Degradation of 2-chlorobenzoate by Pseudomonas cepacia 2CBS

A bacterium was isolated from water by enrichment on 2-chlorobenzoate as sole source of carbon and energy. Based on morphological and physiological properties, this microorganism was assigned to the species Pseudomonas cepacia. The organism was designated Pseudomonas cepacia 2CBS. During growth on 2-chlorobenzoate, the chlorine substituent was released quantitatively, and a small amount of 2,3-dihydroxybenzoate accumulated in the culture medium. Mutants of Pseudomonas cepacia 2CBS were induced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. Some of these mutants produced catechol from 2-chlorobenzoate. Other mutants accumulated the meta-cleavage product of catechol, 2-hydroxy-cis,cis-muconic acid semialdehyde. In crude cell-free extracts of Pseudomonas cepacia 2CBS, an enzyme was detected which catalysed the conversion of 2-chlorobenzoate to catechol. Molecular oxygen, NADH and exogenous Fe2+ were required for activity. Stoichiometric amounts of chloride were released. Experiments with 18O2 revealed that both oxygen atoms in the hydroxyl groups of the product were derived from molecular oxygen. Thus, the enzyme catalysing the conversion of 2-chlorobenzoate was identified as 2-chlorobenzoate 1,2-dioxygenase (1,2-hydroxylating, dehalogenating, decarboxylating). 2-Chlorobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS was shown to be a multicomponent enzyme system. The activities of catechol 2,3-dioxygenase and catechol 1,2-dioxygenase were detected in crude cell-free extracts. The activity of catechol 2,3-dioxygenase was 60 times higher than the activity of catechol 1,2-dioxygenase, indicating that catechol is mainly degraded via meta-cleavage in Pseudomonas cepacia 2CBS. No enzyme was found which converted 2,3-dihydroxybenzoate, suggesting that this compound is a dead-end metabolite of 2-chlorobenzoate catabolism. A pathway for the degradation of 2-chlorobenzoate by Pseudomonas cepacia 2CBS is proposed.     

Resolution of 2-nitroalcohols by Burkholderia cepacia lipase-catalyzed enantioselective acylation

Racemic 2-nitro-1-phenylethanol was resolved by via enantioselective transesterification catalyzed by Burkholderia cepacia lipase. The reaction afforded excellent E values (E > 200) and enantioselectivity (up to >99% enantiomeric excesses [ee]) of both remaining substrates and acetylated product. Moreover, the lipase displayed high enantioselectivity in the resolution of additional 2-nitroalcohols (E up to >200). This method provides an efficient alternative for obtaining enantiopure 2-nitroalcohols.     

Resolution of N-(2-ethyl-6-methylphenyl) alanine by using microgel beads containing Pseudomonas cepacia lipase

Abstract

Pseudomonas cepacia lipase (PCL) was immobilized in alginate microgel beads by electrostatic dispersion. The high electrical potential applied in the immobilization process could significantly decrease the droplet size. The optimum conditions for lipase immobilization were 2% (w/v) alginate, 100 mM CaCl₂, 8 mg/mL enzyme, 4 kV electrical potential and 200 μm mean bead size. Under these conditions, 78.2 U/g of immobilized PCL activity was obtained with 39.1% retained activity and 57.2% immobilization efficiency. The immobilized PCL (PCL-CA) was subsequently used in the enantioselective hydrolysis of (R, S)-N-(2-ethyl-6-methylphenyl) alanine methyl ester. Although PCL-CA exhibited slightly lower activity than free PCL, it preserved the high enantioselectivity (E-value >?200), which afforded enantiomerically pure (R)-acid (99% e.e._p). Furthermore, PCL-CA exhibited higher thermal stability, storage and medium stability than that of free PCL. Batch-wise operational stability studies demonstrated that PCL-CA retained its initial activity for at least 10 cycles of hydrolysis.     

Degradation of 2-methylbenzoic acid by Pseudomonas cepacia MB2.

