Production Of Catechol Research Articles

Isolation of a benzoate-utilizing Pseudomonas strain from soil and production of catechol from benzoate by transpositional mutants

Pseudomonas sp. Ba-0511 was isolated from soil by enrichment cultivation on a medium containing 6 mg/ml of sodium benzoate. The bacterium could grow on a medium containing 20 mg/ml of sodium benzoate by a successive enrichment culture. One hundred and twelve transpositional mutants of the bacterium produced catechol from benzoate and accumulated it outside of the cells. Among the mutants, strain BA+63 produced a maximal amount of catechol (2.3 mg/ml) from 6 mg/ml of sodium benzoate after growing for 10.5 h. The conversion rate of benzoate to catechol was 50% on a molar basis. The catechol production by the resting cells increased in the presence of glycerol, and the maximal amount of catechol produced from 6 mg/ml of sodium benzoate reached 3.3 mg/ml at the conversion rate of 72% after 5 h of incubation. The resting cells converted m-methylbenzoic acid to 3- and 4-methylcatechol and m-chlorobenzoic acid to 3- and 4-chlorocatechol.

Production of catechol from benzoate by the wild strain Ralstonia species Ba-0323 and characterization of its catechol 1,2-dioxygenase.

Ralstonia sp. Ba-0323, a wild strain isolated from soil, produced catechol from benzoate and accumulated it outside the cells. The bacterium produced a maximal amount of catechol (1.6 mg/ml) from 3 mg/ml of sodium benzoate in a 20-h growing culture. The conversion rate of benzoate to catechol was 70% on a molar basis. The catechol production by the resting cells increased in the presence of glycerol, and the maximal amount of catechol produced from 3 mg/ml of sodium benzoate reached 1.9 mg/ml at the conversion rate of 83% after 8 h of incubation. Catechol 1,2-dioxygenase, which catalyzed the ring cleavage of catechol, was purified to homogeneity from a cell extract of Ralstonia sp. Ba-0323 growing on benzoate and characterized. The specific activity of the purified enzyme was much lower than those of the dioxygenases from other microorganisms reported. The Km for catechol of the purified enzyme was much higher than those of other dioxygenases. In addition, the NH2-terminal amino acid sequence of the enzyme was less similar to the other catechol 1,2-dioxygenases than they are to each other.

Differences in host use efficiency of larvae of a generalist moth, Operophtera brumata on three chemically divergent Salix species.

The food selection, growth, and fecundity of insect herbivores are largely dictated by the chemical composition and nutritive values of plant foliage. We studied the host-use efficiency of larvae of the generalist moth, Operophtera brumata (Lepidoptera: Geometridae) on three chemically divergent but nutritively similar willows (Salix spp.). The 4th instars were able to use the salicylate-free leaves of S. phylicifolia efficiently. Growth was slightly reduced on S. pentandra, which contained a moderate level of acetylated salicylates. The high concentration of salicylates found in the leaves of S. myrsinifolia seemed to provide efficient protection against non-specialized O. brumata. We also studied assimilation of nutrients and degradation of salicylates and other secondary compounds in the digestive tract of O. brumata larvae. Neither the assimilation of nitrogen nor of carbon were affected by secondary chemicals of ingested food. Salicylates were shown to be degraded to salicin and catechol, while further degradation of salicin to saligenin was rather slow. In an artificial diet experiment, we showed that two degradation products of salicylates, catechol and saligenin markedly reduced the growth of the larvae. Neither salicin nor chlorogenic acid affected larval growth. We conclude that salicylates reduced the growth of the generalist winter moth mainly by feeding deterrence caused by 6-hydroxy-2-cyclohexenone and catechol. Compared to the deleterious effects of salicylates the effects of other secondary compounds were minor.

