7,10-Dihydroxy-8(E)-octadecenoic acid produced by Pseudomonas 42A2: evaluation of different cultural parameters of the fermentation

A fermentation process for the microbial production of a new lipid surface-active compound, 7,10-dihydroxy-8 (E)-octadecenoic acid (OCD), has been established using a vegetable oil as carbon source in a coordinated carbon/nitrogen feed strategy. The surfactant was produced during the logarithmic growth phase. Aeration was the most critical parameter for product formation. Up to 7 g product/l was produced.The authors are with the Laboratorio de Microbiologia, Facultad de Farmacia, Avenida Diagonal 643, Universidad de Barcelona, 08028 Barcelona, Spain     

Isolation and characterization of a lipoxygenase from Pseudomonas 42A2 responsible for the biotransformation of oleic acid into (S)-(E)-10-hydroxy-8-octadecenoic acid

The isolation of a new lipoxygenase-like (LOX-like) enzyme from Pseudomonas 42A2 and its characterization is described. The enzyme, located in the periplasm of the cell, which contained 0.55 mol of Fe2+ per mol of protein, is monomeric and has a molecular mass of 45 kDa. In the presence of oxygen, the enzyme converts oleic acid into (E)-10-hydroperoxy-8-octadecenoic acid (HPOD), which decomposes to the corresponding (E)-10-hydroxy-8-octadecenoic acid (HOD). The absolute configuration of this acid was determined as S on the basis of exciton-coupled CD data, and specific rotation and NMR analysis of the corresponding p -bromobenzoate derivative. The reaction in vivo leads to the dihydroxy derivative (E)-7,10-dihydroxy-8-octadecenoic acid (DHOD), so that the three hydroxy-fatty acids can be isolated from the culture medium. The activity of the enzyme was optimal between 25 and 30 degrees C and 44% of its activity still remained at 55 degrees C. Its optimal pH is 8.5-9; and the presence of magnesium ions increased LOX activity by 1.5. The activity of the LOX is highest in unsaturated fatty acids containing double bonds in position 9 (oleic, linoleic and linolenic acids), linoleic acid being preferred (100% activity) over linolenic (60.4%) and oleic acids (46%). However, kinetic studies showed that the affinity of the enzyme is similar for the three substrates.     

Optical resolution of RS-(±)-mandelic acid by Pseudomonas sp.

For the optical resolution of R-(–)-mandelic acid from (±)-mandelic acid, Pseudomonas sp. MA02, which assimilated S-(+)-mandelic acid as carbon and energy source, was isolated from soil. Using the fed-batch culture under optimal condition, R-(–)-mandelic acid was accumulated up to the maximum theoretical yield of 50% (30 g l^–1) and entiomeric excess of 99.4%.     

Conversion of substituted naphthalenesulfonates by Pseudomonas sp. BN6

The range of substituted naphthalenesulfonates which are metabolized by Pseudomonas sp. BN6 were investigated. Resting cells from strain BN6 oxidized 1- and 2-naphthalenesulfonate, 1-hydroxynaphthalene-2-sulfonate, 2,6-naphthalenedisulfonate and all monosulfonated naphthalene-2-sulfonates which carry one or two substitutents in the positions 4-, 5-, 6-, 7- or 8- of the naphthalene ring-system. With the exception of (substituted) 4- or 5-amino- and 4-hydroxynaphthalene-2-sulfonates these compounds were converted to the corresponding salicylates. Strain BN6 did not oxidize substituted naphthalene-1-sulfonates, 3-substituted naphthalenesulfonates and substituted naphthalenedisulfonates. Turnover of 4-amino- or 4-hydroxynaphthalene-2-sulfonates resulted in the accumulation of the corresponding naphthoquinones in the culture medium. Thus, degradation of 4-amino- and 4-hydroxynaphthalenesulfonates was restricted by the rapid autoxidation of the substituted 1,2-dihydroxynaphthalenes formed as metabolites. Catabolic activities of strain BN6 for naphthalenesulfonates were induced by salicylate, 3- or 6-hydroxysalicylate, and 3-, 4- or 5-aminosalicylate but not by 4- and 5-hydroxysalicylate. All naphthalenesulfonates that were not converted into the corresponding salicylates, were found to be inefficient as effectors. It was therefore concluded that (substituted) salicylates are the inducers of the relevant enzymes. The degradation of 2-naphthalene-sulfonate by a pure culture of strain BN6 was prevented by the toxicity of the dead-end product salicylate. Substituted salicylates were less toxic and allowed growth of strain BN6 in axenic culture with various substituted naphthalenesulfonates.Abbreviations AB aminobenzoate - ANS aminonaphthalenesulfonate - DHN dihydroxynaphthalene - DHNC dihydroxynaphthalene-carboxylate - DHNDO 1,2-dihydroxynaphthalene dioxygenase - HBPA 2-hydroxybenzalpyruvate aldolase - HNS hydroxynaphthalenesulfonate - HS hydroxysalicylate - Ind-C indolecarboxylate - Ind-S indolesulfonate - MANS N-methylaminonaphthalenesulfonate - NC naphthalenecarboxylate - NDS naphthalenedisulfonate - NQ naphthoquinone - NS naphthalenesulfonate - NSDO naphthalenesulfonate dioxygenase - R_t retention time - SADH salicylaldehyde dehydrogenase - THN trihydroxynaphthalene (hydroxy-1,2-dihydroxynaphthalene)     

