Bench-scale biosynthesis of isonicotinic acid from 4-cyanopyridine by Pseudomonas putida

Pseudomonas putida CGMCC3830 harboring nitrilase was used in isonicotinic acid production from 4-cyanopyridine. This nitrilase showed optimum activities towards 4-cyanopyridine at pH 7.5 and 45°C. The half-life of P. putida nitrilase was 93.3 h, 33.9 h, and 9.5 h at 30°C, 38°C, and 45°C, respectively. 4-Cyanopyridine (100 mM) was fully converted into isonicotinic acid within 20 min. The bench-scale production of isonicotinic acid was carried out using 3 mg of resting cells per mL in a 1 L system at 30°C and finally, 123 g L^?1 of isonicotinic acid were obtained within 200 min without any by-products. The conversion reaction suffered from the product inhibition effect after the tenth feeding. The volumetric productivity was 36.9 g L^?1 h^?1. P. putida shows significant potential in nitrile hydrolysis for isonicotinic acid production. This paper is the first report on isonicotinic acid biosynthesis using Pseudomonas putida and it represents the highest isonicotinic acid production reported so far.     

NMR spectroscopy as a tool to investigate the degradation of aromatic compounds by a Pseudomonas putida strain

Pseudomonas putida strain KT2442, harbouring the pWW0 TOL plasmid, was grown with a number of different homologous aromatic acids as carbon sources. Small samples of liquid culture supernatant were collected and directly analysed by 2D NMR spectroscopy. In all cases similar compounds with olefinic signals were observed to accumulate. To elucidate the structures of these compounds, 2D NMR experiments with 500 and 600 MHz spectrometers equipped with a CryoProbe (Bruker BioSpin) were performed on samples obtained from a culture growing on 4‐methylbenzoate and, for ¹³C spectroscopy, on ¹³C‐labelled 4‐methylbenzoate. In all cases a 1,2‐dihydroxycyclohexa‐3,5‐diene‐carboxylate derivative was identified. The use of this technique helped us to identify easily some metabolites that were released into the solution by bacteria and to follow their secretion as a function of time. The high sensitivity of the present approach allowed a clear and rapid acquisition of spectra, notwithstanding the low concentration of the compounds. The benefits of introducing the use of NMR cryoprobes to perform metabolic pathway studies is demonstrated. Copyright © 2003 John Wiley & Sons, Ltd.     

Tuning the sensitivity of a foldamer-based mercury sensor by its folding energy

A fluorescent sensor for Hg2+ was obtained by incorporation of two methionine units into a cholate hexamer and by attaching a Dansyl group at the chain end. Folding could be made highly favorable or unfavorable in different solvents. Because folding was required to bring the sulfur groups together to chelate the mercury ion, binding affinity of the foldamer and, thus, its sensitivity as a sensor, could be tuned over broad ranges (at least 5 orders of magnitude) by solvent changes.     

Detection of phenolic compounds by thick film sensors based on Pseudomonas putida

Amperometric biosensors using bacterial cells were developed for the determination of phenolic compounds and the measurement was based on the respiratory activity of the cells. For this purpose, Pseudomonas putida DSM 50026 which is one of the well-known phenol degrading organisms, was used as a biological component. The cells were grown in the presence of phenol as the sole source of organic carbon. As well as phenol adapted cells, the bacterium which used the glucose as the major carbon source, was also used to obtain another type of biosensor for the comparison of the responses and specificities towards different xenobiotics. The commercial oxygen electrode was used as a transducer to test the sensor responses for both induced and non-induced cells. Our results showed that the adaptation step enable us to obtain biosensor devices with different substrate specificity. Moreover, P. putida was immobilized on the surface of thick film working electrodes made of gold by using gelatin membrane cross-linked with glutaraldehyde. The biosensors were calibrated for different phenolic substances. Furthermore, phenol detection was performed in synthetic wastewater samples.     

Adhesion of Pseudomonas putida NCIB 9816-4 to a naphthalene-contaminated soil

The effect of a soil contaminant on the initial adhesion to the soil of a contaminant-degrading soil microorganism in the exponential phase was investigated using naphthalene as the soil contaminant and Pseudomonas putida strain NCIB 9816-4 as the naphthalene-degrading bacteria. P. putida strain DK-1, which is not capable of degrading naphthalene, was used as a control. P. putida NCIB 9816-4 in the exponential phase showed the more adhesion to the soil than that in the stationary phase. In contrast, P. putida DK-1 showed the increased adhesion to the soil when it was in the stationary phase. P. putida NCIB 9816-4 in the exponential phase showed the preferred adhesion to the naphthalene-contaminated soil, whereas the adhesion of P. putida DK-1 was not affected by naphthalene. From the data of surface hydrophobicities of the cells and the soil, the microbial adhesion, especially the initial adhesion to the naphthalene-contaminated soil, takes place through the hydrophobic interaction. We suspect that the surface hydrophobicity of P. putida NCIB 9816-4 in the exponential phase might be increased during the uptake of naphthalene, which caused the preferred adhesion to the naphthalene-contaminated soil.     

