Growth factor requirements of two strains ofSphaerotilus discophorus

The relatively complex growth-factor requirements of two strains ofSphaerotilus discophorus, strains 31 and 32, have been elucidated. In addition to thiamin and biotin which are required by other strains ofS. discophorus, the two strains must be supplied also with adenine or guanine and with cyanocobalamin for growth in a glucose — Casamino Acids — mineral salts medium. The cyanocobalamin can be replaced by methionine but only if relatively large amounts of this amino acid, 100 µg or more per ml, are added to the medium. The methionine requirement of the two strains is approximately 3 times greater than that of otherS. discophorus strains.     

3-Ketoglutarate generation in pancreatic B-cell mitochondria regulates insulin secretory action of amino acids and 2-keto acids

The various neutral amino acids and aliphatic 2-keto acids exhibit differential effects on insulin secretion. The common denominator for all these effects is the 2-ketoglutarate generation in the pancreatic B-cell mitochondria. The neutral amino acidsl-leucine andl-norvaline and the aliphatic ketomonocarboxylic acids 2-ketoisocaproate, 2-ketocaproate, 2-ketovalerate, and 2-keto-3-methylvalerate all stimulate insulin secretion and increase 2-ketoglutarate generation in pancreatic B-cell mitochondria through activation of glutamate dehydrogenase and transamination withl-glutamate andl-glutamine, respectively. The neutral amino acidsl-valine,l-norleucine, andl-alanine and the aliphatic 2-keto acids 2-ketoisovalerate and pyruvate do not stimulate insulin secretion and do not increase 2-ketoglutarate generation in pancreatic B-cell mitochondria. Inhibition of 2-keto acid induced insulin secretion byl-valine andl-isoleucine is accompanied by reduced 2-ketoglutarate generation in pancreatic B-cell mitochondria. Thus intramitochondrial 2-ketoglutarate generation in pancreatic B-cells may regulate the insulin secretory potency of amino acids and 2-keto acids.     

Living cell reaction process forl-isoleucine andl-valine production

Summary A new process (Living Cell Reaction Process) forl-isoleucine production using viable, non-growing cells ofBrevibacterium flavum AB-07 was optimised using ethanol as the energy source and -ketobutyric acid (-KB) as precursor.l-valine also could be produced from glucose at high yield by this process. This process differs from the usual fermentation method in that non-growing cells are used, and the production ofl-isoleucine andl-valine were carried out under conditions of repressed cell division and growth. Minimal medium missing the essential growth factor, biotin was employed as the reaction mixture for the production ofl-isoleucine andl-valine. The productivity ofl-isoleucine andl-valine were 200 mmol·l^–1 · day^–1 (molecular yield to -KB: 95%) and 300 mmol · l^–1 · day^–1 (molecular yield to glucose: 80%) respectively. The content ofl-isoleucine andl-valine in total amino acids produced in the each mixture were 97% and 96% respectively.     

Stimulation of L-asparaginase production inEscherichia coli by organic and amino acids

The effect of 18 amino acids and 7 organic acids on the production ofl-asparaginase EC-2 by a strain ofEscherichia coli in a chemically defined medium was investigated under moderate aeration. All the amino acids and some of the organic acids stimulated the enzyme production. The specific activity without stimulants was about 0.16 nkat per mg dry weight, with stimulants it lay between 1 and 6 nkat per mg dry weight but withl-leucine andl-methionine the values were 12 nkat and 17 nkat per mg, respectively. When two organic or amino acids were added simultaneously at concentrations that were suboptimal for stimulation, the stimulating effects were cumulative in most cases. When cells were grown under conditions approaching anaerobiosis, the specific activity reached, even in the absence of stimulants, values as high as 5 nkat per mg; under these conditions, a further substantial increase in specific activity was only caused byl-leucine andl-methionine. Stimulating effects ofdl-lactate and of some amino acids were also found in other strains ofEscherichia coli. The ability to grow on a medium withl-asparagine as the sole source of both nitrogen and carbon was found in two strains; growth took place even when there was no measurable activity ofl-asparaginase EC-2.     

