to decreased binding (relative to the level characteristic of aliphatic compounds). The degree of binding depended considerably on the type of functional group in the aromatic compounds. Cryotextures were more potent than granules of native cornstarch in binding aromatic compounds.     

Binding of aromatic compounds to cornstarch polysaccharides

Sorption of aromatic compounds from aqueous solutions by cryotextures and suspensions of native cornstarches was studied by capillary gas chromatography. Acetophenone and benzyl alcohol were not sorbed by cryotropic-cornstarch gel and native-cornstarch suspension. A linear concentration dependence was found for aldehydes. Phenylethyl alcohol was characterized by a nonlinear concentration dependence. The presence of a benzene ring contributed to decreased binding (relative to the level characteristic of aliphatic compounds). The degree of binding depended considerably on the type of functional group in the aromatic compounds. Cryotextures were more potent than granules of native cornstarch in binding aromatic compounds.     

A para-site-specific hydroxylation of various aromatic compounds by Mycobacterium sp. strain 12523

A screening of microorganisms with para-site-specific hydroxylation activity of aromatic compounds has been carried out by a three-step screening procedure involving a newly established micro-plate assay method. About 1300 strains isolated from about 5000 soils showed the activity. The hydroxylation of aromatic compounds by one of the isolates, designated Mycobacterium sp. strain 12523, which had the highest activity, was examined in detail. It produced p-phenols with high selectivity without o-or m-site hydroxylation. The yield of hydroquinone from phenol by strain 12523 was 97 mol%.     

Degradation of polycyclic aromatic hydrocarbon compounds under various redox conditions in soil-water systems

This study evaluated the microbial degradation of naphthol, naphthalene, and acenaphthene, under aerobic, anaerobic, and denitrification conditions in soil-water systems. Chemical degradation of naphthol and naphthalene in the presence of a manganese oxide was also studied. Naphthol, naphthalene, and acenaphthene were degraded microbially under aerobic conditions from initial aqueous-phase concentrations of 9, 7, and 1 mg/liter to nondetectable levels in 3, 10, and 10 days, respectively. Under anaerobic conditions naphthol degraded to nondetectable levels in 15 days, whereas naphthalene and acenaphthene showed no significant degradation over periods of 50 and 70 days, respectively. Under denitrification conditions naphthol, naphthalene, and acenaphthene were degraded from initial aqueous-phase concentrations of 8, 7, and 0.4 mg/liter to nondetectable levels in 16, 45, and 40 days, respectively. Acclimation periods of approximately 2 days under aerobic conditions and 2 weeks under denitrification conditions were observed for both naphthalene and acenaphthene. Abiotic degradation of naphthalen and naphthol were evaluated by reaction with manganese oxide, a minor soil constituent. In the presence of a manganese oxide, naphthalene showed no abiotic degradation over a period of 9 weeks, whereas the aqueous naphthol concentration decreased from 9 mg/liter to nondetectable levels in 9 days. The results of this study show that low-molecular-weight, unsubstituted, polycyclic aromatic hydrocarbons are amenable to microbial degradation in soil-water systems under denitrification conditions.     

Biotransformation of indole derivatives by mycelial cultures

Biotransformation of tryptophan to tryptamine and 3-methyl-indole by Psilocybe coprophila was performed. On the other hand, Aspergillus niger was able to transform tryptophan to 5-hydroxy-tryptophan. P. coprophila biotransformed 5-hydroxy-tryptophan to 5-hydroxytryptamine. These results prove once more that fungi are good tools to establish hydroxyindole derivatives.     

Biotransformation of phenolic compounds by Bacillus aryabhattai

Bioprocess and Biosystems Engineering - Phenolic compounds could pose environmental problems if they are in excess, although they could be a renewable resource of substances with industrial...     

Differential expression of enzyme activities initiating anoxic metabolism of various aromatic compounds via benzoyl-CoA

The regulation of the expression of enzyme activities catalyzing initial reactions in the anoxic metabolism of various aromatic compounds was studied at the whole cell level in the denitrifying Pseudomonas strain K 172. The specific enzyme activities were determined after growth on six different aromatic substrates (phenol, 4-hydroxybenzoate, benzoate, p-cresol, phenylacetate, 4-hydroxyphenylacetate) all being proposed to be metabolized anaerobically via benzoyl-CoA. As a control cells were grown on acetate, or aerobically on benzoate. The expression of the following enzyme activities was determined.Phenol carboxylase, as studied by the isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate; 4-hydroxybenzoyl-CoA reductase (dehydroxylating); p-cresol methylhydroxylase; 4-hydroxybenzyl alcohol dehydrogenase; 4-hydroxybenzaldehyde dehydrogenase; coenzymeA ligases for the aromatic acids benzoate, 4-hydroxybenzoate, phenylacetate, and 4-hydroxyphenylacetate; phenylglyoxylate: acceptor oxidoreductase and 4-hydroxyphenylglyoxylate: acceptor oxidoreductase; aromatic alcohol and aldehyde dehydrogenases.The formation of most active enzymes is strictly regulated; they were only induced when required, the basic activities being almost zero. The observed whole cell regulation pattern supports the postulate that the enzyme activities play a role in anoxic aromatic metabolism and that the compounds are degraded via the following intermediates: Phenol 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; benzoate benzoyl-CoA; p-cresol 4-hydroxybenzaldehyde 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; phenylacetate phenylacetyl-CoA phenylglyoxylate benzoyl-CoA plus CO₂; 4-hydroxyphenylacetate 4-hydroxyphenylacetyl-CoA 4-hydroxyphenylglyoxylate 4-hydroxybenzoyl-CoA plus CO₂ benzoyl-CoA.     

Degradation of polycyclic aromatic hydrocarbon compounds under various redox conditions in soil-water systems.

This study evaluated the microbial degradation of naphthol, naphthalene, and acenaphthene, under aerobic, anaerobic, and denitrification conditions in soil-water systems. Chemical degradation of naphthol and naphthalene in the presence of a manganese oxide was also studied. Naphthol, naphthalene, and acenaphthene were degraded microbially under aerobic conditions from initial aqueous-phase concentrations of 9, 7, and 1 mg/liter to nondetectable levels in 3, 10, and 10 days, respectively. Under anaerobic conditions naphthol degraded to nondetectable levels in 15 days, whereas naphthalene and acenaphthene showed no significant degradation over periods of 50 and 70 days, respectively. Under denitrification conditions naphthol, naphthalene, and acenaphthene were degraded from initial aqueous-phase concentrations of 8, 7, and 0.4 mg/liter to nondetectable levels in 16, 45, and 40 days, respectively. Acclimation periods of approximately 2 days under aerobic conditions and 2 weeks under denitrification conditions were observed for both naphthalene and acenaphthene. Abiotic degradation of naphthalen and naphthol were evaluated by reaction with manganese oxide, a minor soil constituent. In the presence of a manganese oxide, naphthalene showed no abiotic degradation over a period of 9 weeks, whereas the aqueous naphthol concentration decreased from 9 mg/liter to nondetectable levels in 9 days. The results of this study show that low-molecular-weight, unsubstituted, polycyclic aromatic hydrocarbons are amenable to microbial degradation in soil-water systems under denitrification conditions.