Restricted oxygen supply and excretion of metabolites

Summary The effect of restricted oxygen supply on the excretion of metabolites was studied in Pseudomonas acidovorans (DSM 39), P. delafieldii (DSM 64) and a mutant strain of Paracoccus denitrificans unable to accumulate poly-3-hydroxybutanoic acid. Different metabolites were produced at distinct submaximum respiration rates by these strains. These metabolites were, in order of decreasing respiration rates; 2-oxoglutarate, 2-oxo-3-methylbutanoate, cisaconitate, 3-hydroxybutanoate, succinate, hydrogen gas, formate, acetate, butanoate, acetoin, meso- and D,L-2,3-butanediol, and ethanol. Poly-3-hydroxy-butanoic acid (PHB) accumulated intracellularly at almost the same respiration rates at which the excretion of 3-hydroxybutanoate occurred.The production of ethanol, 2,3-butanediol, butanoate, formate, and hydrogen gas indicate the function of enzymes such as ethanol and butanediol dehydrogenases, pyruvate formate lyase, formate hydrogen lyase, and butanoyl-CoA dehydrogenase. These enzymes are not expected to be present in strict aerobes at different degrees of restricted oxygen supply.Excreted metabolites are indicators of the degree to which the oxygen demand of cells is met. On the other hand, a fermentation process designed for the production of a distinct metabolite can be controlled by maintaining the appropriate oxygen supply.     

A new member of the short-chain dehydrogenases/reductases superfamily: Purification, characterization and substrate specificity of a recombinant carbonyl reductase from Pichia stipitis

A novel short-chain dehydrogenases/reductases superfamily (SDRs) reductase (PsCR) from Pichia stipitis that produced ethyl (S)-4-chloro-3-hydroxybutanoate with greater than 99% enantiomeric excess, was purified to homogeneity using fractional ammonium sulfate precipitation followed by DEAE-Sepharose chromatography. The enzyme purified from recombinant Escherichia coli had a molecular mass of about 35 kDa on SDS–PAGE and only required NADPH as an electron donor. The K_m value of PsCR for ethyl 4-chloro-3-oxobutanoate was 4.9 mg/mL and the corresponding V_max was 337 μmol/mg protein/min. The catalytic efficiency value was the highest ever reported for reductases from yeasts. Moreover, PsCR exhibited a medium-range substrate spectrum toward various keto and aldehyde compounds, i.e., ethyl-3-oxobutanoate with a chlorine substitution at the 2 or 4-position, or α,β-diketones. In addition, the activity of the enzyme was strongly inhibited by SDS and β-mercaptoethanol, but not by ethylene diamine tetra acetic acid.     

Production of D(-)-lactic acid from cellulose by simultaneous saccharification and fermentation using Lactobacillus coryniformis subsp. torquens

D(–)-Lactic acid was produced from cellulose by simultaneous saccharification and fermentation (SSF) in media containing cellulolytic enzymes and Lactobacillus coryniformis subsp. torquens ATCC 25600 at 39 °C and pH 5.4, yielding 0.89 g D(–)-lactic acid g^–1 cellulose at a mean volumetric productivity of 0.5 g l^–1 h^–1. No L(+)-lactic acid was found in the medium.     

Biphasic nitrogenase activity in Azospirillum brasilense in long lasting batch cultures

Long lasting batch cultures of Azospirillum brasilense SP 7 ATCC 29145 grown in liquid malate medium for 8–14 days without any fixed nitrogen source exhibited a biphasic nitrogenase activity, when incubated under gas atmospheres of 99.0% N₂ and 1.0% O₂ or 99.5% N₂ and 0.5% O₂ respectively. Maximum specific nitrogenase activity was 1100 nmol C₂H₄·mg protein^-1·h^-1. Poly-3-hydroxybutanoic acid (PHBA) synthesis and growth of the cells also showed two phases. Maxima and minima of glutamine synthetase activity developed synchronously with nitrogenase activity, whereas those of glutamate dehydrogenase and alanine aminotransferase were reverse. During a 192 h period of growth protein increased 3–4-fold and PHBA 25 fold. At maximum accumulation of the polymer the PHBA-nitrogen ratio was 6:1 or 8:1. Azospirillum brasilense was also able to fix nitrogen on agar surfaces exposed to air, but nitrogen fixation was monophasic under these conditions during a 14 d period. Specific nitrogenase activity was dependent on the type and concentration of the source of fixed nitrogen (leucine, ammonia) in solidified media. With 1 mM leucine maximum specific nitrogenase activity was 110 nmol C₂H₄·mg protein^-1·h^-1.Non-Standard Abbreviations PHBA poly-3-hydroxybutanoic acid - TAPS tris(hydroxymethyl)methylaminopropane sulfonic acid - TES N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid - TRICINE N-tris(hydroxymethyl)methylglycine - TRIS tris(hydroxymethyl)aminomethane     

