Kinetic determination of tryptophan by using the B-Z oscillating chemical system

A simple and rapid method was devised for determination of tryptophan, based on the Belousov-Zhabotinskii (B-Z) oscillating chemical system. Changes in oscillating period and amplitude were linearly proportional to the negative logarithm of l-tryptophan concentration over the range of 6.44 × 10⁻⁷–2.55 × 10⁻⁴ M, with the regression coefficients of near unity and a lower detection limit of 6.5 × 10⁻⁸ M. d-tryptophan was also examined although it is rarely found in most biological fluids, and perhaps not at all in natural proteins. The change of period against to negative logarithm of d-tryptophan concentration over the range of 4.9 × 10⁻⁵–8.24 × 10⁻⁴ M is linear. Because the optimum conditions for determination of l- and d-tryptophan are not the same, a little amount of d-tryptophan does not affect the determination of l-tryptophan. Various influences were studied and a possible mechanism of perturbation to the B-Z oscillator by tryptophan was also discussed. Spectrophotometry and fluorescence spectrophotofluorimetry were used for comparision and confirmation of the results.     

Synthesis of a coumarin based fluorescent amino acid

Summary l-2-amino-3-(6,7-dimethoxy-4-coumaryl)-propionic acid (l-Adp), as a non-proteinogenic fluorescent amino acid has been synthesized by a highly stereoselective routine (>99.5%). This fluorescent amino acid, as fluorophorequencher pair, may be used to study peptide assays. For enantiomeric excess determination, the racemicdl-Adp (dl-2-amino-3-(6,7-dimethoxy-4-coumaryl)-propionic acid) has also been synthesized.     

The determination of micro-organisms in air

Summary A brief discussion has been given of various devices available for the determination of micro-organisms in air. In order to investigate the efficiency of some of these devices it proved desirable to work with artificially contaminated air. Hereto clouds of spores ofBacillus cereus were produced with the aid of a modified model of aDe Vilbiss 40 nebulizer.Comparative tests were made with theFolin-bubbler, theWheeler-bubbler, theMoulton-atomizer and the capillary impinger as used byRosebury for the determination of the spore concentration in the contaminated air. The latter device proved to be by far the most efficient, 99% or more of the spores being retained by this simple apparatus.     

An apparatus for the continuous culture of bacteria at constant generation times

Summary An apparatus for continuous culture of bacteria at constant generation times is described, analogous toNovick andSzilard's “chemostat” and the apparatus ofMonod. Detailed instructions for the use of the apparatus are given and a method for the determination of the vessel constant is described. The theory and the interesting possibilities of the apparatus for a number of fundamental problems in bacteriology are shortly discussed.     

Evaluation of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose, a new fluorescent derivative of glucose, for viability assessment of yeast Candida albicans

A new fluorescent derivative of d-glucose, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), which had been previously developed for the analysis of glucose uptake activity by living cells, was investigated to evaluate its applicability for assaying the viability of yeast Candida albicans. Lineweaver-Burk plots showed the uptake of 2-NBDG to be competitively inhibited by d-glucose and not by l-glucose, which suggested the involvement of the glucose transporting system of C. albicans in the uptake of 2-NBDG. A good correlation was obtained between the yeast viability, determined by the plate-count method, and the 2-NBDG uptake activity of yeast cells (correlation constant: r=0.97). This is expected to lead to the development of a new fluorescent probe for the determination of yeast cell viability.     

A semi-quantitative high-throughput screening method for microbial <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine production in microtiter plates

In this study, we developed a fluorescence-based assay for quantifying the aromatic amino acid l-tyrosine in small sample volumes employing 96-well microtiter plates. The method was based on the specific derivatisation of l-tyrosine with 1-nitroso-2-naphthol and the formation of a stable complex for the fluorescent determination of l-tyrosine concentrations. The original procedure for l-tyrosine measurements in blood or tissue samples (Waalkes and Udenfriend in J Lab Clin Med 50:733–736, 1957) was modified to a simple assay suitable for high-throughput screening of l-tyrosine producing microorganisms such as engineered Escherichia coli.     