Degradation of dibenzofuran by Pseudomonas putida Ph-01

The ability of a Pseudomonas putida PH-01 to degrade the dioxin-like compound dibenzofuran was characterized and the effect of extracellular substance produced during the incubation was determined. The strain grew aerobically on dibenzofuran as the sole source of carbon and energy, and the solubility of dibenzofuran was rapidly increased with the appearance of those extracellular substances. During the incubation, production of salicylic acid, catechol, gentisic acid, and other unidentified trace metabolic products was observed. After six days of incubation, catechol accumulated mainly in the culture medium.

Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells

3,4-Dihydroxyphenylethanol (hydroxytyrosol; DPE) is the major phenolic antioxidant present in extra virgin olive oil, either in a free or esterified form. Despite its relevant biological effects, no data are available on its bioavailability and metabolism. The aim of the present study is to examine the molecular mechanism of DPE intestinal transport, using differentiated Caco-2 cell monolayers as the model system. The kinetic data demonstrate that [ 14C]DPE transport occurs via a passive diffusion mechanism and is bidirectional; the calculated apparent permeability coefficient indicates that the molecule is quantitatively absorbed at the intestinal level. The only labelled DPE metabolite detectable in the culture medium by HPLC (10% conversion) is 3-hydroxy-4-methoxyphenylethanol, the product of catechol- O-methyltransferase; when DPE is assayed in vitro with the purified enzyme a K m value of 40 μM has been calculated.

A Commentary on Experiments with Tyrosinase

Research Article| September 01 1999 A Commentary on Experiments with Tyrosinase Rob L. Dean Rob L. Dean Search for other works by this author on: This Site PubMed Google Scholar The American Biology Teacher (1999) 61 (7): 523–527. https://doi.org/10.2307/4450755 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Rob L. Dean; A Commentary on Experiments with Tyrosinase. The American Biology Teacher 1 September 1999; 61 (7): 523–527. doi: https://doi.org/10.2307/4450755 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu nav search search input Search input auto suggest search filter All ContentThe American Biology Teacher Search This content is only available via PDF. Copyright The National Association of Biology Teachers Article PDF first page preview Close Modal You do not currently have access to this content.

Rapid evaluation of biocidal activity using a transposon-encoded catechol 2,3-dioxygenase from Pseudomonas putida.

Pseudomonas putida (UWC1), containing a genetically-engineered plasmid (pQM899), that encodes for the production of catechol 2,3-dioxygenase (C230), was used as a potential means of rapidly estimating bactericidal activity of chlorhexidine diacetate (CHA), phenol, cetylpyridinium chloride (CPC) and phenylmercuric nitrate (PMN). Enzyme C230 converts catechol to 2-hydroxymuconic semialdehyde (2-HMS), which is yellow in colour, via a meta cleavage pathway. Ideal conditions for production and measurement spectrophotometrically of 2-HMS were determined. However, the correlation between this method and viable plate counts was not sufficiently accurate to enable 2-HMS production to provide a sufficiently sensitive determination of biocidal activity. An alternative method, synchronous scanning fluorimetry, in which the decrease in catechol concentration was measured under standardized conditions, provided a good dose-response histogram for all the biocides tested. Although, in comparison with plate counts, there was an underestimation of the bactericidal effects of phenol an PMN, the results of this study suggest that this method has potential in determining the bactericidal efficacy of agents such as CHA and CPC.

An integrated process for the production of toxic catechols from toxic phenols based on a designer biocatalyst.