Determination of carnitine, butyrobetaine, and betaine as 4′-bromophencyl ester derivatives by high-performance liquid chromatography

A method for determination of carnitine, 4-(N,N,N-trimethylammonio)butanoate (butyrobetaine), and 2-(N,N,N-trimethylammonio)acetate (betaine) is described. These ω-trimethylammonio carboxylates and the chemically analogous internal standards 4-(N,N-dimethyl-N-propylammonio)-3-hydroxybutanoate or 5-(N,N,N-trimethylammonio)hexanoate were derivatized by reaction wiht 4′-bromophenacyl triflate in the presence of N,N-diisopropylethylamine. The trialkylammonio carboxylate 4′-bromophenacyl ester derivatives were separated from other sample constituents by reversed-phase ion-pair high-performance liquid chromatography with spectrophotometric detection at 254 nm. Standard curves were linear over a sample concentration range of 10–100 nmol/ml. Quantities of 2.5 nmol of ω-trialkylammonio acid derivatives injected into the chromatography were detected with signal-to-noise ratios greater than 50.     

Kinetic resolution of 2-hydroxybutanoate racemic mixtures by NAD-independent L-lactate dehydrogenase

Optically active d-2-hydroxybutanoate is an important building block intermediate for medicines and biodegradable poly(2-hydroxybutanoate). Kinetic resolution of racemic 2-hydroxybutanoate may be a green and desirable alternative for d-2-hydroxybutanoate production. In this work, d-2-hydroxybutanoate at a high concentration (0.197 M) and a high enantiomeric excess (99.1%) was produced by an NAD-independent l-lactate dehydrogenase (l-iLDH) containing biocatalyst. 2-Oxobutanoate, another important intermediate, was co-produced at a high concentration (0.193 M). Using a simple ion exchange process with the macroporous anion exchange resin D301, d-2-hydroxybutanoate was separated from the biotransformation system with a high recovery of 84.7%.     

The production of hemicellulose-degrading enzymes byBacillus macerans in anaerobic culture

Summary The cell-associated and exocellular hemicellulolytic polysaccharide depolymerase and glycoside hydrolase activity ofBacillus macerans NCDO 1764 was monitored over a range of anaerobic growth conditions in batch and continuous culture. The enzymes were detectable throughout the complete growth cycle in batch culture reaching and maintaining maximum levels in the stationary phase. In continuous culture enzyme activity was largely independent of growth rate (D=0.025–0.1 h^-1) although the activity was reduced at higher dilution rates (0.125–0.15 h^-1). Although activity was detectable over a wide pH range (pH 5.5–7.5) it was pH dependent, and maximum activities of both the cell-associated and exocellular enzymes were measured in cultures maintained at pH 6.5–7.0±0.1.The principal metabolites formed anaerobically from xylose byB. macerans in batch and continuous culture were acetic acid, formic acid and ethanol which represented 95–99% of the products formed. Smaller amounts of acetone,d,l-lactic acid and succinic acid were formed together with traces of butyric acid (<5 nmol/ml) and isovaleric acid (<25 nmol/ml). The proportions of the metabolites produced varied with growth conditions and were influenced by the pH of the culture and the rate and stage of growth of the microorganism.     