Development of a chemically defined medium for growth ofNeisseria gonorrhoeae

A chemically defined medium satisfactory for growth of a number of laboratory strains and recent isolates ofNeisseria gonorrhoeae has been devised. It contains inorganic salts, dextrose, guanine, cytosine, B-vitamin supplement, and the following amino acids:l-arginine,l-aspartic acid,l-cystine,l-isoleucine,l-leucine,l-proline,l-threonine, andl-valine.Nine of the eleven strains grew satisfactorily in this medium without being provided supplemental CO₂ during incubation, and a tenth strain grew in the medium supplemented with glutamine. No single B-vitamin or purine or pyrimidine base was essential for growth of any of the strains, but some combinations of them were stimulatory. Riboflavin, however, was inhibitory. The strains showed variations in requirements for amino acids. The amino acids which were either essential or stimulatory for one or more of the strains were included in the medium. Those to which the strains responded differently were used at concentrations intermediate between those optimal for growth of one strain and inhibitory for another. Conventional agar was inhibitory, but a purified agar, having a gel strength twice that of conventional agar, was satisfactory. An aqueous solution of 0.1% cysteine and 0.86% NaCl was satisfactory for preparation of inocula.This investigation was supported by a Public Health Service Predoctoral Fellowship (F-FI-GM-24-755-01A1) from the National Institute of General Medical Sciences of the United States Public Health Service to the senior author.     

Differential effects of amino acid analogs on growth and heterocyst differentiation in two nitrogen-fixing blue-green algae

Effects of a few amino acid analogs on growth and heterocyst differentiation have been studied in two nitrogen-fixing species ofAnabaena. All the analogs except α-methyl-dl-aspartic acid inhibited growth. Exposure ofAnabaena doliolum, todl-5-fluorotryptophan anddl-p-fluorophenylalanine caused pronounced fragmentation of filaments into single cells. At low concentrations (0.01 mM), α-methyl-dl-aspartic acid stimulated growth of the strain ofA. doliolum as well as the strain of the second (unidentified)Anabaena species. Ethionine,dl-p-fluorophenylalanine,dl-5-fluorotryptophan, and canavanine blocked heterocyst differentiation, whereas α-methyl-dl-aspartic acid, α-methyl-dl-methionine,N-o-nitrophenylsulfenyl-l-tryptophan, norleucine, andS-2-aminoethyl-l-cysteine did not show any significant effect. Treatment with 7-azatryptophan,dl-β-hydroxynorvaline,l-methionine-dl-sulfoximine,l-methionine sulfone, and β-2-thienyl-dl-alanine led to a twofold increase in heterocyst frequency. Possible modes of action of the analogs in growth inhibition and changes in heterocyst frequency are discussed.     

Amino Acid-Induced Inhibition and Stimulation of Saccharomyces carlsbergensis: I. Variations in the Response to Pantothenic Acid Induced by Casein Hydrolysate

The response pattern of Saccharomyces carlsbergensis (ATCC 9080) to pantothenic acid in Atkin's medium was changed dramatically by adding small amounts of casein hydrolysate (0.032 to 0.32 mg/ml) to the assay medium. Under static, mildly anaerobic conditions, growth at low pantothenic acid levels was reduced by 54 to 69%, whereas at saturating or near saturating pantothenate concentrations marked stimulation of growth (up to 41%) was observed. Under aerobic conditions, inhibition but not stimulation of growth occurred. It is recommended that Atkin's medium for the assay of pantothenic acid with S. carlsbergensis (ATCC 9080) be modified to include 0.6% acid-hydrolyzed casein (Vitamin Free Casamino Acids, Difco) to prevent erroneous growth responses, which may result if significant amounts of amino acids are present in natural materials being assayed for this vitamin.     

Racemic Resolution of some <Emphasis Type="SmallCaps">dl</Emphasis>-Amino Acids using <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-Amino Acid Oxidase

The ability of Aspergillus fumigatus l-amino acid oxidase (l-aao) to cause the resolution of racemic mixtures of dl-amino acids was investigated with dl-alanine, dl-phenylalanine, dl-tyrosine, and dl-aspartic acid. A chiral column, Crownpak CR+ was used for the analysis of the amino acids. The enzyme was able to cause the resolution of the three dl-amino acids resulting in the production of optically pure d-alanine (100% resolution), d-phenylalanine (80.2%), and d-tyrosine (84.1%), respectively. The optically pure d-amino acids have many uses and thus can be exploited industrially. This is the first report of the use of A. fumigatus l-amino acid oxidase for racemic resolution of dl-amino acids.     