We describe the biocatalytic production of 3-phenylcatechol from 2-phenylphenol with the whole cell biocatalyst Escherichia coli JM101 (pHBP461). The recombinant produces 2-hydroxybiphenyl 3-monooxygenase, an enzyme from Pseudomonas azelaica HBP1. This enzyme introduces a hydroxyl-group at the C3-position of a variety of 2-substituted phenols, such as 2-phenylphenol. This permits the biocatalytic production of 3-substituted catechols, which are difficult to synthesize chemically. Both 2-phenylphenol and 3-phenylcatechol are highly toxic to E. coli. The toxic effects of 2-phenylphenol were minimized by feeding this substrate to the reactor at a rate slightly below the maximum biooxidation rate. As a result, the substrate concentration in the reactor remained below toxic levels during the bioconversion. The toxic product formed was removed by continuous adsorption on the solid resin Amberlite XAD-4. To this end the reaction mixture, containing the biocatalyst, was pumped continuously through an external loop with a fluidized bed of the resin. This resin efficiently and quantitatively adsorbed both 3-phenylcatechol and the remaining trace amounts of 2-phenylphenol. Consequently, the concentrations of these compounds were kept at subtoxic levels (below 100 mg L-1) and gram amounts of 3-phenylcatechol were produced with space-time yields of up to 0.39 g L-1 h-1. The product was recovered from the resin by acidic methanol elution and purified by recrystallization from n-hexane resulting in overall yields exceeding 59%. The optimized system served as a surprisingly simple and efficient integrated process, that allows the bioconversion of toxic substrates to toxic products with whole cell biocatalysts.

Biotransformation of Phenol to Catechol by Recombinant Phenol Hydroxylase

The phenol hydroxylase gene from Pseudomonas putida NCIB10015 was cloned and expressed in a high-copy number plasmid in E. coli DH5α. Expression of the gene was found to be positively regulated. Induction of the phenol hydroxylase activity was shown to be maximal after 3 h of culture. Phenol and cresol isomers were shown to be good inducers of the recombinant phenol hydroxylase and, to a lesser extent, other aromatic compounds such as catechol, 2-amino-phenol and 2-chlorophenol were also inducers of gene expression. In optimal culture conditions, catechol was produced at concentrations up to 4.1mM in cultures of E. coli DH5α carrying the recombinant phenol hydroxylase gene. Phenol hydroxylase activity was strongly inhibited in vivo by high levels of both phenol and catechol. Catechol stability in the culture medium was strictly dependent on the pH of the medium, being optimal at pH 6.0. Several membrane-permeabilizing compounds were tested for their effect on catechol production, although none of them showed a positive effect on the process.

Preparative scale production of 3-substituted catechols using a novel monooxygenase from Pseudomonas azelaica HBP 1

Pseudomonas azelaica HBP 1 contains a 2-hydroxybiphenyl 3-monooxygenase (E.C. 1.14.13.44, HbpA) which produces 3-substituted catechols from 2-substituted phenols. The tetrameric enzyme (subunit mass 60 kDa) carries FAD as a prosthetic group, uses NADH as cofactor and is homologous to a number of NADPH dependent flavin containing phenol monooxygenases. HbpA regio-selectively oxidizes 2-substituted phenols and has a broad substrate-range. As ortho-substituent it accepts various alkyl-residues, halogen atoms and substituted phenyl-residues. We have used this monooxygenase for the production of 3-substituted catechols, which are useful synthons that are difficult to obtain by chemical means. A recombinant E. coli JM101, containing the hbpA gene, was used for whole-cell biotransformations. Since the phenols as well as the corresponding catechols produced are toxic to cells, a process with a limited feed of starting-material combined with in situ product recovery was used. 3-Phenyl-, 3-chloro-, 3-bromo-, 3-ethyl-, 3-propyl-, 3- i-propyl-, and 3- sec-butyl-catechol could thus be produced in gram amounts with space-time yields up to 0.45 g l −1h −1. The products were recovered and further purified by recrystallization. Authenticity of all products, some of which had not been described before, was verified by 1 H NMR, 13 C NMR, IR spectroscopy and GC/MS.

Purification and characterization of catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 and cloning and sequencing of its catA gene.