We report the isolation of Pseudomonas cepacia MB2, believed to be the first microorganism to utilize 2-methylbenzoic acid as the sole carbon source. Its growth range included all mono- and dimethylbenzoates (with the exception of 2,5- and 2,6-dimethylbenzoates) and 3-chloro-2-methylbenzoate (but not 4- or 5-chloro-2-methylbenzoate) but not chlorobenzoates lacking a methyl group. 2-Chlorobenzoate, 3-chlorobenzoate, and 2,3-, 2,4-, and 3,4-dichlorobenzoates inhibited growth of MB2 on 2-methylbenzoate as a result of cometabolism to the corresponding chlorinated catechols which blocked the key enzyme catechol 2,3-dioxygenase. A metapyrocatechase-negative mutant, MB2-G5, showed accumulation of dimethylcatechols from 2,3- and 3,4-dimethylbenzoates, and phenols were detected in resting-cell transformation extracts bearing the same substitution pattern as the original substrate, presumably following thermal degradation of the intermediate dihydrodiol. 2-Methylphenol was also found in extracts of the mutant cells with 2-methylbenzoate. These observations suggested a major route of methylbenzoate metabolism to be dioxygenation to a carboxy-hydrodiol which then forms a catechol derivative. In addition, the methyl group of 2-methylbenzoate was oxidized to isobenzofuranone (by cells of MB2-G5) and to phthalate (by cells of a separate mutant that could not utilize phthalate, MB2-D2). This pathway also generated a chlorinated isobenzofuranone from 3-chloro-2-methylbenzoate.     

Microbial Resolution of 1-Phenoxy-2-propanol by Stereospecific Decomposition and Asymmetric Hydrolysis of 1-Phenoxy-2-propyl Acetate

We screened microorganisms for those that resolved a stereoisomer from rac-1-phenoxy-2-propyl acetate (PPAc). Several species of Nocardia and Rhodococcus hydrolyzed rac-PPAc to rac-1-phenoxy-2-propanol (PPol), and then selectively decomposed an isomer of PPol, leading to an accumulation of (R)-PPol. Several yeasts and bacteria hydrolyzed PPAc asymmetrically, affording (R)-PPol and the ester of (S)-PPol. An enzyme that catalyzed the asymmetric hydrolysis of PPAc was partially purified from Corynebacterium glutamicum ATCC 13059 and characterized. The enzymatic hydrolysis was highly specific for (R)-PPAc.     

Degradation of 2-methylbenzoic acid by Pseudomonas cepacia MB2.

We report the isolation of Pseudomonas cepacia MB2, believed to be the first microorganism to utilize 2-methylbenzoic acid as the sole carbon source. Its growth range included all mono- and dimethylbenzoates (with the exception of 2,5- and 2,6-dimethylbenzoates) and 3-chloro-2-methylbenzoate (but not 4- or 5-chloro-2-methylbenzoate) but not chlorobenzoates lacking a methyl group. 2-Chlorobenzoate, 3-chlorobenzoate, and 2,3-, 2,4-, and 3,4-dichlorobenzoates inhibited growth of MB2 on 2-methylbenzoate as a result of cometabolism to the corresponding chlorinated catechols which blocked the key enzyme catechol 2,3-dioxygenase. A metapyrocatechase-negative mutant, MB2-G5, showed accumulation of dimethylcatechols from 2,3- and 3,4-dimethylbenzoates, and phenols were detected in resting-cell transformation extracts bearing the same substitution pattern as the original substrate, presumably following thermal degradation of the intermediate dihydrodiol. 2-Methylphenol was also found in extracts of the mutant cells with 2-methylbenzoate. These observations suggested a major route of methylbenzoate metabolism to be dioxygenation to a carboxy-hydrodiol which then forms a catechol derivative. In addition, the methyl group of 2-methylbenzoate was oxidized to isobenzofuranone (by cells of MB2-G5) and to phthalate (by cells of a separate mutant that could not utilize phthalate, MB2-D2). This pathway also generated a chlorinated isobenzofuranone from 3-chloro-2-methylbenzoate.     