Phenanthrene mineralization by Pseudomonas sp. UG14

A phenanthrene-mineralizing Pseudomonas sp., designated UG14, was isolated from creosote-contaminated soil. It contained two plasmids, of approximately 77 kb and 76 kb, the smaller of which contained DNA sequences that hybridized with probes specific for ndoB and xylE, genes involved in catabolism of aromatic hydrocarbons. At initial phenanthrene concentrations of 10, 50, 200 and 1000 mg/l broth, 27%, 19%, 7.7% and 3.3%, respectively, of the [9-¹⁴C]phenanthrene was recovered as ¹⁴CO₂ after 36 days' incubation at 30°C. Most ¹⁴C-label was converted to a water-soluble metabolite tentatively identified as 1-hydroxy-2-naphthoic acid. Rhamnolipid biosurfactants produced by P. aeruginosa UG2 enhanced mineralization of 50 mg phenanthrene/l by Pseudomonas sp. UG14. With the biosurfactant at 0, 25 and 250 mg rhamnose equivalents/l, 6.5%, 8.2% and 9.8%, respectively, of the phenanthrene was mineralized after 35 days.M.A. Providenti, H. Lee and J.T. Trevors are with the Department of Environmental Biology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada; C.W. Greer is with the National Research Council Canada, Biotechnology Research Institute, 6100 Royalmount Ave, Montreal, Quebec, H4P 2R2, Canada.     

Short communication: Methylparathion degradation by Pseudomonas sp. A3 immobilized in sodium alginate beads

An organophosphate-degrading soil isolate of Pseudomonas sp. A3, immobilized at 5% (wet wt/v) cell mass in 3% (w/v) sodium alginate beads, detoxified 99% of 1 mm methylparathion in 48 h. The beads were re-usable for five batches, the sixth batch only giving 73% methylparathion removal.     

A novel EDTA-degrading Pseudomonas sp.

A bacterial strain LPM-410 capable of utilizing ethylenediaminetetraacetate (EDTA) as the sole source of energy, carbon, and nitrogen was isolated from sewage sludge and identified as a Pseudomonas sp. on the basis of its phenotypic characteristics. Suspensions of exponential-phase cells degraded EDTA, Mg–, Ca–, Ba–, and Mn–EDTA at constant specific rates ranging from 0.363 to 0.525 mmol EDTA/(g cells h). The more stable chelate, Zn–EDTA, was degraded at a lower rate (0.195 ± 0.030 mmol EDTA/(g cells h)), and here was no degradation of Co–, Cu–, Pb–, and Fe(III)–EDTA.     

Metabolism of benzalphthalide by Pseudomonas sp.

A Pseudomonas sp. degraded benzalphthalide to o-phthalate and benzoate. A tentative pathway for the metabolism of benzalphthalide in this Pseudomonas sp. is proposed on the basis of isolated metabolites, oxygraphic assay and enzymatic studies.     

