Effect of amino acids on growth ofSphaerotilus discophorus

The effect of casein hydrolysate, of mixtures of amino acids and of individual amino acids on the growth of 4 strains ofSphaerotilus discophorus was determined. Growth was virtually completely inhibited by 1.0% Bacto Casamino Acids, 0.54% simulated casein hydrolysate and 0.2% of a uniform mixture of 18 amino acids. The latter were prepared withl amino acids except thatdl-serine,dl-valine anddl-threonine were present in the uniform amino acid mixture.Experiments designed to test the toxicity of the 18 individual amino acids at 0.018 – 0.36% concentration indicated that arginine, glutamic acid, leucine, lysine and proline were non-toxic. However, aspartic acid and methionine were moderately toxic; growth was greatly repressed at a concentration of 0.36%. The remaining 11 amino acids which included alanine, cystine, glycine, tyrosine, histidine, isoleucine, phenylalanine, serine, threonine, tryptophane and valine were the most toxic of the group. They prevented growth partially or completely, at a concentration of 0.18% or 0.36%.dl-Serine anddl-valine were especially toxic and prevented growth at a concentration of 0.018%. The toxicity of the individuall-amino acids can account for the toxicity of Casamino Acids and simulated casein hydrolysate. l-Methionine or cyanocobalamin (vitamin B₁₂) is required for the growth ofS. discophorus. Alsod- anddl-methionine can replace cyanocobalamin although they completely repress growth when used at the relatively high concentration of 200 µg per ml of medium.     

The conversion of amino acids in soils

Summary In an investigation on the conversion of amino acids in percolated soils, it was found that during the breakdown of glutamic acid to ammonia micro-organisms developed in the soil capable of denitrifying nitrite and nitrate to gaseous nitrogen. The enrichment of a soil with these micro-organisms was studied.Drying of the enriched soil had a deleterious effect on the activity of these micro-organisms.The interaction between denitrification and soil nitrification processes was studied in soil subjected to various percolation treatments. When the denitrifying micro-organisms and their metabolites were present in the soil the amount of nitrogen lost by denitrification depended on the availability of nitrite and nitrate. When this was supplied externally, in glutamate—nitrite or glutamate—nitrate mixtures, considerable reduction occurred. Losses were less severe where nitrite and nitrate entered the system internall y by nitrification of the ammonia produced from the breakdown of the amino acid. In fresh soils there were indications that the amount of nitrification occurring during amino-acid breakdown was the important factor.All the data appeared to be consistent with the hypothesis that during the conversion of amino acids in soils a delicate balance is established between nitrification and denitrification reactions by different types of soil micro-organisms.     

The effect of oxygen concentration on the decomposition of organic materials in soil

Summary 1. Techniques are described for relating the oxygen concentrations in the soil water on the surfaces of micro-organisms to their metabolizing activities.2. Studies were made on the decomposition of organic materials in water-saturated crumbs (mean radius 1.55 × 10^–1 cm) of a loam soil.3. Respiration of water-saturated crumbs was not inhibited unless the oxygen concentration was less than about 10^–6 M. Evidence was obtained that above a similar low oxygen concentration there was no inhibition of respiration in soils of widely different type.4. Anaerobic decomposition of the soil organic matter was very slow. Anaerobic decomposition of casein digest was more rapid than that of any other material tested; the products were water soluble and included 83 µ-equivalents of volatile fatty acid per mg of -amino-N decomposed.5. Casein digest percolation of soil crumbs under air resulted in the formation of micro-organisms that respired at 70 per cent of their maximum rate when the oxygen concentration was about 2.7 × 10^–6 M.6. No products of anaerobic casein digest decomposition could be detected on percolating casein digest through soil crumbs when 80 per cent of the soil contained no oxygen and the maximum concentration in any part of the soil was about 3 × 10^–5 M.7. The kinetics of oxygen uptake consequent on the decomposition of casein digest and of other simple organic compounds in soil crumbs were similar and were only slightly affected by reduction of oxygen partial pressure in the atmosphere from 15 to 1.7 cm of mercury.8. It is concluded that change-over from aerobic to anaerobic metabolism of organic materials takes place in widely different soils at an oxygen concentration less than about 3 × 10^–6 M.     

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.     

