Control of pyrimidine nucleotide formation in <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">fulva</Emphasis>

Control of pyrimidine formation was examined in Pseudomonas fulva ATCC 31418. Pyrimidine supplementation lowered pyrimidine biosynthetic pathway enzyme activities in cells grown on glucose or succinate as a carbon source indicating possible repression of enzyme synthesis. Pyrimidine limitation experiments were conducted using an orotidine 5′-monophosphate decarboxylase mutant strain isolated in this study. Compared to uracil-supplemented, glucose-grown mutant cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities to increase about 6-, 13-, 3-, 15-fold, respectively, which confirmed regulation of enzyme synthesis by pyrimidines. At the level of enzyme activity, transcarbamoylase activity in Ps. fulva was strongly inhibited by pyrophosphate, CTP, GTP and GDP under saturating substrate concentrations.     

Antioxidative response of <Emphasis Type="Italic">Hordeum maritimum</Emphasis> L<Emphasis Type="Italic">.</Emphasis> to potassium deficiency

The objective of the present study was to determine the influence of potassium deprivation on the halophyte species Hordeum maritimum grown in hydroponics for 2 weeks. Treatments were with potassium (+K) or without potassium (−K). Growth, water status, mineral nutrition, parameters of oxidative stress [malondialdehyde (MDA), carbonyl groups (C=O), and hydrogen peroxide concentration (H₂O₂) contents], antioxidant enzyme activities [superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPX, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate peroxidase (MDHAR, EC 1.6.5.4), dehydroascorbate peroxidase (DHAR, EC 1.8.5.1), and glutathione reductase (GR, EC 1.6.4.2)], and antioxidant molecules [ascorbate (ASC), and glutathione (GSH)] were determined. Results showed that the growth of vegetative organs decreased owing to potassium deficiency with roots (−36%) more affected than shoots (−12%). Water status was only diminished in roots (reduction of 24%). Potassium deprivation decreased potassium concentration in both organs, this decrease was more pronounced in roots (−81%) than in shoots (−55%). In contrast to carbonyl groups, MDA content increased owing to potassium deprivation. Except for CAT activity that remained unaffected; SOD, GPX, APX, GR, MDHAR, and DHAR activities were significantly increased. H₂O₂ concentration was negatively correlated with the activities of enzymes and the accumulation of non-enzymatic antioxidants implicated in its detoxification. In conclusion, a cooperative process between the antioxidant systems is important for the tolerance of H. maritimum to potassium deficiency.     

Synthesis and biotransformation of 2-alkyl-4(1<Emphasis Type="Italic">H</Emphasis>)-quinolones by recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

2-Alkyl-4(1H)-quinolones (AQs) and related derivatives, which exhibit a variety of biological properties, are secondary metabolites produced by, e.g., Pseudomonas and Burkholderia spp. Due to their main role as signaling molecules in the quorum sensing system of Pseudomonas aeruginosa, 2-heptyl-4(1H)-quinolone (HHQ) and its 3-hydroxy derivative, termed the “Pseudomonas quinolone signal” (PQS), have received considerable attention. Since chemical synthesis of different AQs is complex, we assessed the applicability of recombinant P. putida KT2440 strains for the biosynthetic production of AQs. In mineral salts medium supplemented with octanoate and anthranilate, batch cultures of P. putida KT2440 [pBBR-pqsABCD] produced about 45 μM HHQ, 30% and 70% of which were localized in the culture supernatant and methanolic cell extract, respectively. 2,4-Dihydroxyquinoline and minor amounts of C₃- to C₁₃-saturated and C_7:1 to C_13:1 monounsaturated AQs were formed as by-products. Mass spectrometry and nuclear magnetic resonance analyses spectroscopy indicated that unsaturated AQs having the same molecular mass are cis and trans isomers rather than position isomers, with the double bond located between the α and β carbon of the alkyl chain. Supplementing the cultures with hexanoate instead of octanoate shifted the AQ profile towards increased formation of C₅-AQ. Individual AQs can be prepared from concentrated methanolic extracts by preparative high-performance liquid chromatography (HPLC). Regioselective hydroxylation of HHQ to PQS can be achieved in >90% yield by biotransformation with P. putida KT2440 [pBBR-pqsH]. PQS can be isolated from methanolic cell extracts by HPLC, or be precipitated as Fe(III)-PQS complex. Preparation of a library of AQs will facilitate studies on the biological functions of these compounds.     