A fluorescent electrophilic reagent, 9-fluorenone-4-carbonyl chloride (FCC), for the enantioresolution of amino acids on a teicoplanin phase under the elution of the methanol-based solvent mixture

Summary. A fluorescent electrophilic reagent, 9-fluorenone-4-carbonyl chloride (FCC), is chosen to functionalize amino acids in alkaline medium before their HPLC resolution. FCC reacts with both primary and secondary amino acids to produce stable and highly fluorescent derivatives suitable for sensitive and efficient chromatographic determination and resolution on a teicoplanin chiral stationary phase (CSP) using the methanol-based solvent mixture as the mobile phase. The detection limit is in the picomole range and approximately 0.01% of the d-enantiomer in an excess of the l-enantiomer is detectable. However, the resolution is not reproducible under the elution of either the water- or the acetonitrile-based mobile phase. The increase in solubility of analyte in the mobile phase seems to be responsible. Upon comparison under the optimal chromatographic conditions, the resolution is better than that for the 9-fluorenylmethyl chloroformate (FMOC) or 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives reported previously.     

Improvement of α-l-arabinofuranosidase production by Talaromyces thermophilus and agro-industrial residues saccharification

This study is an application of an experimental design methodology for the optimization of the culture conditions of α-l-arabinofuranosidase production by Talaromyces thermophilus. Wheat bran and yeast extract were first selected as the best carbon and nitrogen sources, respectively, for enzyme production. A Plackett–Burman design was then used to evaluate the effects of eight variables. Statistical analyses showed that while pH had a negative effect on α-l-arabinofuranosidase production, wheat bran and MgSO₄ had a significantly positive effect. The values of the latter three parameters were further optimised using a central composite design and a response surface methodology. The experimental results were fitted to a second-order polynomial model that yielded a determination coefficient of R ² = 0.91. The statistical output showed that the linear and quadric terms of the three variables had significant effects. Using optimal conditions, the experimental value of α-l-arabinofuranosidase activity produced was very close to the model-predicted value. The optimal temperature and pH of enzyme activity were 55 °C and 7.0, respectively. This enzyme was very stable over a considerable pH range from 4 to 9. The crude enzyme of T. thermophilus rich in α-l-arabinofuranosidase was also used for saccharification of lignocellulosic materials and arabinose production.     

Studies on the growth of Ophicephalus striatus Bloch

Summary Growth ofOphicephalus striatus was studied from zones on the scales as these were found to be suitable for age determination. Lenght estimates obtained from the scale reading were further substantiated by back-calculations. Thevon Bertalanffy equation was employed to find out theoretical values of length in respect of various year classes. The theoretical values agreed with the observed values very closely.Length-weight relationship showed that the weight of fish increases slightly faster than the cube of the length. Thevon Bertalanffy equation gave a good fit to weight-age data.     

A photometer for the extinctiometric determination of the density of suspensions of microorganisms with minimal deviation from the law of Beer-Lambert

Summary The theory and construction of a photometer for the extinctiometric determination of the density of suspensions of bacteria are given. The application of the principle of directional selectivity and the use of sodium light leads to a satisfactory accuracy in the determination of the density of suspensions of bacteria. The results are in conformity with the law ofBeer-Lambert. The advantages of the proposed method are discussed.     

Microbial sensor for aspartame

Summary A microbial amperometric sensor using immobilized Bacillus subtilis cells was developed for the determination of the dipeptide sweetener aspartame (l-aspartyl-l-phenylalaninemethylester). From 0.07 to 0.6 mmol/l aspartame, a linear dependence of the initial current change (i.e., change in respiration rate) was obtained. The sensitivity for aspartame was one order of magnitude higher than for its amino acid constituents. The microbial sensor was stable for 8 weeks.     