Streptomyces rimosus extracellular proteases 3. Isolation and characterization of leucine aminopeptidase

Summary A leucine aminopeptidase was purified to homogeneity fromStreptomyces rimosus culture filtrates, which are waste broth of oxytetracycline bioproduction process. Purification procedure includes ultrafiltration and chromatography on CM-Sephadex, AH-Sepharose and FPLC Mono S column. The enzyme is a monomer with molecular weight of 27,500 Daltons and pI of 7.3, stable in broad pH range and up to 70°C. It is a metallo enzyme dependent on Ca²⁺ ions for its full activity. By its specificity it is a true aminopeptidase active on amino acid amide, arylamide, peptide and ester bonds. The hydrolysing activity shows preference for leucine at the N-terminal position of substrates, also acts on aromatic acids and methionine, but does not release glycine, proline, acidic amino acids orD-amino acid residues.     

Separation of protein and fatty acids from tuna viscera using supercritical carbon dioxide

Supercritical carbon dioxide extraction was investigated as a method for removing lipids and bad flavor from tuna viscera. To find the optimum conditions, different experimental variables, such as pressure, temperature, flow rate of solvent and sample size, were evaluated for the effective removal of lipids and the undesirable smell. Ethanol was used as the entrainer, with a 3% by vol CO₂ flow rate. By increasing the pressure at constant temperature, the efficiency of the lipid removal was improved and the protein was concentrated without denaturalization. The main fatty acids extracted from the tuna viscera were palmitic acid (16∶0), heptadecanoic acid (17∶1), oleic acid (18∶1) and docosahexaenoic acid (22∶6). The major amino acids in the tuna viscera treated by supercritical carbon dioxide were glutamic acid, leucine and lysine, and the free amino acids werel-proline, taurine andl-α-aminoadipic acid.     

D-Amino acids in protein de novo design. II. Protein-diastereomerism versus protein-enantiomerism

Summary With a few exceptions, proteins in our biosphere are based exclusively onl-amino acids. The inversion of configuration of all the stereogenic centers in a protein leads to anall-d compound with ‘mirror image’ properties and ‘mirror image’ structure. We propose to use the termprotein-enantiomerism to describe the relationship between two proteins that have the same sequence but whose amino acids have opposite configuration. We will use the termprotein-diastereomerism to define the relationship between two proteins that have the same sequence in which some amino acids have opposite configurations. A classification of type I, II, III, and IV protein-diastereomerism is proposed. By extension, a diastereoprotein is a protein where some amino acids have the same configuration (l ord) while others have the opposite one (d orl). A particular case of diastereoproteins aremesoproteins, also analyzed in this article. In addition to the goal of making proteins resistant to protease degradation, the use ofd-amino acids in protein de novo design may give rise to proteins with structures, and perhaps properties, very different to those of nativeall-l-proteins.     

Characteristics of amino acid uptake in barley

Plants have the ability to take up organic nitrogen (N) but this has not been thoroughly studied in agricultural plants. A critical question is whether agricultural plants can acquire amino acids in a soil ecosystem. The aim of this study was to characterize amino acid uptake capacity in barley (Hordeum vulgare L.) from a mixture of amino acids at concentrations relevant to field conditions. Amino acids in soil solution under barley were collected in microlysimeters. The recorded amino acid composition, 0–8.2 μM of l-Serine, l-Glutamic acid, Glycine, l-Arginine and l-Alanine, was then used as a template for uptake studies in hydroponically grown barley plants. Amino acid uptake during 2 h was studied at initial concentrations of 2–25 μM amino acids and recorded as amino acid disappearance from the incubation solution, analysed with HPLC. The uptake was verified in control experiments using several other techniques. Uptake of all five amino acids occurred at 2 μM and below. The concentration dependency of the uptake rate could be described by Michaelis–Menten kinetics. The affinity constant (K _m) was in the range 19.6–33.2 μM. These K _m values are comparable to reported values for soil micro-organisms.     

Studies on the decomposition of amino acids in soils

Summary 1. The kinetics of decomposition of 17 common amino acids in a garden soil have been followed by percolation techniques that permitted the measurement of O₂-uptake and CO₂-output consequent upon decomposition.2. Most of the amino acids decomposed rapidly; methionine and threonine decomposed much more slowly.3. After decomposition of amino acids in soil, the material residual in the soil had a C : N ratio of about 3 : 1; this possibly represents microbial protein.4. The significance of the results, and the potentialities of the manometric techniques used, are discussed.     