Comparison of the Arylamine <Emphasis Type="Italic">N-</Emphasis>Acetyltransferase from <Emphasis Type="Italic">Mycobacterium marinum</Emphasis> and <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Arylamine N-acetyltansferase (NAT) from Mycobacterium tuberculosis (TBNAT) is a potential drug target for anti-tubercular therapy. Recombinant TBNAT is much less soluble and is produced in lower yields than the closely related NAT from Mycobacterium marinum (MMNAT). In order to explore MMNAT as a model for TBNAT in drug discovery, we compare the two mycobacterial NAT enzymes. Two site-directed mutants of MMNAT have been prepared and characterised: MMNAT71, Tyr → Phe and MMNAT209, Met → Thr, in which residues within 6 Å of the active-site cysteine have been replaced with the corresponding residue from TBNAT. Two chimeric proteins have also been produced in which the third domain of MMNAT has been replaced by the third domain of TBNAT and vice versa. The activity profile of the chimeric proteins suggests a role for the third domain in the evolutionary divergence of NAT between these closely related mycobacterial species.     

Expression,Characterization, and Site-Directed Mutation of a Multiple Herbicide-Resistant Acetohydroxyacid Synthase (rAHAS) from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. Lm10

A multiple herbicide-resistant acetohydroxyacid synthase (rAHAS) gene was cloned from Pseudomonas sp. Lm10. Sequence analysis showed that the rAHAS regulatory subunit was identical to that of Pseudomonas putida KT2440 (sensitive AHAS, sAHAS), whereas six different sites [H134→N (rAHAS→sAHAS), A135→P, S136→T, I210→V, F264→Y, and S486→W] were found in the catalytic subunit. The rAHAS and sAHAS were over expressed, purified and characterized. rAHAS showed higher resistance to four kinds of AHAS-inhibitor herbicides than sAHAS. The resistance factor of rAHAS was 56.0-fold, 12.6-fold, 6.5-fold, and 9.2-fold as compared with sAHAS when metsulfuron-methyl, imazethapyr, flumetsulam, and pyriminobac-methyl used as inhibitor, respectively. The specific activity of rAHAS was lower than that of sAHAS and the K _m value of rAHAS for pyruvate was approximately onefold higher than the corresponding value for sAHAS. Data from site-directed mutagenesis demonstrated that alteration at A135, F264, and S486 resulted in resistance reduction, while the mutation at H134, S136, and I210 has little effect on the resistance. A135 was mainly responsible for resistance to imidazolinone; F264 conferred resistance to sulfonylurea and triazolopyrimidine sulfonamide; and S486 showed multiple herbicides resistance to the four herbicides.     

Structure-function relationship of substrate length specificity of dextran glucosidase from <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Dextran glucosidase from Streptococcus mutans (SMDG), an exo-type glucosidase of glycoside hydrolase (GH) family 13, specifically hydrolyzes an α-1,6-glucosidic linkage at the non-reducing ends of isomaltooligosaccharides and dextran. SMDG shows the highest sequence similarity to oligo-1,6-glucosidases (O16Gs) among GH family 13 enzymes, but these enzymes are obviously different in terms of substrate chain length specificity. SMDG efficiently hydrolyzes both short-and long-chain substrates, while O16G acts on only short-chain substrates. We focused on this difference in substrate specificity between SMDG and O16G, and elucidated the structure-function relationship of substrate chain length specificity in SMDG. Crystal structure analysis revealed that SMDG consists of three domains, A, B, and C, which are commonly found in other GH family 13 enzymes. The structural comparison between SMDG and O16G from Bacillus cereus indicated that Trp238, spanning subsites +1 and +2, and short β → α loop 4, are characteristic of SMDG, and these structural elements are predicted to be important for high activity toward long-chain substrates. The substrate size preference of SMDG was kinetically analyzed using two mutants: (i) Trp238 was replaced by a smaller amino acid, alanine, asparagine or proline; and (ii) short β → α loop 4 was exchanged with the corresponding loop of O16G. Mutant enzymes showed lower preference for long-chain substrates than wild-type enzyme, indicating that these structural elements are essential for the high activity toward long-chain substrates, as implied by structural analysis.     