Uptake and Incorporation of Amino Acids and Peptides by Bacteroides amylophilus

Bacteroides amylophilus H-18 demonstrated a higher growth yield, a slightly higher growth rate, and a diminished lag period when Tryptose was added to the basal medium. This uptake of labeled amino acids was concentration-dependent, as the contribution of exogenous amino acid to the cell protein increased from 15.4 to 24.1% when the concentration of Casamino Acids in the medium was increased from 1.4 to 2.8 mg/ml. There was considerable redistribution of (14)C-label to other amino acids. Tryptic peptides of casein competed effectively with the amino acids for uptake. The (14)C-label from a protein was incorporated into B. amylophilus H-18 cells presumably after breakdown of the protein by the B. amylophilus H-18 protease.     

Condensed phosphate deposition,sulfur amino acid use,and unidirectional transsulfuration in Synechococcus leopoliensis

Cells of the cyanobacterium, Synechococcus leopoliensis, have previously been shown to exhibit diminished growth, increased condensed phosphate accumulation, enlarged polyphosphate bodies, and severe chlorosis when cultured under conditions of sulfur deficiency. These characteristics were used to identify which of several sulfur amino acids and a tripeptide served as a sole sulfur source for this unicellular microorganism. Completely serving sulfur compounds were l-cystine, dl-lanthionine, l-djenkolic acid, and glutathione. Sulfur amino acids serving poorly or not at all were l-cystathionine, dl-homocystine, l-methionine, l-cysteic acid, and taurine. This pattern of use suggests that the unidirectional transsulfuration pathway demonstrated in enteric bacteria and green plants, i.e. l-cysteine to l-homocysteine, operates as well in cyanobacteria of the Synechococcus type.     

Effect of carbon and nitrogen sources on the production of penitrem B byPenicillium aurantiogriseum

Effect of different carbon and nitrogen sources on the production of penitrem B was studied.d-Xylose induced maximum penitrem B production, while melibiose, glycerol, citric acid and succinic acid were poor substrates. Potassium nitrate,l-asparagine, sodium nitrate, glycine,dl-aspartic acid andl-tryptophan supported good production of penitrem B. Conversely zirconyl nitrate, barium nitrate, aluminum nitrate, acetanilide, 4-aminobenzoic acid, 4-nitrobenzoic acid and 4-nitroaniline were toxic and did not even permit the growth of the fungus.     

Enzymatic production of <Emphasis Type="SmallCaps">l</Emphasis>-amino acids from the corresponding <Emphasis Type="SmallCaps">dl</Emphasis>-5-substituted hydantoins by <Emphasis Type="Italic">Bacillus fordii</Emphasis> MH602

Bacillus fordii MH602 was newly screened from soil at 45 °C and exhibited high activities of hydantoinase and carbamoylase, efficiently yielding l-amino acids including phenylalanine, phenylglycine and tryptophan with the bioconversion yield of 60–100% from the corresponding dl-5-substituted hydantoins. Hydantoinase activity was found to be cell-associated and inducible. The optimal inducer was dl-5-methylhydantoin with concentration of 0.014 mol L⁻¹ and added to the fermentation medium in the exponential phase of growth. In the production of optically pure amino acids from dl-5-benylhydantoin, the optimal temperature and pH of this reaction were 45–50 °C and 7.5 respectively. The hydantoinase was non-stereoselective, while carmbamoylase was l-selective. The hydantoinase activity was not subject to substrate inhibition, or product inhibition by ammonia. In addition, The activities of both enzymes from crude extract of the strain were thermostable; the hydantoinase and carbamoylase retained about 90% and 60% activity after 6 h at 50 °C, respectively. Since reaction at higher temperature is advantageous for enhancement of solubility and for racemization of dl-5-substituted hydantoins, the relative paucity of l-selective hydantoinase systems, together with the high level of hydantoinase and carbamoylase activity and unusual substrate selectivity of the strain MH602, suggest that it has significant potential applications.     