Microbial sensor for selective determination of sulphide

A microbial sensor consisting of immobilized Thiobacillus thiooxidans, a gas-permeable membrane, and an O₂ electrode was prepared for the determination of sulphide. When a sample solution containing sulphide was passed into the flow cell, the output of the microbial sensor decreased markedly with time until a steady state was reached. The total time required for an assay was 20–30 min by the steady-state method. In the pulse method, the total time required for an assay was about 5 min. A linear relationship was obtained between the sensor output and the concentration of sodium sulphide below 0.40 mm. The minimum detectable concentration of sodium sulphide was 0.02 mm. Selectivity of the sensor was satisfactory. The microbial sensor was applied to the determination of sulphide in spring water. A good agreement was obtained between the microbial sensor and the methylene blue method. The regression coefficient was 0.97 for five experiments. The activity of the microbial membrane was stable for more than 25 days. The response was reproducible with 2.5% of the relative standard deviation when a sample solution containing 0.2 mm sodium sulphide was employed. *** DIRECT SUPPORT *** AG903053 00005     

Bacterial siderophores: structure elucidation, and 1H, 13C and 15N two-dimensional NMR assignments of azoverdin and related siderophores synthesized by Azomonas macrocytogenes ATCC 12334

Two major azoverdins were isolated from the cultures of Azomonas macrocytogenes ATCC 12334 grown in irondeficient medium. Their structures have been established using fast atom bombardment-mass spectroscopy, homonuclear and heteronuclear two-dimensional ¹⁵N, ¹³C and ¹H NMR, and circular dichroism techniques. These siderophores are chromopeptides possessing at the N-terminal end of their peptide chain the chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline common to pyoverdins. The linear peptide chain (l)-Hse-(d)-AcOHOrn-(d)-Ser-(l)-AcOHOrn-(d)-Hse-(l)-CTHPMD has at its C-terminal end a new natural amino acid which is the result of the condensation of 1 mol of homoserine and 1 mol of 2,4-diaminobutyric acid forming a cyclic amidine belonging to the tetrahydropyrimidine family: 2-homoseryl-4-carboxyl-3,4,5,6-tetrahydropyrimidine. The azoverdins differ only by a substitutent bound to the nitrogen on C-3 of the chromophore: azoverdin, the most abundant one, possesses a succinamide moiety, whereas azoverdin A bears a succinic acid moiety. ¹⁵N-labelled azoverdin afforded readily, after the complete assignment of the ¹⁵N spectrum of the siderophore, a sequence determination of the peptidic part of the molecule and gave evidence for the presence of two tetrahydropyrimidine groups on the molecule: one on the chromophore and the second at the C-terminal end of the siderophore.     

Determination of the isotope distribution in malate-14C with fumarase-negative lactic acid bacteria

No fumarase activity could be found in whole cells or in cell-free crude extracts from Leuconostoc mesenteroides or Lactobacillus curvatus. The degradation of l-malate-4-¹⁴C by these organisms yielded more than 95% of the label as ¹⁴CO₂. It is therefore recommended that these organisms, rather than Lactobacillus plantarum, should be used in the determination of isotope distribution in l-malate-¹⁴C, since L. plantarum exhibits a significant fumarase activity and thus randomizes malate prior to the decarboxylation of this substance by the malolactic enzyme.     

Synthesis of <Emphasis Type="SmallCaps">dl</Emphasis>-tryptophan by modified broad specificity amino acid racemase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IFO 12996