Functional characterization of a gene encoding a dual domain for uridine kinase and uracil phosphoribosyltransferase in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) are enzymes catalyzing the formation of uridine 5′-monophosphate (UMP) from uridine and adenine 5′-triphosphate (ATP) and from uracil and phosphoribosyl-α-1-pyrophosphate (PRPP), respectively, in the pyrimidine salvage pathway. Here, we report the characterization and functional analysis of a gene AtUK/UPRT1 from Arabidopsis thaliana. Sequencing of an expressed sequence tag clone of this gene revealed that it contains a full-length open reading frame of 1461 nucleotides and encodes a protein with a molecular mass of approximately 53 kDa. The sequence analysis revealed that the N-terminal region of AtUK/UPRT1 contains a UK domain and the C-terminal region consists of a UPRT domain. Expression of AtUK/UPRT1 in upp and upp-udk mutants of Escherichia coli supplied with 5-fluorouracil (5-FU) and 5-fluorouridine (5-FD) led to growth inhibition. Identical results were obtained with 5-FD and 5-FU treatments when the UK and UPRT domains were separated by the introduction of translation initiation and stop codons prior to complementation into the upp-udk and upp mutants. These results suggest that the AtUK/UPRT1 product can use uracil and uridine as substrates for the production of UMP. We also investigated the function of AtUK/UPRT1 in an Arabidopsis mutant. The wild-type Arabidopsis plants showed drastic growth retardation when they were treated with 5-FU and 5-FD while the growth of atuk/uprt1 mutant plants was not significantly affected. These findings confirm that AtUK/UPRT1 has a dual role in coding for both uridine kinase and uracil phosphoribosyltransferase that form UMP through the pyrimidine salvage pathway in Arabidopsis.     

Kinetics of styrene biodegradation by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. E-93486

The research into kinetics of styrene biodegradation by bacterial strain Pseudomonas sp. E-93486 coming from VTT Culture Collection (Finland) was presented in this work. Microbial growth tests in the presence of styrene as the sole carbon and energy source were performed both in batch and continuous cultures. Batch experiments were conducted for initial concentration of styrene in the liquid phase changed in the range of 5–90 g m⁻³. The Haldane model was found to be the best to fit the kinetic data, and the estimated constants of the equation were: μ _m = 0.1188 h⁻¹, K _S = 5.984 mg l⁻¹, and K _i = 156.6 mg l⁻¹. The yield coefficient mean value Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} for the batch culture was 0.72 g_{dry cells weight} (g_substrate)⁻¹. The experiments conducted in a chemostat at various dilution rates (D = 0.035–0.1 h⁻¹) made it possible to determine the value of the coefficient for maintenance metabolism m _d = 0.0165 h⁻¹ and the maximum yield coefficient value Y_\textxs^\textM = 0.913 Y_{\text{xs}}^{\text{M}} = 0.913 . Chemostat experiments confirmed the high value of yield coefficient Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} observed in the batch culture. The conducted experiments showed high activity of the examined strain in the styrene biodegradation process and a relatively low sensitivity to inhibition of its growth at higher concentrations of styrene in the solution. Such exceptional features of Pseudomonas sp. E-93486 make this bacterial strain the perfect candidate for technical applications.     

Characterization of the <Emphasis Type="Italic">Pragia fontium</Emphasis> Lipopolysaccharides

Lipopolysaccharides (LPSs) of two strains Pragia fontium 97U116 and 27480 were isolated and characterized; they were close to those of other representatives of the family Enterobacteriaceae in fatty acid composition and contained, respectively, 3-hydroxytetradecanoic acid as the predominant component (45.8 and 45.1%), tetradecanoic (23.5 and 28.9%), hexadecanoic (12.6 and 7.9%), hexadecenoic (12.6 and 7.9%), and dodecanoic (4.9 and 4.2%) fatty acids. The O-specific polysaccharides consisted of linear penta- and tetrasaccharide repeating units: →2)-α-D-Galf-(1→3)-α-L-Rhap2Ac-(1→4)-α-D-GlcpNAc-(1→2)-α-L-Rhap-(1→3)-β-D-GlcpNAc-(1→ →4)-β-D-ManpNAc3NAcA-(1→2)-α-L-Rhap-(1→3)-β-L-Rhap-(1→4)-α-D-GlcpNAc-(1→ The LPSs of P. fontium 97U116 and 27480 were serologically active and belonged to different serogroups; they were less toxic than those of strain E. coli O55:B5, but more pyrogenic than the Pyrogenal preparation.     