Mineralization of phenol and its derivatives by Pseudomonas sp. strain CP4

A Pseudomonas sp. strain, CP4, was isolated that used phenol up to 1.5 g/l as sole source of carbon and energy. Optimal growth on 1.5 g phenol/l was at pH 6.5 to 7.0 and 30°C. Unadapted cells needed 72 h to decrease the chemical oxygen demand (COD) of about 2000 mg/l (from 1 g phenol/l) to about 200 mg/l. Adapted cells, pregrown on phenol, required only 65 h to decrease the COD level to below 100 mg/l. Adaptation of cells to phenol also improved the degradation of cresols. Cell-free extracts of strain CP4 grown on phenol or o-, m- or p-cresol had sp. act. of 0.82, 0.35, 0.54 and 0.32 units of catechol 2,3-dioxygenase and 0.06, 0.05, 0.05 and 0.03 units of catechol 1,2-dioxygenase, respectively. Cells grown on glucose or succinate had neither activity. Benzoate and all isomers of cresol, creosote, hydroxybenzoates, catechol and methyl catechol were utilized by strain CP4. No chloroaromatic was degraded, either as sole substrate or as co-substrate.The authors are with the Department of Microbiology and Bioengineering, Central Food Technological Research Institute, Mysore-570 013, India     

Studies on the anaerobic degradation of benzoic acid and 2-aminobenzoic acid by a denitrifying Pseudomonas strain

The growth of a denitrifying Pseudomonas strain on benzoic acid and 2-aminobenzoic acid (anthranilic acid) has been studied. The organism grew aerobically on benzoate, 2-aminobenzoate, and gentisate, but not on catechol or protocatechuic acid. These and other findings suggest that aerobic degradation of benzoic acid was via gentisic acid. Under completely anaerobic conditions in the presence of nitrate, benzoate and 2-aminobenzoate (5 mM each) were oxidized to CO₂ with the concurrent reduction of NO ₃ ^- to NO ₂ ^- . Only after complete NO ₃ ^- consumption was NO ₂ ^- reduced to N₂. Cells contained a NADP-specific 2-oxoglutaate dehydrogenase, in contrast to a NAD-specific pyruvate dehydrogenase. During anaerobic metabolism of [carboxyl-¹⁴C]benzoic acid, 16% of the label of metabolized benzoic acid was incorporated into cell material; this excludes intermediary decarboxylation during anaerobic metabolism. Extracts catalysed the activation of benzoic acid and a variety of its derivatives to the respective aryl-coenzyme A thioesters, ATP being cleaved to AMP and PP_i; two synthetase activites were present. Extracts from 2-aminobenzoate-grown cells catalyzed a NADH-dependent reduction of 2-aminobenzoyl-CoA (100 nmol·min^-1·mg^-1 cell protein) to an unidentified CoA thioester, with a stoichiometric release of NH₃ and a stoichiometry of 3 mol NADH oxidized per mol 2-aminobenzyol-CoA reduced when tested under aerobic conditions. The 2-aminobenzoyl-CoA reductase activity was lacking in anaerobic benzoate-grown cells and in aerobic cells. This is taken as evidence that 2-aminobenzoyl-CoA reductase is a key enzyme in a novel reductive pathway of anaerobic 2-aminobenzoic acid metabolism.Dedicated to Prof. Charles W. Evans     

A novel S-enantioselective amidase acting on 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide from Arthrobacter sp. S-2

A novel S-enantioselective amidase acting on 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide was purified from Arthrobacter sp. S-2. The enzyme acted S-enantioselectively on 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide to yield (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropanoic acid. Based on the N-terminal amino acid sequence of this amidase, the gene coding S-amidase was cloned from the genomic DNA of Arthrobacter sp. S-2 and expressed in an Escherichia coli host. The recombinant S-amidase was purified and characterized. Furthermore, the purified recombinant S-amidase with the C-His₆-tag, which was expressed in E. coli as the C-His₆-tag fusion protein, was used in the kinetic resolution of (±)-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide to obtain (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropanoic acid and (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide.     

Las-like quorum-sensing system negatively regulates both pyoluteorin and phenazine-1-carboxylic acid production in Pseudomonas sp. M18

A las-like quorum-sensing system in Pseudomonas sp. M18 was identified, which consisted of lasI and lasR genes encoding LuxI-LuxR type regulator. Several functions of the las system from strain M18 were investigated in this study. The chromosomal inactivation of either lasI or lasR by recombination increased the production of both pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA) by 4－5 fold and 2－3 fold over that of the wild type strain of M18, respectively. Production of both antibiotics was restored to wild-type levels after in trans complementation with the wild-type lasI or lasR gene. Ex-pression of the translational fusions pltA׳-׳lacZ and phzA׳-׳lacZ further confirmed the negative effect of lasI or lasR on both biosynthetic operons, and it was also demonstrated that the las system was related to the ability of swarming motility and the inhibition of cell growth.     