The Structure of Uncommon Lipid A from the Marine Bacterium <Emphasis Type="Italic">Marinomonas communis</Emphasis> ATCC 27118<Superscript>T</Superscript>

Lipid A was obtained in a high yield (27%) by the hydrolysis of lipopolysaccharide from the marine gamma proteobacterium Marinomonas communis ATCC 27118^T with 1% AcOH. Using chemical analysis and 1D and 2D NMR spectroscopic and fast atom bombardment mass spectrometric methods, it was shown to be β-1′,6-linked D-glucosaminobiose 1-phosphate acylated with (R)-3-dodecanoyl- or (R)-3-decanoyloxydecanoic acid, (R)-3-{(R)-3-hydroxydecanoyloxy)]decanoic acid and (R)-3-hydroxydecanoic acid at the C2, C2′ and C3 positions, respectively. Uncommon structural peculiarities (a low acylation and phosphorylation degree) of the M .communis lipid A in comparison with those of terrestrial bacteria may be of pharmacological interest. The potential physiological meaning of this lipid A and compounds of similar structure are discussed.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 404–413.Original Russian Text Copyright © 2005 by Vorob’eva, A. Dmitrenok, P. Dmitrenok, Isakov, Krasikova, Solov’eva.The article was translated by the authors.     

Continuous production of poly(3-hydroxybutyrate-co-3-hyhroxyvalerate) byAlcaligenes eutrophus

Summary Copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) were produced in a continuous culture ofAlcaligenes eutrophus with 17.5 gl ^–1 of fructose and 2.5gl ^–1 of pentanoic acid in the feed. The P(3HB-co-3HV) productivity was maximal at a dilution rate of 0.17h^–1 and yielded 0.31gl ^–1h^–1 under an ammonium-limited condition. The 3HV content in copolymers increased from 11 to 79 mol% as the dilution rate of cells was increased from 0.06 to 0.32h^–1.     

Production of propionic acid by mixed cultures ofPropionibacterium shermanii andLactobacillus casei in autoclave-sterilized whey

Summary Pure cultures ofPropionibacterium freudenreichii ss.shermanii did not grow in autoclave-sterilized cheese whey (121°C, 15 psi, 20 min) at whey concentrations greater than 2% (w/v) spray-dried sweet dairy whey. Propionic acid was produced from autoclave-sterilized whey by growingP. shermanii in mixed culture withLactobacillus casei. In medium containing 5–12% autoclaved whey solids and 1% yeast extract, the mixed culture produced 1.3–3.0% propionic acid, 0.5–1.0% acetic acid, and 0.05–0.80% lactic acid. All the lactose was consumed. Using pH-controlled fermentors (pH=7.0), mixed cultures produced at least 30% more propionic acid than cultures in which pH was not controlled.     

Quantification of carnitine and specific acylcarnitines by high-performance liquid chromatography: application to normal human urine and urine from patients with methylmalonic aciduria, isovaleric acidemia or medium-chain acyl-CoA dehydrogenase deficiency

This paper describes the development of a high-performance liquid chromatographic method for the quantitation of free carnitine, total carnitine, acetylcarnitine, propionylcarnitine, isovalerylcarnitine, hexanoylcarnitine and octanoylcarnitine in human urine. Carnitine and acylcarnitines were isolated from 10 or 25 μl of urine using 0.5-ml columns of silica gel, derivatized with 4'-bromophenacyl trifluoromethanesulfonate and separated by high-performance liquid chromatography. Using 4-(N,N-dimethyl-N-ethylammonio)-3-hydroxybutanoate (“e-carnitine”) as the internal standard, standard curves (10–300 nmol/ml) were generated. Carnitine and acylcarnitines were quantified (when they were present) in normal human urine and the urine of patients diagnosed with one of three different disorders of organic acid metabolism: methylmalonic aciduria, isovaleric acidemia, and medium-chain acyl-CoA dehydrogenase deficiency.     

Fermentative production of P(3HB-co-3HV) from propionic acid by Alcaligenes eutrophus in fed-batch culture with pH-stat continuous substrate feeding method

The feeding of propionic acid for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by Alcaligenes eutrophus ATCC17697 was optimized using a fed-batch culture system. The concentration of propionic acid was maintained at 3 g l^–1 as growth was inhibited by propionic acid in the broth. A pH-stat substrate feeding system was used in which propionic acid was fed automatically to maintain a pH of the culture broth at 7.0. By feeding a substrate solution containing 20% (w/v) propionic acid, 4.9% (w/v) ammonia water [at a molar ratio of carbon to nitrogen (C/N molar ratio) of 10] in cell growth phase, the concentration of propionic acid in the broth was maintained at 3 g l^–1 giving a specific growth rate of 0.4 h^–1. To promote P(3HB-co-3HV) production, two stage fed-batch culture which consisted of the stage for the cell growth and the stage for the P(3HB-co-3HV) accumulation was carried out. When the substrate solution whose C/N molar ratio was 50 was fed in P(3HB-co-3HV) accumulation phase, the cell concentration and the P(3HB-co-3HV) content in the cells reached 64 g l^–1 and 58% (w/w) in 55.5 h, respectively.     