Inhibition of hydrogen uptake in <Emphasis Type="Italic">Escherichia coli</Emphasis> by expressing the hydrogenase from the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis>sp. PCC 6803

Background  

Molecular hydrogen is an environmentally-clean fuel and the reversible (bi-directional) hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 as well as the native Escherichia coli hydrogenase 3 hold great promise for hydrogen generation. These enzymes perform the simple reaction 2H⁺ + 2e^- ↔ H₂ (g).     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

Microcalorimetric Study of <Emphasis Type="Italic">Tetrahymena</Emphasis> Growth Affected by Copper(II) Complexes

By means of microcalorimetry, the effect of four copper(II) complexes on Tetrahymena growth was investigated. The extent and duration of the inhibitory effect on the metabolism, judged by the rate constant, k, and the half inhibition concentration, IC₅₀, varied with the different complexes. The results showed that the half inhibition concentrations IC₅₀ of CuCl₂, (C₉H₆NO)₂Cu and [Cu(phen)₂]Cl₂⋅6H₂O were 9.9 × 10⁻⁴, 2.0 × 10⁻⁴, and 2.6 × 10⁻⁴ mol/L, respectively. The sequence of antibiotic activity of these three complexes was: (C₉H₆NO)₂Cu > [Cu(phen)₂]Cl₂⋅6H₂O > CuCl₂. The growth rate constants of [Cu(phen)₃]Cl₂⋅6H₂O did not change obviously with the increase of concentrations, but [Cu(phen)₃]Cl₂⋅6H₂O also can prolong the time of Tetrahymena growth.     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

The peroxidase activity of cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from spinach chloroplasts

The cytochrome b ₆ f (Cyt b ₆ f) complex, which functions as a plastoquinol-plastocyanin oxidoreductase and mediates the linear electron flow between photosystem II (PSII) and photosystem I (PSI) and the cyclic electron flow around PSI, was isolated from spinach (Spinacia oleracea L.) chloroplasts using n-octyl-β-D-glucopyranoside (β-OG). The preparation was also able to catalyze the peroxidase-like reaction in the presence of hydrogen peroxide (H₂O₂) and guaiacol. The optimal conditions for peroxidase activity of the preparation included: pH 3.6, ionic strength 0.1, and temperature 35°C. The apparent Michaelis constant (K _m) values for H₂O₂ and guaiacol were 50 mM and 2 mM, respectively. The bimolecular rate constant (k _obs) was about 26 M⁻¹ s⁻¹ and the turnover number (K _cat) was about 60 min⁻¹ (20 mM guaiacol, 100 mM sodium phosphate, pH 3.6, 25°C, [H₂O₂]<100mM). These parameters were similar to those of several other heme-containing proteins, such as myoglobin and Cyt c.     

The <Emphasis Type="Italic">b</Emphasis><Subscript>arg36</Subscript> contributes to efficient coupling in F<Subscript>1</Subscript>F<Subscript>O</Subscript> ATP synthase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In Escherichia coli, the F₁F_O ATP synthase b subunits house a conserved arginine in the tether domain at position 36 where the subunit emerges from the membrane. Previous experiments showed that substitution of isoleucine or glutamate result in a loss of enzyme activity. Double mutants have been constructed in an attempt to achieve an intragenic suppressor of the b _arg36→ile and the b _arg36→glu mutations. The b _arg36→ile mutation could not be suppressed. In contrast, the phenotypic defect resulting from the b _arg36→glu mutation was largely suppressed in the b _{arg36→glu,glu39→arg} double mutant. E. coli expressing the b _{arg36→glu,glu39→arg} subunit grew well on succinate-based medium. F₁F_O ATP synthase complexes were more efficiently assembled and ATP driven proton pumping activity was improved. The evidence suggests that efficient coupling in F₁F_O ATP synthase is dependent upon a basic amino acid located at the base of the peripheral stalk.     

Effect of trace metals on ethanol production from synthesis gas by the ethanologenic acetogen, <Emphasis Type="Italic">Clostridium</Emphasis><Emphasis Type="Italic">ragsdalei</Emphasis>