Degradation of carbazole and its derivatives by a Pseudomonas sp.

Carbazole, carbazoles with monomethyl or dimethyls substituted on different positions (C₁-carbazoles or C₂-carbazoles), and benzocarbazoles, as toxic and mutagenic components of petroleum and creosote contamination, were biodegradable by an isolated bacterial strain Pseudomonas sp. XLDN4-9. C₁-carbazoles were degraded in preference to carbazole and C₂-carbazoles. The biodegradation of C₁-carbazoles or C₂-carbazoles was influenced by the positions of methyl substitutions. Among C₁-carbazole isomers, 1-methyl carbazole was the most susceptible. C₂-carbazole isomers with substitutions on the same benzo-nucleus were more susceptible at a concentration of less than 3.4 μg g⁻¹ petroleum, especially when harboring one substitution on position 1. In particular, 1,5-dimethyl carbazole was the most recalcitrant dimethyl isomer.     

Cometabolic biosynthesis of copolyesters consisting of 3-hydroxyvalerate and medium-chain-length 3-hydroxyalkanoates by Pseudomonas sp. DSY-82

A newly isolated strain, designated as Pseudomonas sp. DSY-82, synthesized medium-chain-length polyhydroxyalkanoate (MCL-PHA) copolyesters when grown on alkanoates from hexanoate to undecanoate as the sole carbon source. When used alone, butyrate and valerate supported the growth of the isolate but not PHA production. However, unusual polyesters containing 3-hydroxyvalerate, as well as various MCL 3-hydroxyalkanoate monomeric units, were synthesized when valerate was cofed with either nonanoate or 10-undecenoate, suggesting the formation of monomer units from both substrates. Concentrations of 3-hydroxyvalerate, 3-hydroxyoctanoate, and 3-hydroxydecanoate in the PHAs produced were significantly elevated by the addition of valerate, indicating that the incorporation of these monomer units to PHA occurred primarily through cometabolism. The total amount of these monomer units in the PHAs reached up to 30%. The PHAs produced in this study were most likely random copolyesters as determined by differential scanning calorimetric analysis. This is the first case of microbial synthesis of copolyesters consisting of 3-hydroxyvalerate and MCL 3-hydroxyalkanoate monomer units through cometabolism.     

Biosurfactant production and hydrocarbon-degradation by halotolerant and thermotolerant Pseudomonas sp.

A hydrocarbon degrading and biosurfactant producing, strain DHT2, was isolated from oil-contaminated soil. The organism grew and produced biosurfactant when cultured in variety of substrates at salinities up to 6 g l⁻¹ and temperatures up to 45°C. It was capable of utilizing crude oil, fuels, alkanes and PAHs as carbon source across the wide range of temperature (30–45°C) and salinity (0–6%). Over the range evaluated, the salinity and temperature did not influence the degradation of hydrocarbon and biosurfactant productions. Isolate DHT2 was identified as Pseudomonas aeruginosa by analysis of 16S rRNA sequences (100% homology) and biochemical analysis. PCR and DNA hybridization studies revealed that enzymes involved in PAH metabolism were related to the naphthalene dioxygenase pathway. Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by DHT2 during growth on both, water miscible and immiscible substrates, including PAH. The biosurfactants lowered the surface tension of medium from 54.9 to 30.2 dN/cm and formed a stable emulsion. The biosurfactant produced by the organism emulsified a range of hydrocarbons with hexadecane as best substrate and toluene was the poorest. These findings further indicate that the isolate could be useful for bioremediation and bio-refining application in petroleum industry.     

Degradation of 2-methylnaphthalene by free and immobilized cells of Pseudomonas sp. strain NGK1

A Pseudomonas sp. strain NGK1 (NCIM 5120) capable of utilizing 2-methylnaphthalene (2-MN) was immobilized in various matrices namely, polyurethane foam (PUF), alginate, agar and polyvinyl alcohol (PVA) (1.5 × 10¹² c.f.u. g^–1 beads). The degradation rates of 25 and 50 mM 2-MN by freely suspended cells (2 × 10¹¹ c.f.u. ml^–1) and immobilized cells in batches, semi-continuous with shaken culture and continuous degradation in a packed-bed reactor were compared. The PUF-immobilized cells achieved higher degradation of 25 and 50 mM of 2-MN than freely suspended cells and the cells immobilized in alginate, agar or PVA. The PVA- and PUF-immobilized cells could be reused for more than 30 and 20 cycles respectively, without losing any degradation capacity. The effect of dilution rates on the rate of degradation of 25 and 50 mM 2-MN with freely suspended and immobilized cells were compared in the continuous system. Increase in dilution rate increased the degradation rate only up to 1 h^–1 in free cells with 25 mM 2-MN and no significant increase was observed with 50 mM 2-MN. With immobilized cells, the degradation rate increased with increase in dilution rate up to 1.5 h^–1 for 25 mM and 1 h^–1 for 50 mM 2-MN. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for biodegradation of 2-MN.     