Degradation of diphenylether by Pseudomonas cepacia

The microbial degradation of hard coal implies the cleavage of diaryl ether linkages in the coal macromolecule. We investigated the biodegradation of diphenylether as a model compound representing this substructure of coal. A bacterial strain isolated from soil and identified as Pseudomonas cepacia, was able to grow with diphenylether as sole source of carbon. During microbial growth, three metabolites were detected in the culture supernatant by high pressure liquid chromatography. As product of ring hydroxylation and subsequent rearomatization, 2,3-dihydroxydiphenylether was identified by UV, mass and nuclear magnetic resonance spectrometry and gas chromatography analyses. The cleavage of the ether linkage led to the formation of phenol and 2-pyrone-6-carboxylic acid, the latter being not further degraded by Pseudomonas cepacia. The possible cleavage mechanism of the ether linkage is discussed.Non-standard abbreviations DPE diphenylether - PCA 2-pyrone-6-carboxylic acid - GC gas chromatography - MS mass spectrometry - HPLC high pressure liquid chromatography     

Monitoring trichloroethylene mineralization by Pseudomonas cepacia G4 PR1

To analyze the extent of mineralization of trichloroethylene (TCE) without disturbing an actively growing biofilm, a minimal growth medium was formulated that reduces the concentration of chloride ions to the extent that the chloride ions generated from TCE mineralization may be detected with a chloride-ion-specific electrode. By substituting chloride salts with phosphates and nitrates, a chloride-free minimal medium was produced that yields a specific growth rate for Pseudomonas cepacia G4 PR1 which was 93% of that in chloride-ion-containing minimal medium. Furthermore, TCE degradation by resting cell suspensions was similar in both media (85% of 75 M TCE degraded in 6 h), and complete mineralization of TCE was slightly superior in the chloride-free minimal medium (77% compared to 60% of 75 M TCE mineralized in 6 h). In addition, indole-containing, minimal-medium agar plates were developed to indicate the presence of the TCE-degrading enzyme toluene ortho-monooxygenase (fire-engine-red colonies) as well as to distinguish this enzyme from other TCE-degrading enzymes (toluene dioxygenase and toluene para-monooxygenase).     

Biotransformation of Benzyl Alcohol by Pseudomonas cepacia

To cDNAs encoding class I chitinases of rice were expressed in Escherichia coli. The cDNAs were fused to the MS2-polymerase gene in an expression vector, pEx31. The fusion proteins, expressed under the control of the λP_L-promoter, showed the chitinase activity independent of the existence of the hevein domain. The enzymatic hydrolysis of colloidal chitin by the fusion proteins showed that the proteins were endo-type enzymes.     

Production of Poly-beta-Hydroxyalkanoic Acid by Pseudomonas cepacia

The possibility of using the nutritionally versatile bacterium Pseudomonas cepacia to produce poly-beta-hydroxyalkanoic acid was evaluated. Chemostat culture showed that growth of P. cepacia became nitrogen limited when the molar carbon-to-nitrogen ratio of the medium fed into the fermentor was above 15. When grown under nitrogen limitation in batch culture with fructose as the sole source of carbon, P. cepacia accumulated poly-beta-hydroxybutyric acid (PHB) in excess of 50% of the dry weight of its biomass. In batch culture, almost no PHB was produced until the onset of nitrogen limitation. After this point, PHB was produced at a linear rate of 0.12 g liter h (from a constant value of 1.6 g of cellular protein liter). PHB produced by P. cepacia had a weight-average molecular weight of 5.37 x 10 g mol and a polydispersivity index of 3.9. Poly(beta-hydroxybutyric acid-beta-hydroxyvaleric acid) copolymer was produced with a poly-beta-hydroxybutyric acid-poly-beta-hydroxyvaleric acid ratio of up to 30% by weight when propionic acid was added to the medium.     