The effect of trace metal ions (Co²⁺, Cu²⁺, Fe²⁺, Mn²⁺, Mo⁶⁺, Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻) on growth and ethanol production by an ethanologenic acetogen, Clostridium ragsdalei was investigated in CO:CO₂-grown cells. A standard acetogen medium (ATCC medium no. 1754) was manipulated by varying the concentrations of trace metals in the media. Increasing the individual concentrations of Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻ from 0.84, 6.96, 1.06, and 0.68 μM in the standard trace metals solution to 8.4, 34.8, 5.3, and 6.8 μM, respectively, increased ethanol production from 35.73 mM under standard metals concentration to 176.5, 187.8, 54.4, and 72.3 mM, respectively. Nickel was necessary for growth of C. ragsdalei. Growth rate (μ) of C. ragsdalei improved from 0.34 to 0.49 (day⁻¹), and carbon monoxide dehydrogenase (CODH) and hydrogenase (H₂ase)-specific activities improved from 38.45 and 0.35 to 48.5 and 1.66 U/mg protein, respectively, at optimum concentration of Ni²⁺. At optimum concentrations of WO₄ ⁻ and SeO₄ ⁻, formate dehydrogenase (FDH) activity improved from 32.3 to 42.6 and 45.4 U/mg protein, respectively. Ethanol production and the activity of FDH reduced from 35 mM and 32.3 U/mg protein to 1.14 mM and 8.79 U/mg protein, respectively, upon elimination of WO₄ ⁻ from the medium. Although increased concentration of Zn²⁺ enhanced growth and ethanol production, the activities of CODH, FDH, H₂ase and alcohol dehydrogenase (ADH) were not affected by varying the Zn²⁺ concentration. Omitting Fe²⁺ from the medium decreased ethanol production from 35.7 to 6.30 mM and decreased activities of CODH, FDH, H₂ase and ADH from 38.5, 32.3, 0.35, and 0.68 U/mg protein to 9.07, 7.01, 0.10, and 0.24 U/mg protein, respectively. Ethanol production improved from 35 to 54 mM when Cu²⁺ was removed from the medium. The optimization of trace metals concentration in the fermentation medium improved enzyme activities (CODH, FDH, and H₂ase), growth and ethanol production by C. ragsdalei.     

Degradation of ethylbenzene by free and immobilized <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">fluorescens-</Emphasis>CS2

Pseudomonas fluorescens-CS2 metabolized ethylbenzene as the sole source of carbon and energy. The involvement of catechol as the hydroxylated intermediate during the biodegradation of ethylbenzene was established by TLC, HPLC and enzyme analysis. The specific activity of Catechol 2,3-dioxygenase in the cell free extracts of P. fluorescens-CS2 was determined to be 0.428 μmoles min⁻¹ mg⁻¹ protein. An aqueous-organic, Two-Phase Batch Culture System (TPBCS) was developed to overcome inhibition due to higher substrate concentrations. In TPBCS, P. fluorescens-CS2 demonstrated ethylbenzene utilization up to 50 mM without substrate inhibition on inclusion of n-decanol as the second phase. The rate of ethylbenzene metabolism in TPBCS was found enhance by fivefold in comparison with single phase system. Alternatively the alginate, agar and polyacrylamide matrix immobilized P. fluorescens-CS2 cells efficiently degraded ethylebenzene with enhanced efficiency compared to free cell cultures in single and two-phase systems. The cells entrapped in ployacrylamide and alginate were found to be stable and degradation efficient for a period of 42 days where as agar-entrapped P. fluorescens was stable and efficient a period of 36 days. This demonstrates that alginate and polyacrylamide matrices are more promising as compared to agar for cell immobilization.     

Substrate specificity of a long-chain alkylamine-degrading <Emphasis Type="Italic">Pseudomonas</Emphasis> sp isolated from activated sludge

A bacterium strain BERT, which utilizes primary long-chain alkylamines as nitrogen, carbon and energy source, was isolated from activated sludge. This rod-shaped motile, Gram-negative strain was identified as a Pseudomonas sp. The substrate spectrum of this Pseudomonas strain BERT includes primary alkylamines with alkyl chains ranging from C₃ to C₁₈, and dodecyl-1,3-diaminopropane. Amines with alkyl chains ranging from 8 to 14 carbons were the preferred substrates. Growth on dodecanal, dodecanoic acid and acetic acid and simultaneous adaptation studies indicated that this bacterium initiates degradation through a C_alkyl–N cleavage. The cleavage of alkylamines to the respective alkanals in Pseudomonas strain BERT is mediated by a PMS-dependent alkylamine dehydrogenase. This alkylamine dehydrogenase produces stoichiometric amounts of ammonium from octylamine. The PMS-dependent alkylamine was found to oxidize a broad range of long-chain alkylamines. PMS-dependent long-chain aldehyde dehydrogenase activity was also detected in cell-free extract of Pseudomonas strain BERT grown on octylamine. The proposed pathway for the oxidation of alkylamine in strain BERT proceeds from alkylamine to alkanal, and then to the fatty acid.