A method was developed for the purification of catechol 1, 2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 that had been grown in the presence of benzyl alcohol. The enzyme has very similar apparent Km (1-2 microM) and Vmax (13-19 units/mg of protein) values for the intradiol cleavage of catechol, 3-methylcatechol and 4-methylcatechol and it is optimally active at pH9. Cross-linking studies indicate that the enzyme is a homodimer. It contains 0.6 atoms of Fe per subunit. The enzyme was crystallized with 15% (w/v) poly(ethylene glycol) 4000/0.33 M CaCl2/25 mM Tris (pH7.5) by using a microseeding technique. Preliminary X-ray characterization showed that the crystals are in space group C2 with unit-cell dimensions a=111.9 A, b=78.1 A, c=134.6 A, beta=100 degrees. An oligonucleotide probe, made by hemi-nested PCR, was used to clone the gene encoding catechol 1,2-dioxygenase (catA). The deduced 282-residue sequence corresponds to a protein of molecular mass 31539 Da, close to the molecular mass of 31558 Da obtained by electrospray MS of the purified enzyme. catA was subcloned into the expression vector pTB361, allowing the production of catechol 1,2-dioxygenase to approx. 40% of the total cellular protein. The deduced amino acid sequence of the enzyme has 56% and 75% identity with the catechol 1, 2-dioxygenases of Arthrobacter mA3 and Rhodococcus erythropolis AN-13 respectively, but less than 35% identity with intradiol catechol and chlorocatechol dioxygenases of Gram-negative bacteria.

Flow injection amperometric detection of catechol using dual-band poly(3-methylthiophene) electrodes

Abstract This work reports the electrochemical detection of catechol (10 −4 to 10 −6 M) in the presence of ascorbic acid (10 −4 to 10 −6 M) using dual-band poly(3-methylthiophene) (P3MT) electrode flow-injection amperometry without prior separation. The selectivity involved in this method is based on the differences in electrochemical behavior of catechol and ascorbic acid. A variety of dual-band Pt, Au, glassy carbon, and P3MT electrodes were constructed and used as the electrochemical detector. The upstream electrode of the series dual-band electrode unit is used for detection of catechol and ascorbic acid and the downstream electrode for detection of the oxidized catechol. The band dimensions range from 0.1×2.5 to 1×5.5 mm with the interelectrode gaps varying from 0.1 to 0.5 mm. Although this method is effective for dilute solutions of catechol ( −5 M) in the presence of ascorbic acid ( −5 M) under both neutral and acidic conditions, it is not efficient for more concentrated mixtures (>10 −5 M) unless the measurements are made under acidic conditions such as at pH 1.6. A negative deviation from the ideal calibration curve of the oxidized catechol reduction is found in the more concentrated mixtures (>10 −5 M) at physiological pH 7.4. Charge–dipole interaction and hydrogen bonding between the oxidized products of catechol and ascorbic acid in the concentrated neutral solutions are proposed to explain the suppression of the current signals. Variation of the flow rates from 0.5 to 3.0 ml/min has no effect on the performance of the detector. The dual-band P3MT electrode has less positive oxidation potentials for catechol and ascorbic acid oxidation compared to the bare Pt, Au, and glassy carbon electrodes because of the catalytic activity of the P3MT electrode surface. Dual-band P3MT as well as glassy carbon electrodes have the best (highest) collection efficiency for catechol detection. The collection efficiency also remains constant when the interelectrode gaps vary from 0.1 to 0.5 mm.

Two nearly identical aromatic compound hydrolase genes in a strong polychlorinated biphenyl degrader, Rhodococcus sp. strain RHA1.