Broad specificity amino acid racemase (E.C. 5.1.1.10) from Pseudomonas putida IFO 12996 (BAR) is a unique racemase because of its broad substrate specificity. BAR has been considered as a possible catalyst which directly converts inexpensive l-amino acids to dl-amino acid racemates. The gene encoding BAR was cloned to utilize BAR for the synthesis of d-amino acids, especially d-Trp which is an important intermediate of pharmaceuticals. The substrate specificity of cloned BAR covered all of the standard amino acids; however, the activity toward Trp was low. Then, we performed random mutagenesis on bar to obtain mutant BAR derivatives with high activity for Trp. Five positive mutants were isolated after the two-step screening of the randomly mutated BAR. After the determination of the amino acid substitutions in these mutants, it was suggested that the substitutions at Y396 and I384 increased the Trp specific racemization activity and the racemization activity for overall amino acids, respectively. Among the positive mutants, I384M mutant BAR showed the highest activity for Trp. l-Trp (20 mM) was successfully racemized, and the proportion of d-Trp was reached 43% using I384M mutant BAR, while wild-type BAR racemized only 6% of initial l-Trp.     

Rapid cytofluorimetric determination of leaf nuclear DNA content in the polyploid seriesRanunculus marsicus (R. auricomus agg.,Ranunculaceae)

A cytofluorimetric method for DNA amount determination on nuclei released from fixed leaves has been successfully tested on samples belonging to the polyploid series ofRanunculus marsicus Guss & Ten. (sect.Auricomus). The procedure was devised to screen rapidly for different ploidy levels within topodemes and progenies.     

Continuous production of L-arginine using immobilized growing Serratia marcescens cells: Effectiveness of supply of oxygen gas

Summary The immobilized growing cell system using Serratia marcescens was applied to continuous L-arginine production. From the determination of oxygen uptake rate, it was shown that the cells entrapped in carrageenan gel were in an oxygen-limited state due to the diffusion barrier to oxygen transport created by the gel layer. This limited state in gel was relieved by supply of oxygen-enriched gas instead of air into the medium. The maximum population of immobilized cells increased to five times that of free cells with the supply of pure oxygen gas. The L-arginine-producing activity of the immobilized growing cells was proportional to the concentration of oxygen gas supplied and was 6 mg/h per millilitre in gel supplied with pure oxyges gas. The continuous L-arginine containing production was constantly maintained by controlling the medium penicillin G at pH 6.5 and more than 10 mg/ml of L-arginine were obtained at 10h of residence time for at least 12 days.     

Nutritional and medicinal aspects of <Emphasis Type="SmallCaps">d</Emphasis>-amino acids

This paper reviews and interprets a method for determining the nutritional value of d-amino acids, d-peptides, and amino acid derivatives using a growth assay in mice fed a synthetic all-amino acid diet. A large number of experiments were carried out in which a molar equivalent of the test compound replaced a nutritionally essential amino acid such as l-lysine (l-Lys), l-methionine (l-Met), l-phenylalanine (l-Phe), and l-tryptophan (l-Trp) as well as the semi-essential amino acids l-cysteine (l-Cys) and l-tyrosine (l-Tyr). The results show wide-ranging variations in the biological utilization of test substances. The method is generally applicable to the determination of the biological utilization and safety of any amino acid derivative as a potential nutritional source of the corresponding l-amino acid. Because the organism is forced to use the d-amino acid or amino acid derivative as the sole source of the essential or semi-essential amino acid being replaced, and because a free amino acid diet allows better control of composition, the use of all-amino-acid diets for such determinations may be preferable to protein-based diets. Also covered are brief summaries of the widely scattered literature on dietary and pharmacological aspects of 27 individual d-amino acids, d-peptides, and isomeric amino acid derivatives and suggested research needs in each of these areas. The described results provide a valuable record and resource for further progress on the multifaceted aspects of d-amino acids in food and biological samples.     

Beiträge zur Kenntnis der europäischen Raupenfliegen (Dipt. Tachinidae)

Summary In this revision of the European species of the genusMyxexoristops t. t. (parasites of sawflies) six species, including a new one (abietis n. sp., parasite ofCephalcia), are described by their distinguishing characters. Separate keys for the determination of males and females are given. For five species, hosts are recorded on the basis of bred material examined by the author himself.Zenillia nox hall, described from Japan is identical to the European speciesM. stolida stein (nov. syn.).