Losses and biogeochemical cycling of soil organic nitrogen with prolonged arable cropping in the South African Highveld – evidence from - and -amino acids

We know little about the mechanisms that cause rapid losses in the soil organic N pool during cropping. As the analysis of amino acid enantiomers can provide insight into both the fate of microbial N and the ageing of cells in the environment, we used this technique as a tool to examine how the pool of protein-bound N in subtropical Plinthosols responds to increasing duration of arable cropping. The samples comprised bulk soils (0–20 cm) and clay fractions from each of three agro-ecosystems in semiarid South Africa; the sites have been cropped for periods varying from 0 to 98 years. The amino acid enantiomers contributed 34% to the total N content. With increasing number of years a piece of land has been cropped, the amino acid concentrations declined bi-exponentially to about 30% of their initial level in the native grasslands. Changes of the remaining soil protein-N pool were indicated by alterations in the d-content of individual amino acids. As the years of arable cropping increased, the proportions of d-alanine and d-glutamic acid increased relative to the respective l-enantiomers. This was attributed to an accumulation of N in residues of bacterial cell walls. In contrast, the d/l-ratios of leucine and aspartic acid declined in the long-term cultivated plots, probably reflecting losses of old amino acid-N reserves at the most degraded arable land.     

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.     

Inhibitory action of serine on growth of bacteria of the genusBacillus on mineral synthetic media

l-Serine added to minimal synthetic media stops the growth ofBacillus subtilis, B. megaterium, B. mycoides andB. pantothenticus. All tested subspecies ofBacillus thuringiensis appear resistant to this amino acid. Serine acts bacteriostatically onB. subtilis strain B 003 and this effect depends on the concentrations of both the amino acid and the plated bacterium. Growth of serine-sensitiveBacillus strains can be restored by simultaneous addition of some other amino acids (e.g. l-threonine,l-arginine,l-aspartate orl-alanine) to the minimal media. This alleviating effect depends on the kind of amino acid. Some amino acids (e.g. l-threonine,l-tyrosine orl-tryptophan) are only effective when the serine concentration is not higher than 125 μmol/L, others (l-arginine,l-proline,l-alanine,l-aspartate orl-glutamate) are effective even when the serine concentration is as high as 500 μmol/L.     

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.     

Organic matter transformations in soils

Summary Post-incubation fractionation of soils incubated with C₁₄-tagged oat roots under aerobic and anaerobic conditions and chromatographic separation of hydrolysates of different organic matter fractions indicated the incorporation of C¹⁴-labelled amino acids in soil organic matter. Anaerobic incubation leads to the formation of hydrolysable heavily C¹⁴-labelled organic substances in greater quantity. The amino acid composition of the different fractions revealed not a very significant qualitative difference. The significance and causes of stabilization of amino acids in soil organic matter are discussed.     

Studies on aspergilli. VI. The utilization of amino acids in mixtures by some ascosporic members of the aspergillus nidulans group

Summary The utilization of certain amino acids when supplied in three different combinations, (a) glycine, serine, valine, histidine (b) methionine, valine, alanine, arginine (c) leucine, tryptophane, glutamic acid, aspartic acid, byA. nidulans (Eidam)Wint;A. rugulosus Thom andRaper;A. variecolor (Berk. andBr.)Thom andRaper;A. quadrilineatus Thom andRaper andA. violaceus Fennel andRaper was studied through circular partition chromatography. It was found that these Aspergilli which are so closely related morphologically exhibited different rate of assimilation of amino acids. Amino acids in mixtures were utilized better than when supplied singly. Although different species had their own preference to certain amino acids yet there was a simultaneous utilization of both good and poor amino acids.     