Iron(III) coordination properties of a pyoverdin siderophore produced by Pseudomonas putida ATCC 33015

The iron complexation of a fluorescent green pyoverdin siderophore produced by the environmental bacterium Pseudomonas putida was characterized by solution thermodynamic methods. Pyoverdin binds iron through three bidentate chelate groups, a catecholate, a hydroxamate, and an alpha-hydroxycarboxylic acid. The deprotonation constants of the free pyoverdin and Fe(III)-pyoverdin complex were determined through a series of potentiometric and spectrophotometric experiments. The ferric complex of pyoverdin forms at very low pH (pH < 2), but full iron coordination does not occur until neutral pH. The calculated pM value of 25.13 is slightly lower than that for pyoverdin PaA (pM = 27), which coordinates iron by a catecholate and two hydroxamate groups. The redox potential of Fe-pyoverdin was found to be very pH sensitive. At high pH (approximately pH 9-11) where pyoverdin coordinates Fe in a hexadentate mode the redox potential is -0.480 V (NHE); however, at neutral pH where full Fe coordination is incomplete, the redox potential is more positive (E(1/2) = -0.395 V). The positive shift in the redox potential and the partial dissociation of the Fe-pyoverdin complex with pH decrease provides a path toward in vivo iron release.     

Enzymatic synthesis of cyclic amino acids by N-methyl-l-amino acid dehydrogenase from Pseudomonas putida

A new enzymatic system for the synthesis of enantiomerically pure cyclic amino acids (CAA) from the corresponding diamino acids or racemic CAA is described. α,ω-Diamino acids were oxidized to α-keto acids with amino acid oxidases (AAO). The α-keto acids were spontaneously transformed into cyclic imino acids in the reaction medium. The resulting imines were reduced to the l-form CAA with N-methyl-l-amino acid dehydrogenase (NMAADH) from Pseudomonas putida ATCC12633 using NADPH as a cofactor. l-Form CAA were also obtained from racemic CAA using d-amino-acid oxidase and NMAADH. Using this method, a new compound [1,4]-thiazepane-3-carboxylic acid (Fig. 1) was synthesized from aminopropylcystein.     

Bioreduction of ionic mercury from wastewater in a fixed-bed bioreactor with activated carbon

Wide industrial use of mercury led to significant mercury pollution of the environment. It requires development of cleanup technologies which would allow treating large volumes of mercury contaminated water in a cost effective and environmentally friendly way. A novel bio-technology, developed from laboratory to industrial scale in Germany at HZI (former GBF), is based on enzymatic reduction of highly toxic Hg(II) to water-insoluble and relatively non-toxic Hg(0) using live mercury resistant bacteria immobilized on a porous carrier material in a fixed-bed bioreactor. Improvement of the original method was based on the use of activated carbon as a carrier for microorganisms and an adsorbent for mercury. Such integration of the process should increase the technology efficiency. In order to compare different carrier materials, activated carbon and pumice stones were used. The strain Pseudomonas putida was immobilized in bioreactors continuously fed with solutions of HgCl₂ enriched with nutrients. Simultaneously, experiments in two more reactors were run in the absence of microorganisms to investigate the influence of nutrients on the adsorption process. In the bioreactor with activated carbon, the outlet mercury concentration was approximately 50 % of that supplied with pumice. It may be concluded that the use of activated carbon in a fixed-bed bioreactor enables improvement of the technology by process integration. Presented at the 34th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 21–25 May 2007.     

Enhancement of the conversion of toluene by Pseudomonas putida F-1 using organic cosolvents

Pseudomonas putida F-1 (ATCC700007) was used as a model organism in stirred tank reactors, to study conversion enhancement of poorly soluble substrates by organic cosolvents. After a literature study, silicone oil was used as a solvent system to enhance the mass transfer rate. To study the benefits of the organic solvent addition, batch experiments were conducted in two side-by-side fermentation vessels (experimental and control) at three different levels of silicone oil (10, 30, and 50%). Results showed that the presence of silicone oil resulted in a 100% increase in the toluene mass transfer compared to the control. Experiments in continuous stirred-tank reactors showed that improved conversion could beobtained, at higher agitation rates.     

