Determination of d- and l-aspartate in amino acid mixtures by high-performance liquid chromatography after derivatization with a chiral adduct of o-phthaldialdehyde

A sensitive and convenient method for the simultaneous determination of d- and l-aspartic acid in amino acid mixtures is described. The method involves derivatization of the mixture with a chiral fluorogen, followed by high-performance liquid chromatography on a reverse-phase column. The fluorogen used is an adduct of o-phthaldialdehyde with an optically active thiol, N-acetyl-l-cysteine. The sensitivity and accuracy of this method is similar to that using adducts of o-pthaldialdehyde with the achiral thiol, 2-mercaptoethanol. Five picomoles of d-aspartate can be accurately detected in the presence of a 100-fold excess of l-aspartate with a total analysis time (including derivatization) of 10 min.     

Alteration of substrate specificity of aspartase by directed evolution

Aspartase (l-aspartate ammonia-lyase, EC 4.3.1.1), which catalyzes the reversible deamination of l-aspartic acid to yield fumaric acid and ammonia, is highly selective towards l-aspartic acid. We screened for enzyme variants with altered substrate specificity by a directed evolution method. Random mutagenesis was performed on an Escherichia coli aspartase gene (aspA) by error-prone PCR to construct a mutant library. The mutant library was introduced to E. coli and the transformants were screened for production of fumaric acid-mono amide from l-aspartic acid-alpha-amide. Through the screening, one mutant, MA2100, catalyzing deamination of l-aspartic acid-alpha-amide was achieved. Gene analysis of the MA2100 mutant indicated that the mutated enzyme had a K327N mutation. The characteristics of the mutated enzyme were examined. The optimum pH values for the l-aspartic acid and l-aspartic acid-alpha-amide of the mutated enzyme were pH 8.5 and 6.0, respectively. The K(m) value and V(max) value for the l-aspartic acid of the mutated enzyme were 28.3 mM and 0.26 U/mg, respectively. The K(m) value and V(max) value for the l-aspartic acid-alpha-amide of the mutated enzyme were 1450 mM and 0.47 U/mg, respectively. This is the first report describing the alteration of the substrate specificity of aspartase, an industrially important enzyme.     

Biochemical and molecular characterization of poly(aspartic acid) hydrolase-2 from sphingomonas sp. KT-1

Poly(aspartic acid) (PAA) hydrolase-2 was purified from crude soluble cellular extracts of Sphingomonas sp. KT-1 (JCM10459) and characterized to elucidate the mechanism of alpha,beta-poly(d,l-aspartic acid) (tPAA) biodegradation. The molecular mass of PAA hydrolase-2 was 42 kDa, and the isoelectric point was 9.6. The optimum values of pH and temperature for the hydrolysis of alpha-di(l-aspartic acid) by PAA hydrolase-2 were 7.0 and 55 degrees C, respectively. The effect of inhibitors on the hydrolysis of alpha-di(l-aspartic acid) showed that the activity of PAA hydrolase-2 was significantly inhibited by EDTA. Thermally synthesized tPAA was hydrolyzed in the presence of two enzymes, PAA hydrolase-1 and PAA hydrolase-2, to generate aspartic acid. The PAA hydrolase-2 was capable of hydrolyzing alpha-poly(l-aspartic acid) of high molecular weights but had limited activity for tPAA. These results lead us to propose the following mechanism. First, PAA hydrolase-1 hydrolyzes tPAA to yield oligo(aspartic acid) via an endo-mode cleavage, and subsequently, PAA hydrolase-2 hydrolyzes the resultant oligo(aspartic acid) to yield aspartic acid. Analysis of hydrolyzed products from alpha- and beta-penta(l-aspartic acid) revealed that PAA hydrolase-2 catalyzed the exo-mode hydrolysis of alpha- and beta-penta (l-aspartic acid). The gene encoding PAA hydrolase-2 from Sphingomonas sp. KT-1 was cloned, and genetic analysis showed that the deduced amino acid sequence of PAA hydrolase-2 is similar to a putative peptidase, which belongs to the M20/M25/M40 family of proteins, from Caulobacter crescentus CB15.     

Decreased levels of free d-aspartic acid in the forebrain of serine racemase (Srr) knock-out mice

d-Serine, an endogenous co-agonist of the N-methyl-d-aspartate (NMDA) receptor is synthesized from l-serine by serine racemase (SRR). A previous study of Srr knockout (Srr-KO) mice showed that levels of d-serine in forebrain regions, such as frontal cortex, hippocampus, and striatum, but not cerebellum, of mutant mice are significantly lower than those of wild-type (WT) mice, suggesting that SRR is responsible for d-serine production in the forebrain. In this study, we attempted to determine whether SRR affects the level of other amino acids in brain tissue. We found that tissue levels of d-aspartic acid in the forebrains (frontal cortex, hippocampus and striatum) of Srr-KO mice were significantly lower than in WT mice, whereas levels of d-aspartic acid in the cerebellum were not altered. Levels of d-alanine, l-alanine, l-aspartic acid, taurine, asparagine, arginine, threonine, γ-amino butyric acid (GABA) and methionine, remained the same in frontal cortex, hippocampus, striatum and cerebellum of WT and mutant mice. Furthermore, no differences in d-aspartate oxidase (DDO) activity were detected in the forebrains of WT and Srr-KO mice. These results suggest that SRR and/or d-serine may be involved in the production of d-aspartic acid in mouse forebrains, although further detailed studies will be necessary to confirm this finding.     