Hydrocarbon pseudosolubilizing and emulsifying proteins produced by Pseudomonas cepacia N1

Significant specific pseudosolubilizing activity of the n-hexadecane pseudosolubilizing factor (C₁₆PSF), n-octadecane pseudosolubilizing factor (C₁₈PSF) and pristane pseudosolubilizing factor (PrisPSF) was found. From the partial chemical analysis of the chromatographically purified samples the pseudosolubilizing factors (PSFs) and n-hexadecane emulsifying factor (HEF-C₁₆) were tentatively identified as glycoprotein and lipoprotein, respectively. Each of the purified PSFs showed molecular weight 14,000 in SDS-PAGE but they had different amino acid compositions. The carbohydrate component of the C₁₆PSF consists of 93% glucose residues. C₁₂-, C₁₃-, C₁₄- and C₁₆- are the major fatty acids constituting 64% of the total fatty acids of HEF-C₁₆.     

Insertion-sequence-dependent rearrangements of Pseudomonas cepacia plasmid pTGL1.

Pseudomonas cepacia 249 (ATCC 17616) harbors a 170-kilobase (kb) plasmid designated pTGL1. We identified three insertion sequences, IS405, IS408, and IS411, on this plasmid. Various prototrophic and auxotrophic derivatives in our collection contained variants of pTGL1 formed by accretion and deletion of other elements. Plasmid pTGL6, the variant in one prototroph, evolved from pTGL1 by the addition of three copies of IS401 (1.3 kb) and one of IS402 (1 kb), to generate pTGL5, and recombination between two of the copies of IS401 on pTGL5 to form pTGL6. The latter event entailed loss of one copy of IS401 and an additional 5.4 kb of plasmid DNA. Derivatives of the broad-host-range plasmid pRP1 carrying the above insertion sequences and recombinant plasmids carrying fragments of plasmids pTGL6 and pTGL5 were used as probes to ascertain the extent of reiteration of the various elements in the P. cepacia genome. The data indicate a high frequency of genomic rearrangements which presumably contributes to the extraordinary adaptability of this bacterium.     

Production of Poly-β-Hydroxyalkanoic Acid by Pseudomonas cepacia

The possibility of using the nutritionally versatile bacterium Pseudomonas cepacia to produce poly-β-hydroxyalkanoic acid was evaluated. Chemostat culture showed that growth of P. cepacia became nitrogen limited when the molar carbon-to-nitrogen ratio of the medium fed into the fermentor was above 15. When grown under nitrogen limitation in batch culture with fructose as the sole source of carbon, P. cepacia accumulated poly-β-hydroxybutyric acid (PHB) in excess of 50% of the dry weight of its biomass. In batch culture, almost no PHB was produced until the onset of nitrogen limitation. After this point, PHB was produced at a linear rate of 0.12 g liter⁻¹ h⁻¹ (from a constant value of 1.6 g of cellular protein liter⁻¹). PHB produced by P. cepacia had a weight-average molecular weight of 5.37 × 10⁵ g mol⁻¹ and a polydispersivity index of 3.9. Poly(β-hydroxybutyric acid-β-hydroxyvaleric acid) copolymer was produced with a poly-β-hydroxybutyric acid-poly-β-hydroxyvaleric acid ratio of up to 30% by weight when propionic acid was added to the medium.     

Surface characteristics of Pseudomonas cepacia

Two major surface characteristics of Pseudomonas cepacia were examined in this study: reactivity with lectins and hydrophobicity. The results indicated that the surfaces of P. cepacia strains are heterogeneous with regard to the distribution of lectin receptors. Only lima bean agglutinin was found to strongly agglutinate all strains. The strains were also heterogeneous with regard to hydrophobicity as determined by adhesion to hexadecane. The degree of hydrophobicity, however, was not significantly altered when selected strains were mixed with either fibronectin or bovine serum albumin. In addition, the strains exhibited no apparent affinities for buccal epithelial cells and gave no evidence for an ability to haemagglutinate human red cells.     