Chemical and enzymatic approaches to the synthesis of optically pure ethyl (R)-4-cyano-3-hydroxybutanoate

Ethyl (R)-4-cyano-3-hydroxybutanoate (HN) is an important chiral synthon for side chain of the cholesterol-lowering drug atorvastatin (Lipitor), which is the hydroxymethylglutaryl CoA reductase inhibitor. HN is also used as a synthon in the production of l-carnitine and (R)-4-amino-3-hydroxybutanoic acid. It is necessary to have a clear understanding of the synthesis process of HN for its extensive use. This review gives an overview of different synthetic strategies of optically active HN, including chemical and enzymatic approaches. The emphasis is focused mainly on the synthetic routes using biocatalysts, such as halohydrin dehalogenase, nitrilase, carbonyl reductase, and lipase.     

In vitro regeneration of Camellia sinensis (L.) O. Kuntze by somatic embryogenesis

Within 3 weeks of culture, excised cotyledon expiants of Camellia sinensis (L.) O. Kuntze produced somatic embryos without intermediate callus when cultured in Murashige and Skoog's basal medium with 30 g^–1 sucrose. In medium without plant growth regulators, up to 60% of the cultures developed somatic embryos. Embryogenic competence was reduced by increasing concentrations of plant growth regulators tested (i.e. kinetin, 6-benzylaminopurine, and indole butyric acid). The somatic embryos developed, grew to maturity without being subcultured within 6–8 weeks. Secondary embryogenesis was not observed. Germination of isolated mature somatic embryos was low in medium without plant growth regulators. Up to 53% and 60% germination occurred when medium impregnated with kinetin at 1.8 mgl^–1 or 1.0 mgl^–1 6-benzylaminopurine were used respectively. Callus was also routinely produced when cotyledons were cultured in MS basal medium with auxins (2,4-dichlorophenoxyacetic acid and indole acetic acid). Callus induction was however, also achieved in plant growth regulator free medium. Indirect somatic embryogenesis was not induced in the present study.Abbreviations K kinetin - BAP 6-benzylaminopurine - IBA indole butyric acid - IAA indole acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA Naphthalene acetic acid - Fe-EDTA Ethylenediaminetetra-acetic acid (Ferric monosodium salt)     

Effects of ascorbic acid on axillary shoot induction in Tylophora indica (Burm. f.) Merrill.

A procedure for rapid in vitro multiplication of Tylophora indica (Burm. f.) Merrill., an important indigenous medicinal plant, has been developed. Addition of ascorbic acid was essential to induce sprouting of axillary buds. Optimum multiplication was observed on MS medium containing 6-benzylamino purine (5.0 mg l^–1), -naphathalene-acetic acid (0.5 mg l^–1) and ascorbic acid (100 mg l^–1). Rooting of in vitro produced shoots was readily achieved with indole-3-acetic acid alone (1.0 mg l^–1) in MS. The plantlets thus obtained were successfully transferred to pots in large numbers which grew normally.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - Kn kinetin - MS Murashige & Skoog media - NAA -naphthalene acetic acid     

Shoot multiplication in cultures of mature <Emphasis Type="Italic">Alnus glutinosa</Emphasis>

Shoot cultures were initiated from mature trees of Alnus glutinosa. On medium containing 1–5 μM 6-benzylamino purine (BAP), the shoots elongated without branching, formed heavy callus at the base of the stems and readily formed roots. The possibility that these characteristics could be attributed to the strong influence of endogenous auxin was tested on media that contained two auxin transport inhibitors, 1-N- naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), at concentrations of 0.1–3 μM, in combination with 2 μM BAP. On these media, shoots produced numerous branches, less callus and no roots. After 30 weeks (five subcultures) on this medium, leaves were smaller and showed signs of vitrification. These problems were resolved without detriment to shoot proliferation, by reverting to medium without NPA or TIBA. Shoots rooted readily after transfer to medium without growth regulators and were successfully acclimatised after transfer to soil.     