Contribution of cell-surface components to Cu2+ adsorption by Pseudomonas putida 5-x

The contribution of various cell-surface components to Cu²⁺ adsorption by a Gram-negative bacterium, Pseudomonas putida 5-x, that was isolated from local electroplating effluent with a high capability to accumulate heavy metal ions was studied. The cell superficial layer had a negative effect on Cu²⁺ adsorption of the bacterial cells. Cu²⁺ adsorption capacity of the separated cell envelopes was fivefold more than that of the intact cells, owing to the liberation of more and more binding sites during the separation process. Some main components in the cell envelope, such as the peptidoglycan (PEG) layer, outer membrane, and inner membrane, provide the capability for Cu²⁺ adsorption. The content of the components in the cell envelope is in the order inner membrane > outer membrane > PEG layer, and their Cu²⁺ adsorption capacity was in the order PEG layer > outer membrane > inner membrane. The total contribution of the separated PEG layer material to Cu²⁺ adsorption by the cell envelope was no more than 15%, and the outer membrane and inner membrane contributed about 30–35% and 25–30%, respectively. The relatively high phospholipid content in the outer membrane may be the major reason for the higher adsorption capacity of the outer membrane to Cu²⁺ and, hence, such a high Cu²⁺ adsorption capacity of P. putida 5-x cell envelope.     

Immobilization of heavy metals by Pseudomonas putida CZ1/goethite composites from solution

Bacterial–mineral composites are important in the retention of heavy metals due to their large sorption capacity under a wide range of environmental conditions. This study provides the first quantitative comparison of the metal-binding capacities of P. putida CZ1–goethite composite to its individual components. When the same amount (on a dry weight basis) of living and nonliving cells of P. putida CZ1, goethite or their composites was separately exposed to solutions of 0.5 mM Cu(II) and Zn(II) in 0.01 M KNO₃, the living cells removed the largest quantity of heavy metals. The results of calculated metal retention values indicated that the adsorption of goethite to bacteria has not mask or neutralize chemically reactive adsorption sites normally available to metal ions. Moreover, the nonliving cells–goethite composite retained approximately 82% more Zn than that predicted by their individual behavior. The preferential association of Zn with P. putida CZ1 was observed by TEM and EDS analyses of a mixture consisting of the bacteria and goethite. Desorption of Cu and Zn with 1.0 M CH₃COOK solution from P. putida CZ1 and goethite indicated the differences in the functional groups able to bind heavy metals.     

Surface sensitivity of esca for sub-monolayer quantities of mercury adsorbed on a gold substrate

A study of the in tensity changes in the X-ray photoelectron 4f_7/2 lines during the formation of multilayers of Hg on Au at low temperature (< 100° K) gives an escape depth, λ_e, of ca. 9A for ≈ 1400 eV electrons, 0.2% of a monolayer of mercury can be detected.     

Initial characterization of a blue-light sensing, phototropin-related protein from Pseudomonas putida: a paradigm for an extended LOV construct

The open reading frame PP2739 from Pseudomonas putida KT2440 encodes a 151 amino acid protein with sequence similarity to the LOV domains of the blue-light sensitive protein YtvA from Bacillus subtilis and to the phototropins (phot) from plants. This sensory box LOV protein, PpSB2-LOV, comprises a LOV core, followed by a C-terminal segment predicted to form an alpha-helix, thus constituting a naturally occurring paradigm for an extended LOV construct. The recombinant PpSB2-LOV shows a photochemistry very similar to that of YtvA and phot-LOV domains, yet the lifetime for the recovery dark reaction, taurec=114 s at 20 degrees C, resembles that of phot-LOV domains (5-300 s) and is much faster than that of YtvA or YtvA-LOV (>3000 s). Time-resolved optoacoustics reveals phot-like, light-driven reactions on the ns-micros time window with the sub-nanosecond formation of a flavin triplet state (PhiT=0.46) that decays into the flavin-cysteine photoadduct with 2 micros lifetime (Phi390=0.42). The fluorescence spectrum and lifetime of the conserved W97 resembles the corresponding W103 in full-length YtvA, although the quantum yield, PhiF, is smaller (about 55% of YtvA) due to an enhanced static quenching efficiency. The anisotropy of W97 is the same as for W103 in YtvA (0.1), and considerably larger than the value of 0.06, found for W103 in YtvA-LOV. Different to YtvA and YtvA-LOV, the fluorescence for W97 becomes larger upon photoproduct formation. These data indicate that W97 is located in a similar environment as W103 in full-length YtvA, but undergoes larger light-driven changes. It is concluded that the protein segment located C-terminally to the LOV core (analogous to an interdomain linker) is enough to confer to the conserved tryptophan the fluorescence characteristics typical of full-length YtvA. The larger changes experienced by W97 upon light activation may reflect a larger conformational freedom of this protein segment in the absence of a second domain.     