Copper biosorption by Pseudomonas cepacia and other strains

Biosorption of copper by Pseudomonas cepacia was found to be dependent on added copper concentration. Copper uptake by the cells was rapid over the range of copper concentrations tested and complete within the first 10 min of incubation time. The effect of pH on copper uptake by P. cepacia was determined using overlapping buffers over the pH range 3–8, and copper biosorption from a 10 mM copper solution was greatest at pH 7. Copper uptake (measured by analysis of cell digests) was unaffected by cyanide and azide (up to 30 mM) and by incubation of cells with a 10 mM copper solution at 4 °C. Evidence from these results suggested that copper uptake by P. cepacia cells involves surface binding and not intracellular accumulation by active transport. Biosorption of copper by various Pseudomonas isolates from metal-contaminated environments agreed well with copper biosorption by Pseudomonas strains from the National Collection of Type Cultures (NCTC).     

Rapid differentiation of Pseudomonas pseudomallei from Pseudomonas cepacia

The Minitek disc system was utilized for the differentiation of Pseudomonas pseudomallei, the causative agent of melioidosis, from Ps. cepacia. The system was simple to use, inexpensive, and furnished rapid, clear-cut test results after 4 h. This procedure is suitable for differentiating soil bacteria presumptively identified as Ps. pseudomallei, Ps. cepacia or flavobacteria, and for the rapid confirmation of the presumptive identification of either Ps. pseudomallei or Ps. cepacia obtained by commercial identification-kit systems in the clinical laboratory.     

Metabolism of Halophenols by 2,4,5-trichlorophenoxyacetic acid-degrading Pseudomonas cepacia

Resting cells of 2,4,5-trichlorophenoxyacetic acid-grown Pseudomonas cepacia AC1100 were able to completely and rapidly dechlorinate several chlorine-substituted phenols, including 2,4,5-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol. Several other trichlorophenols were only partially dechlorinated. The evidence suggests that 2,4,5-trichlorophenol is an intermediate in the degradation of 2,4,5-trichlorophenoxyacetic acid by strain AC1100. Moreover, although strain AC1100 was isolated by selection for growth on a chlorinated aromatic compound, brominated and fluorinated analogs were efficiently dehalogenated by strain AC1100 resting cells, whereas an iodinated analog was poorly dehalogenated.     

Production and composition of phenylpyrrole metabolites prodcued by Pseudomonas cepacia

Summary In shaken cultures, a strain of Pseudomonas cepacia isolated from apple leaves produced pyrrolnitrin and four other phenylpyrrole antibiotics. The concentrations of these metabolites were determined at intervals for 7 days in three different media at two initial pH levels. Optical density measurements revealed maximum cell concentrations after 24 h in nutrient broth, after 48 h in King's B medium, and after 96 h in minimum salts solution. The effects caused by initiating fermentations at pH 5.8 rather than 7.0 were in most cases not dramatic, although in some instances, especially in minimum salts broth, higher concentrations of metabolites were produced with the lower initial pH. Concentrations of the phenylpyrrole antibiotics were greatly affected by choice of culture medium and incubation time. Concentrations of the two nitrophenyl metabolites, pyrrolnitrin and 2-chloropyrrolnitrin, rose throughout the 7-day incubation and were more than 20 times greater in minimum salts medium than in either King's B medium or nutrient broth. The maximum concentrations of each of the three aminophenyl metabolites (dichloroamino, trichloroamino and monochloroamino) occurred in different media, the monochloro compound in nutrient broth, the dichloro compound in Kings B medium and the trichloro compound in minimum salts medium. The time dependence of the concentrations of the five metabolites supports the proposed biosynthesis of these pyrroles from tryptophan by successive chlorinations followed by oxidation of the amino group at the end of the pathway.     

Kinetic resolution of 4-chloro-2-(1-hydroxyalkyl)pyridines using Pseudomonas cepacia lipase

Here we report a detailed procedure for the enzymatic kinetic resolution of 4-chloro-2-(1-hydroxyalkyl)pyridines, valuable precursors for the preparation of enantiomerically pure catalysts derived from 4-(N,N-dimethylamino)pyridine. Pseudomonas cepacia lipase shows excellent enantioselectivity in the acylation of the (R)-enantiomer at 30 degrees C and 250 r.p.m., with vinyl acetate as the acyl donor and tetrahydrofuran as the solvent. The reaction times for resolution of the pyridine derivatives depend on the structure of the selected substrate.