The two 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD) hydrolase genes, etbD1 and etbD2, were cloned from a strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, and their nucleotide sequences were determined. The etbD2 gene was located in the vicinity of bphA gene homologs and encoded an enzyme whose amino-terminal sequence was very similar to the amino-terminal sequence of the HOHD hydrolase which was purified from RHA1. Using the etbD2 gene fragment as a probe, we cloned the etbD1 gene encoding the purified HOHD hydrolase by colony hybridization. Both genes encode a product having 274 amino acid residues and containing the nucleophile motif conserved in alpha/beta hydrolase fold enzymes. The deduced amino acid sequences were quite similar to the amino acid sequences of the products of the single-ring aromatic hydrolase genes, such as dmpD, cumD, todF, and xylF, and not very similar to the amino acid sequences of the products of bphD genes from PCB degraders, including RHA1. The two HOHD hydrolase genes and the RHA1 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HPDA) hydrolase gene, bphD, were expressed in Escherichia coli, and their relative enzymatic activities were examined. The product of bphD was very specific to HPDA, and the products of etbD1 and etbD2 were specific to HOHD. All of the gene products exhibited poor activities against the meta-cleavage product of catechol. These results agreed with the results obtained for BphD and EtbD1 hydrolases purified from RHA1. The three hydrolase genes exhibited similar induction patterns both in an RNA slot blot hybridization analysis and in a reporter gene assay when a promoter probe vector was used. They were induced by biphenyl, ethylbenzene, benzene, toluene, and ortho-xylene. Strain RCD1, an RHA1 mutant strain lacking both the bphD gene and the etbD2 gene, grew well on ethylbenzene. This result suggested that the etbD1 gene product is involved in the meta-cleavage metabolic pathway of ethylbenzene.

A facile synthesis of functionalized catechols

A general route to functionalized catechols is achieved by the reaction of 3, 6-di(chloromethyl)-4, 5-dimethyl-catechol with various nucleophiles in the presence of a base. The reactions are proposed to occur by way of a dehydrochlorinated intermediate which undergoes Michael additions to give the catechol products.

Mathematical modelling and simulation of catechol production from benzoate using resting cells of Pseudomonas putida

A catechol-accumulating mutant of Pseudomonas putida was isolated using NTG ( N-methyl- N′-nitro- N-nitrosoguanidine) mutagenesis. The mutant produced catechol in a medium containing benzoic acid. The use of optimal concentrations of carbon, nitrogen, phosphate and trace elements improved catechol productivity significantly. Spontaneous feeding of energy source and benzoate at a fixed interval prevented the degradation of catechol and thus increased catechol productivity. Among various operation modes, a two-stage biotransformation using resting cells was efficient for catechol production. The mathematical models comprising catabolite repression and enzyme induction were suggested for catechol production under resting cell conditions. The model fitted the experimental data well for various reaction conditions, such as initial substrate and cell concentrations. By mathematical simulation of the system, optimum acetate and benzoate concentrations were found. Acetate concentration was the most critical variable for the performance of catechol production. The model proposed was useful in describing biotransformation under resting cell conditions.

Catechol production by O-demethylation of 2-methoxyphenol using the obligate anaerobe, Acetobacterium woodii

Acetobacterium woodii produced catechol (up to 7.84 mM) by demethylating 2-methoxyphenol during growth in the presence or absence of fructose. The highest product concentrations were obtained when 2-methoxyphenol was the sole energy source but the highest substrate conversion (97%) was obtained in fructose-limited chemostat culture. Growing cells were the most suitable form of the biocatalyst since the catalytic activity was 5-fold higher than in harvested cells.

Palladium or Platinum Exacerbates Hydroxyl Radical Mediated DNA Damage

Strand breakage of supercoiled pBR322 DNA by a Fenton system is increased in the presence of palladium (Pd) or platinum (Pt) ions. Neither Pd nor Pt ions can substitute for iron in the Fenton system. We have obtained several lines of evidence that Pd and Pt ions in the presence of a Fenton system can augment the production of OH , as monitored by a spectrophotometric method quantifying hydroxylated salicylate or by a fluorometric method quantifying catechol production. Furthermore, the promoting effect of both metal ions on OH production was substantiated by the identification of multiple hydroxylated products of salicylate [2,3-dihydroxybenzoate (A), 2,5-dihydroxybenzoate (B), and catechol (C)] using HPLC. The concentrations of A, B, and C produced in the control were 4.5, 8.0, and 2.0 μM, respectively; whereas, their respective concentrations increased to 23.6, 42.0, and 10.0 μM with the addition of Pd ions. The observed phenomenon was further confirmed by the identification of HO–DMPO spin adducts using ESR spectroscopy. Taken together, our data suggest that the mechanism of Pd or Pt ion-mediated exacerbation of DNA damage by a Fenton system is due to the promotion of OH production by these metal ions. © 1997 Elsevier Science Inc.