Biosynthesis of copolyesters consisting of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids from 1,3-butanediol or from 3-hydroxybutyrate by Pseudomonas sp. A33

Pseudomonas sp. A33 and other isolates of aerobic bacteria accumulated a complex copolyester containing 3-hydroxybutyric acid (3HB) and various medium-chain-length 3-hydroxyalkanoic acids (3HA_MCL) from 3-hydroxybutyric acid or from 1,3-butanediol under nitrogen-limitated culture conditions. 3HB contributed to 15.1 mol/100 mol of the constituents of the polyester depending on the strain and on the cultivation conditions. The accumulated polymer was a copolyester of 3HB and 3HA_MCL rather than a blend of poly(3HB) and poly(3HA_MCL) on the basis of multiple evidence. 3-Hydroxyhexadecenoic acid and 3-hydroxyhexadecanoic acid were detected as constituents of polyhydroxyalkanoates, which have hitherto not been described, by¹³C nuclear magnetic resonance or by gas chromatography/mass spectrometric analysis. In total, ten different constituents were detected in the polymer synthesized from 1,3-butanediol by Pseudomonas sp. A33:besides seven saturated (3HB, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydrohexadecanoate) three unsaturated (3-hydroxydodecenoate, 3-hydroxytetradecenoate and 3-hydrohexadecanoate) hydroxyalkanoic acid constituents occured. The polyhydroxyalkanoate synthase of Pseudomonas sp. A33 was cloned, and its substrate specificity was evaluated by heterologous expression in various strains of P. putida, P. oleovorans and Alcaligenes eutrophus.     

A novel biotransformation of 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. for the purification of crude 2,6-naphthalene dicarboxylic acid

Crude 2,6-naphthalene dicarboxylic acid was purified by Pseudomonas sp. HN-72 which biotransformed the major impurity of 2-formyl-6-naphthoic acid into 2,6-naphthalene dicarboxylic acid. The biotransformation yield reached 100% when the reaction was performed at 40°C for 1 h, in 200 ml KH₂PO₄/KOH buffer (50 mM, pH 8.0), with 0.2% (w/v) crude 2,6-naphthalene dicarboxylic acid and 2.5 mg dry cell wt/ml.     

Degradation of chlorosubstituted aromatic compounds by Pseudomonas sp. strain B13: fate of 3,5-dichlorocatechol

The degradation of 3,5-dichlorocatechol by enzymes of 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 was studied. The following compounds were formed from 3,5-dichlorocatechol: trans-2-chloro-4-carboxymethylenebut-2-en-4-olide, cis-2-chloro-4-carboxymethylenebut-2-en-4-olide, and chloroacetylacrylate as the decarboxylation product of 2-chloromaleylacetate. They were identified by chromatographic and spectroscopic methods (UV, MS, PMR). An enzyme activity converting trans-2-chloro-4-carboxymethylenebut-2-en-4-olide into the cis-isomer was observed.Abbreviations 3CB 3-chlorobenzoate - 4CB 4-chlorobenzoate - 3,5DCB 3,5-dichlorobenzoate - 2,4D 2,4-dichlorophenoxyacetate - NOE Nuclear-Overhauser-Effect     

Biotransformation of phenylpyruvic acid to phenyllactic acid by growing and resting cells of a <Emphasis Type="Italic">Lactobacillus</Emphasis> sp.

Phenyllactic acid (PLA) is a novel antimicrobial compound derived from phenylalanine (Phe). Lactobacillus sp. SK007, having high PLA-producing ability, was isolated from Chinese traditional pickles. When 6.1 mM phenylpyruvic acid (PPA) was used to replace Phe as substrate at the same concentration, PLA production increased 14-fold and the fermentation time decreased from 72 h to 24 h with growing cells. With resting cells, however, 6.8 mM PLA could be obtained as optimal yield using the following conditions: 12 mM PPA, 55 mM glucose, pH 7.5, 35°C and 4 h.