Rapid Cycling of Organic Nitrogen in Taiga Forest Ecosystems

ABSTRACT We examined the dynamics of organic nitrogen (N) turnover in situ across a primary successional sequence in interior Alaska, USA, in an attempt to understand the magnitude of these fluxes in cold, seasonally frozen soils. Through a combination of soil extraction procedures and measurements of ¹³C-enriched CO₂ efflux from soils amended in the field with ¹³C-labeled amino acids, we were able to trace the fate of this N form. Amino acid turnover in situ at soil temperatures of 10°C or below show that amino acids represent a highly dynamic soil N pool with turnover times of approximately 3–6 h. The rapid turnover of free amino acids is associated with high soil proteolytic activity, which in turn is tightly correlated with soil protein concentration. Moreover, these estimates of soil amino acid turnover in the field correspond well with measurements of amino acid turnover under equivalent temperatures in the laboratory. The gross flux of amino acid-N over the growing season greatly exceeded the annual vegetation N requirement, suggesting that microbial biomass represent a significant sink for this organic N. Depending on the strength of this sink, N flow via free soil amino acids can potentially account for the entire N demand of vegetation in the absence of net N mineralization. These relationships underscore the important biogeochemical role of labile DON fractions in high-latitude forest ecosystems.     

D-Amino acids of the amino acid pool and occurrence of racemase andD-amino acid oxidase activities inEscherichia coli B

Less than 20 % of the amino acid content of the amino acid pool ofEscherichia coli B exists in theD-form. Alanine, glutamic acid, and valine were shown by gas-chromatography to be partially in theD-form. OnlyD-alanine was formed by racemization in the crude extract of this organism. Alanine racemase was easily released from the membranes or vesicles butD-alanine oxidase activity remained firmly bound to the membrane. Most protein amino acids stimulated proline uptake into the vesicles, and the oxidative deamination activities were verified by the proline uptake stimulating amino acids. It is concluded that the obligatory pathway of L-amino acid -D-amino acid - oxo acid which exists in the oxidation ofL-alanine does not exist with otherL-amino acids. It is likely that otherD-amino acids in the pool are formed in the presence ofD-amino acid oxidase orD-amino acid aminotransferase.     

A common transport system for methionine, L-methionine-DL-sulfoximine (MSX), and phosphinothricin (PPT) in the diazotrophic cyanobacterium Nostoc muscorum

We present evidence, for the first time, of the occurrence of a transport system common for amino acid methionine, and methionine/glutamate analogues l-methionine-dl-sulfoximine (MSX) and phosphinothricin (PPT) in cyanobacterium Nostoc muscorum. Methionine, which is toxic to cyanobacterium, enhanced its nitrogenase activity at lower concentrations. The cyanobacterium showed a biphasic pattern of methionine uptake activity that was competitively inhibited by the amino acids alanine, isoleucine, leucine, phenylalanine, proline, valine, glutamine, and asparagine. The methionine/glutamate analogue-resistant N. muscorum strains (MSX-R and PPT-R strains) also showed methionine-resistant phenotype accompanied by a drastic decrease in ³⁵S methionine uptake activity. Treatment of protein extracts from these mutant strains with MSX and PPT reduced biosynthetic glutamine synthetase (GS) activity only in vitro and not in vivo. This finding implicated that MSX- and PPT-R phenotypes may have arisen due to a defect in their MSX and PPT transport activity. The simultaneous decrease in methionine uptake activity and in vitro sensitivity toward MSX and PPT of GS protein in MSX- and PPT-R strains indicated that methionine, MSX, and PPT have a common transport system that is shared by other amino acids as well in N. muscorum. Such information can become useful for isolation of methionine-producing cyanobacterial strains.     

Amino acids in root exudates of cotton

Summary Cotton-field soils from a wilt-free zone (KPT) and from a wilt-sick zone (PLD), and root exudates of diploid and amphidiploid cotton strains grown in KPT and PLD soils were analyzed for 13 free amino acids employing microbiological assay techniques. Test organisms used wereLactobacillus arabinosus 17/5 andLeuconostoc mesenteroides P. 60.Arginine, proline, lysine, serine, glycine, cystine, and tyrosine occurred in both the soils; amounts of these were higher in PLD soil than in KPT soil.Seven to eleven amino acids were found in the root exudates of cotton. Aspartic acid, threonine (in one), phenylalanine and leucine were found in root exudates but not in the control soils. Diploid strains were found to exude greater amounts of amino acids than the amphidiploid strains. Comparing the effect of the two soils tested, it was found that amino acids in exudates of plants grown in PLD soil were higher except in the case of one diploid strain.In the inoculated series, amino acids in the exudates were markedly lower than the respective healthy controls in the diploid strains. In the exudates of amphidiploid strains also, amino acid contents were reduced but to a lesser extent.Significance of occurrence of cystine in the exudates of some strains is indicated.Part of Doctoral thesis, University of Madras.     

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.