In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium

In vitro plantlet regeneration systems for the seed geranium (Pelargonium x hortorum Bailey) using cotyledon, hypocotyl and root explants were optimized by studying the influence of seedling age, growth regulators and excision orientation on organogenesis. Indole-3-acetic acid combined with zeatin yielded the highest rate of shoot production on cotyledon explants (0.2–2 shoots per explant). More shoots were produced on explants cut from the most basal region of cotyledons from 2 to 4-day-old seedlings than from older seedlings or more distal cut sites. Hypocotyl explants produced the highest number of shoots, up to 40 shoots per explant, on indole-3-acetic acid (2.8–5.6 mM) + zeatin (4.6 mM) or thidiazuron (4.5 mM). Maximum shoot formation (0.3–1.4 shoots per explant) on root explants occurred when they were cultured on medium containing zeatin. Regenerated shoots rooted best on a basal medium containing no growth regulators. There were substantial differences among cultivars in shoot formation from each of the explant systems.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - TDZ thidiazuron     

Batch and continuous production of propionic acid from whey permeate by Propionibacterium acidi-propionici in a three-electrode amperometric culture system

Summary Growth of Propionibacterium acidi-propionici was studied on lactose as substrate and in acid whey permeate in a three-electrode poised-potential system with cobalt sepulchrate as artificial electron donor. In batch culture experiments in a stirred-tank reactor the substrate was fermented completely to propionic acid up to 6.5 g 1^–1 lactose in a supplemented whey permeate medium. No acetic acid was produced during the growth of P. acidi-propionici. An electron flow of 80–100 mA was obtained and the electron balance was 101%. In continuously growing cultures with 3 g 1^–1 of lactose as the substrate, propionate was formed as the only fermentation product up to a dilution rate (D) of 0.04 h^–1. With D>0.04 h^–1 the bacteria immobilized on the working electrode surface. It was examined whether an electron transfer occurred between the platinum working electrode and the immobilized cells. Correspondence to: W. Trösch     

Resolution and quantitation of pentazocine enantiomers in human serum by reversed-phase high-performance liquid chromatography using sulfated β-cyclodextrin as chiral mobile phase additive and solid-phase extraction

A sensitive and stereospecific HPLC method was developed for the analysis of (−)- and (+)-pentazocine in human serum. The assay involves the use of a phenyl solid-phase extraction column for serum sample clean-up prior to HPLC analysis. Chromatographic resolution of the pentazocine enantiomers was performed on a octadecylsilane column with sulfated-β-cyclodextrin (S-β-CD) as the chiral mobile phase additive. The composition of the mobile phase was aqueous 10 mM potassium dihydrogenphosphate buffer pH 5.8 (adjusted with phosphoric acid)–absolute ethanol (80:20, v/v) containing 10 mM S-β-CD at a flow-rate of 0.7 ml/min. Recoveries of (−)- and (+)-pentazocine were in the range of 91–93%. Linear calibration curves were obtained in the 20–400 ng/ml range for each enantiomer in serum. The detection limit based on S/N=3 was 15 ng/ml for each pentazocine enantiomer in serum with UV detection at 220 nm. The limit of quantitation for each enantiomer was 20 ng/ml. Precision calculated as R.S.D. and accuracy calculated as error were in the range 0.9–7.0% and 1.2–6.2%, respectively, for the (−)-enantiomer and 0.8– 7.6% and 1.2–4.6%, respectively, for the (+)-enantiomer (n=3).     

High-performance liquid chromatographic determination of selenocysteine with the fluorescent reagent, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid

The method described is based on derivatization of selenocysteine with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid and responds linearly to selenocysteine spiked into plasma. Recovery is insensitive to inter-individual variation or use of serum versus plasma, but is decreased by hemolysis. The derivative is stable for at least three days. The total imprecision of determinations in plasma was 0.8–2.1% (coefficient of variation) over the range of 6–30 μM selenocysteine, with a detection limit of 0.4 μM (3 × S.D.). There was no significant interference from plasma thiols. This appears to be the first report of the selective reaction of free selenocysteine with a fluorescent reagent. This simple method works well in plasma and serum and may be adaptable to other types of samples.