Metabolism of amino acid amides in Pseudomonas putida ATCC 12633

The metabolism of the natural amino acid l-valine, the unnatural amino acids d-valine, and d-, l-phenyglycine (d-, l-PG), and the unnatural amino acid amides d-, l-phenylglycine amide (d-, l-PG-NH₂) and l-valine amide (l-Val-NH₂) was studied in Pseudomonas putida ATCC 12633. The organism possessed constitutive l-amidase activities towards l-PG-NH₂ and l-Val-NH₂, both following the same pattern of expression, suggesting the involvement of similarly regulated enzymes, or a common enzyme. Quite surprisingly, growth in mineral media with l-PG-NH₂ resulted in variable, long lag phases of growth and strongly reduced l-amidase activities. Conversion of d-PG-NH₂ into d-PG and l-PG also occurred and could be attributed to the presence of an inducible d-amidase and the racemization of the amino acid amide in combination with l-amidase activity, respectively. The further degradation of l-PG and d-PG involved constitutive l-PG aminotransferase and inducible d-PG dehydrogenase activities, respectively, both with a high degree of enantioselectivity. Amino acid racemase activity for d- and l-PG was not detected. Correspondence to: L. Dijkhuizen     

Casamino acids enhance growth of Bacteroides melaninogenicus.

Casamino Acids enhance the growth of Bacteroides melaninogenicus when added to various concentrations of Trypticase. Absence of a peptide, not amino acids, is responsible for the inability of Casamino Acids to support growth.     

Cultivation ofCandida blankii in simulated bagasse hemicellulose hydrolysate

Summary All fourCandida blankii isolates evaluated for growth in simulated bagasse hemicellulose hydrolysate utilized the sugars and acetic acid completely. The utilization ofd-xylose,l-arabinose and acetic acid were delayed by the presence ofd-glucose, but after glucose depletion the other carbon sources were utilized simultaneously. The maximum specific growth rate of 0.36 h^–1 and cell yield of 0.47 g cells/g carbon source assimilate compared with published results obtained withC. utilis. C. blankii appeared superior toC. utilis for biomass production from hemicellulose hydrolysate in that it utilizedl-arabinose and was capable of growth at higher temperatures.     

Improvement of l-valine production at high temperature in Brevibacterium flavum by overexpressing ilvEBNrC genes

Brevibacterium flavum ATCC14067 was engineered for l-valine production by overexpression of different ilv genes; the ilvEBN^rC genes from B. flavum NV128 provided the best candidate for l-valine production. In traditional fermentation, l-valine production reached 30.08 ± 0.92 g/L at 31°C in 72 h with a low conversion efficiency of 0.129 g/g. To further improve the l-valine production and conversion efficiency based on the optimum temperatures of l-valine biosynthesis enzymes (above 35°C) and the thermotolerance of B. flavum, the fermentation temperature was increased to 34, 37, and 40°C. As a result, higher metabolic rate and l-valine biosynthesis enzymes activity were obtained at high temperature, and the maximum l-valine production, conversion efficiency, and specific l-valine production rate reached 38.08 ± 1.32 g/L, 0.241 g/g, and 0.133 g g⁻¹ h⁻¹, respectively, at 37°C in 48 h fermentation. The strategy for enhancing l-valine production by overexpression of key enzymes in thermotolerant strains may provide an alternative approach to enhance branched-chain amino acids production with other strains.     

Biosynthesis of 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane by methanogenic bacteria

In an attempt to establish the nature of the ammonium-assimilation products which mediate the inhibition by ammonium of nitrate uptake in cyanobacteria, the effect of different amino acids on nitrate utilization by intact Anacystis nidulans cells has been assayed. To exclude an indirect inhibition of nitrate uptake through the ammonium which the amino acids might release, the cells were pretreated with l-methionine-d,l-sulfoximine (MSX), a potent inactivator of glutamine synthetase. Under these conditions, several l-amino acids, but not the corresponding d-isomers, affected nitrate utilization to a variable extent, causing inhibitions ranging between 20 and 80% when added at 20 mM concentration.For most of the inhibitory amino acids, including l-isoleucine, l-leucine and l-valine, a correlation was found between their ability to act as amino group donors to -ketoglutarate, in reactions catalyzed by A. nidulans cell-free extracts, and their inhibitory effect on nitrate utilization. l-Glutamine, l-asparagine and glycine, being effective inhibitors of nitrate utilization, were poor substrates for the transaminating activity to -ketoglutarate, however. The possible role of the latter amino acids as mediators in the ammonium-promoted inhibition of nitrate uptake is discussed.Abbreviations MSX l-methionine-d,l-sulfoximine - MTA-5 mixed alkyltrimethylammonium bromide - Mops morpholinopropane sulfonic acid     