Development of a fluoroimmunosensor for theophylline using immobilised antibody

A flowthrough theophylline fluoroimmunosensor with an antibody covalently immobilised on a solid support has been developed. The immobilisation technique proposed in this paper used Protein-A on control pore glass (Protein A-CPG) in an immunoreactor and dimethylsuberimidate as a cross-linking agent. Several supports and cross-linking reagents were tested in order to obtain oriented immobilisation and thus efficiency of the immunological reaction and reusability of the immunosensor. The immunosensor performance characteristics were established. The precision expressed as RSD, was 1.6%; the detection limit was 3 mug l(-1); the immunoreactor lifetime was established in 80 assays and there were no interferences with structurally similar compounds such as aminophylline, dihydroxypropyltheophylline and caffeine in the determination of the analyte. This fluoroimmunosensor was applied to determine theophylline in human serum samples from patients of the Puerta de Hierro Hospital in Madrid. The results obtained show that there are no significant differences between the proposed immunosensor and the high-pressure liquid chromatographic method with UV detection used by the Hospital, thus demonstrating the validity of the method.     

Metabolic engineering of Pseudomonas putida for methylmalonyl-CoA biosynthesis to enable complex heterologous secondary metabolite formation

An operon consisting of three open reading frames, annotated in silico as methylmalonyl-CoA (mm-CoA) epimerase, mm-CoA mutase (MCM), and meaB, was identified in the sequencing project of the myxobacterium Sorangium cellulosum So ce56. This putative MCM pathway operon was subcloned from a bacterial artificial chromosome by Red/ET recombineering onto a minimal replicon derived from p15A. This plasmid was modified for integration and heterologous expression in Pseudomonas putida to enable the production of complex secondary metabolites requiring mm-CoA as precursor. Methylmalonate was identified in the recombinant P. putida strain by an analysis method based on gas chromatography/mass spectrometry. The engineered strain is able to synthesize polyketides requiring mm-CoA as an extender unit, which was demonstrated by the production of myxothiazol after integration of the biosynthetic gene cluster into the chromosome, followed by induction of expression.     

Gene Sequence, Soluble Expression and Homologous Comparison of a D-Hydantoinase from Pseudomonas putida YZ-26

A 1440bp open-reading frame encoding D-hydantoinase from Pseudomonas putida YZ-26 was cloned and sequenced（ GenBank AY387829）. The DNA fragment was inserted into Nde and BamHI sites of vector pET-3a, yielding a recombinant plasmid, pET-HDT. After being transferred into the host strain, the artificial strain, pET-HDT/ E. coli BL21 can express the D-hydantoinase as the soluble form in the Lura-Bertani medium without addition of any inducers. The activity of the enzyme toward substrate DL-hydantoin can reach 3000-4000 IU per cells from one-liter bacterial culture incubated at 30 ℃ for 10-12 h. By the comparison of amino acid sequence homology, hydrophobic residues analysis and secondary structure prediction, it was found that D-hydantoinase reported herein is quite similar to that from Pseudomonas putdia CCRC12857, and alike to that from Pseudomonas putdia DSM84 or other bacteria. A rapid and efficient purification procedure of the enzyme was performed by a three-step procedure： ammonium sulfate fractionation, phenyl Sepharose hydrophobic interaction chromatography and Sephacryl S-200 gel filtration. The molecular mass of the monomeric enzyme is 52042 Da as determined by MALDI-TOF mass spectrometry.     

Substoichiometric determination of mercury by neutron activation analysis

A method has been developed for the substoichiometric determination of mercury by thermal neutron activation analysis, based on the selective extraction of the Hg(II)—Bindschedler's Green complex into 1,2-dichloroethane. The method has been applied for the determination of trace amounts of mercury in geological standards such as W-1, GR, Sye-1, and T-1, meteorite Allende de Publito, and biological materials such as kale, IR1 standard tobacco, and human blood serum.