Degradation of 3-nitrophenol by Pseudomonas putida B2 occurs via 1,2,4-benzenetriol.

Growth of Pseudomonas putida B2 in chemostat cultures on a mixture of 3-nitrophenol and glucose induced 3-nitrophenol and 1,2,4-benzenetriol-dependent oxygen uptake activities. Anaerobic incubations of cell suspensions with 3-nitrophenol resulted in complete conversions of the substrate to ammonia and 1,2,4-benzenetriol. This indicates that P. putida B2 degrades 3-nitrophenol via 1,2,4-benzenetriol, via a pathway involving a hydroxylaminolyase. Involvement of this pathway in nitroaromatic metabolism has previously only been found for degradation of 4-nitrobenzoate. Reduction of 3 nitrophenol by cell-free extracts was strictly NADPH-dependent. Attempts to purify the enzymes responsible for 3-nitrophenol metabolism were unsuccessful, because their activities were extremely unstable. 3-Nitrophenol reductase was therefore characterized in cell-free extracts. The enzyme had a sharp pH optimum at pH 7 and a temperature optimum at 25 degrees C. At 30 degrees C, reductase activity was completely destroyed within one hour, while at 0 degrees C, the activity in cell-free extracts was over 100-fold more stable. The Km values for NADPH and 3-nitrophenol were estimated at 0.17 mM and below 2 microM, respectively. The substrate specificity of the reductase activity was very broad: all 17 nitroaromatics tested were reduced by cell-free extracts. However, neither intact cells nor cell-free extracts could convert a set of synthesized hydroxylaminoaromatic compounds to the corresponding catechols and ammonia. Apparently, the hydroxylaminolyase of P. putida B2 has a very narrow substrate specificity, indicating that this organism is not a suitable biocatalyst for the industrial production of catechols from nitroaromatics.

Metal substituted aluminophosphate molecular sieves as phenol hydroxylation catalysts

Substitution of transition metals for either aluminum and/or phosphorus in the AIPO 4-11 framework is found to afford novel heterogeneous catalysts for liquid phase hydroxylation of phenol with hydrogen peroxide. AlPO 4-11 is more active than SAPO-11 and MgAPO-11 for phenol conversion to hydroquinone. Substitution of transition metal cations, such as Fe, Co and Mn significantly improves the conversion of phenol. The activity follows the order of FeAPO-11 > CoAPO-11 > FeMnAPO-11 > MnAPO-11 ≫ AlPO 4-11. FeAPO-11, FeMnAPO-11 and CoAPO-11 give similar product selectivities of about 1:1 hydroquinone (HQ) to catechol (CT) whereas MnAPO-11 favors the production of catechol. FeAPO-11 show comparable performance to TS-1 (titanium silicate with MFI topology) for phenol conversion, with TS-1 giving higher selectivities toward hydroquinone. Medium pore size CoAPO-11 was more active than larger pore CoAPO-50, -36 and -5. The external surfaces of the catalysts play a significant role in these oxidation reactions.

Role of catechol siderophore synthesis in Vibrio vulnificus virulence.

We isolated a Vibrio vulnificus TnphoA mutant that was unable to produce catechol siderophores or to acquire iron from transferrin. This mutant showed reduced virulence in an infant mouse model. The TnphoA insertion was in an open reading frame designated venB. The venB gene cloned on a plasmid restored catechol production to the mutant. The deduced amino acid sequence of venB is 41% identical to the enzyme isochorismatase of Escherichia coli (EntB), an enzyme involved in the biosynthesis of the catechol siderophore enterobactin.