New ubiquitous translocators: amino acid export by<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis>

Molecular access to amino acid excretion by Corynebacterium glutamicum and Escherichia coli led to the identification of structurally novel carriers and novel carrier functions. The exporters LysE, RhtB, ThrE and BrnFE each represent the protoype of new transporter families, which are in part distributed throughout all of the kingdoms of life. LysE of C. glutamicum catalytes the export of basic amino acids. The expression of the carrier gene is regulated by the cell-internal concentration of basic amino acids. This serves, for example, to maintain homoeostasis if an excess of l-lysine or l-arginine inside the cell should arise during growth on complex media. RhtB is one of five paralogous systems in E. coli, of which at least two are relevant for l-threonine production. A third system is relevant for l-cysteine production. It is speculated that the physiological function of these paralogues is related to quorum sensing. ThrE of C. glutamicum exports l-threonine and l-serine. However, a ThrE domain with a putative hydrolytic function points to an as yet unknown role of this exporter. BrnFE in C. glutamicum is a two-component permease exporting branched-chained amino acids from the cell, and an orthologue in B. subtilis exports 4-azaleucine.     

Transport of glutamine inXenopus laevis oocytes: Relationship with transport of other amino acids

Summary We have investigated transport of the amino acid glutamine across the surface membranes of prophase-arrestedXenopus laevis oocytes. Glutamine accumulation was linear with time for 30 min; it was stereospecific with aK _m of 0.12±0.02mm andV _max of 0.92±0.17 pmol/oocyte · min forl-glutamine. Transport ofl-glutamine was Na⁺-dependent, the cation not being replaceable with Li⁺, K⁺, choline, tris(hydroxymethyl)-aminomethane (Tris), tetramethylammonium (TMA) or N-methyld-glucamine NMDG); external Cl^– appeared to be necessary for full activation of Na⁺-dependent glutamine transport. Two external Na⁺ may be required for the transport of one glutamine molecule.l-glutamine transport (at 50 m glutamine) was inhibited by the presence of other amino acids:l-alanine,d-alanine,l-leucine,l-asparagine andl-arginine (about 60% inhibition at 1mm);l-histidine,l-valine and glycine (25 to 40% inhibition at 1mm);l-serine,l-lysine,l-phenylalanine andl-glutamate (45 to 55% inhibition at 10mm). N-methylaminoisobutyric acid (meAIB) had no effect at 10mm, but 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) inhibited Na⁺/glutamine transport by about 50% at 10mm.l-glutamine was a competitive inhibitor of the Na⁺-dependent transport ofl-alanine,d-alanine andl-arginine; this evidence is consistent with the existence of a single system transporting all four amino acids. Glutamine uptake in oocytes appears to be catalyzed by a transport system distinct from the cotransport Systems A, ASC, N and Gly, although it resembles System B^0,+.     

Influence of l-isoleucine and pantothenate auxotrophy for l-valine formation in Corynebacterium glutamicum revisited by metabolome analyses

The effect of different amounts of supplemented l-isoleucine and pantothenate has been analysed with the auxotrophic strain Corynebacterium glutamicum ΔilvA ΔpanB, showing that the final biomass concentration of this preliminary l-valine production strain can be controlled by the amount of added l-isoleucine. One gramme cell dry weight is formed from 48 μmol l-isoleucine. Different amounts of available pantothenate affect the intracellular pyruvate concentration. By limiting pantothenate supplementation from 0.8 to 0.1 μM, a 35-fold increase of cytoplasmic pyruvate up to 14.2 mM can be observed, resulting in the increased formation of l-valine, l-alanine and organic acids in the presence of low pantothenate concentrations. These findings can be used to redirect the carbon flux from glycolysis via pyruvate to the TCA cycle towards the desired product l-valine.