Enzymatic Production of l-Alanine by Pseudomonas dacunhae

To establish an advantageous method for the production of l-alanine, a procedure was studied for converting l-aspartic acid to l-alanine by microbial l-aspartic beta-decarboxylase. A number of organisms were screened to test their ability to form and accumulate alanine from aspartic acid. Pseudomonas dacunhae was selected as the most advantageous organism. With this organism, enzyme activity as high as 3,910 muliters of CO(2) per hr per ml of medium could be produced by shaking the culture at 30 C in the medium containing ammonium fumarate, sodium fumarate, corn steep liquor, peptone, and inorganic salts. For the enzymatic conversion of l-aspartic acid to l-alanine, the culture broth was employed as the enzyme source. A large amount of l-aspartic acid (as much as 40% of the broth) was converted stoichiometrically to alanine in 72 hr at 37 C. Furthermore, appropriate addition of a surface-active agent to the reaction mixture was found to be highly effective in shortening the time required for the conversion. Accumulated l-alanine was readily isolated in pure form by ordinary procedures with ion-exchange resins. Yields of isolated l-alanine of over 90% from l-aspartic acid were easily attainable.     

Production of L-proline by Kurthia catenaforma

A number of organisms were screened for their ability to produce l-proline. Kurthia catenaforma, which we recently isolated, was selected. A serine-requiring mutant, strain 45, produced about 1.5 times the amount of this amino acid that the parent strain did. In investigations of various media, it was found that approximately 30 ml of l-proline per ml was produced in shaken culture at 30 C in a medium containing glucose, urea, corn steep liquor, casein hydrolysate, l-aspartic acid, and inorganic salts. To study the effect of l-aspartic acid on the production of l-proline, various amino or organic acids were substituted for l-aspartic acid, and the changes during fermentation were investigated. l-Aspartic acid was not replaced by the compounds tested, and this acid appeared to increase growth during the later stages of fermentation with a concurrent increase in the production of l-proline.     

A facile enzymatic method for the location of radioactivity associated with the carbons of L-aspartic acid, L-asparagine, and beta-cyano-L-alanine.

A radiometric enzymatic technique has been devised for locating radioactivity in the carbon skeleton of l-aspartic acid, l-asparagine, and β-cyano-l-alanine. l-Asparaginase at demonstrably discriminant concentrations is used to hydrolyze l-asparagine and/or β-cyano-l-alanine to l-aspartic acid, which in turn is transaminated to oxaloacetic acid by l-glutamate oxaloacetate transaminase. The β-carboxyl group of oxaloacetate is detached by zinc ions, and the radiolabeled CO₂ is collected in alkali after diffusion. The residual pyruvic acid is α-decarboxylated by pyruvate decarboxylase to CO₂, which is collected in a second alkaline trap, and the other product of decarboxylation, acetaldehyde, diffuses into a separate semicarbazide trap. Carbons 4, 1, and 3 plus 2 are located in this order. This method is shown to be facile, sensitive, reliable, and applicable to samples of biological origin.     

Synthesis of tripeptide RGD amide by a combination of chemical and enzymatic methods

The tripeptide Bz-Arg-Gly-Asp(NH₂)OH was synthesized by a combination of chemical and enzymatic methods in this study. Firstly, Gly-Asp-(NH₂)₂ was synthesized by a novel chemical method in three steps including chloroacetylation of l-aspartic acid, esterification of chloroacetyl l-aspartic acid and ammonolysis of chloroacetyl l-aspartic acid diethyl ester. Secondly, the linkage of the third amino acid (Bz-Arg-OEt) to Gly-Asp-(NH₂)₂ was completed by enzymatic method under kinetic control condition. An industrial alkaline protease alcalase was used in water–organic cosolvents systems. The synthesis reaction conditions were optimized by examining the effects of several factors including water content, temperature, pH and reaction time on the yield of the synthesis product Bz-Arg-Gly-Asp(NH₂)OH. The optimum conditions are pH 8.0, 35 °C, in ethanol/Tris–HCl buffer system (85:15, v/v), 8 h with the tripeptide yield of 73.6%.     

A C-NMR study on the influxes into the tricarboxylic acid cycle of a renal epithelial cell line, LLC-PK1/Cl4: the metabolism of [2-C]glycine, l-[3-C]alanine and l-[3-C]aspartic acid in renal epithelial cells

Perchloric acid extracts of LLC-PK₁/Cl₄ cells, a renal epithelial cell line, incubated with either [2-¹³C]glycine l-[3-¹³C]alanine, or d,l-[3-¹³C]aspartic acid were investigated by ¹³C-NMR spectroscopy. All amino acids, except labelled glycine, gave rise to glycolytic products and tricarboxylic acid cycle (TCA) intermediates. For the first time we also observed activity of γ-glutamyltransferase activity and glutathione synthetase activity in LLC-PK₁ cells, as is evident from enrichment of reduced glutathione. Time courseS showed that only 6% of the labelled glycine was utilized in 30 min, whereas 31% of l-alanine and 60% of l-aspartic acid was utilized during the same period. ¹³C-NMR was also shown to be a useful tool for the determination of amino acid uptake in LLC-PK₁ cells. These uptake experiments indicated that glycine alanine and aspartic acid are transported into Cl₄ cells via a sodium-dependent process. From the relative enrichment of the glutamate carbons, we calculated the activity of pyruvate dehydrogenase to be about 61% of when labelled l-alanine was the only carbon source for LLC-PK₁/Cl₄ cells. Experiments with labelled d,l-aspartic, however, showed that about 40% of C-3-enriched oxaloacetate (arising from a de-amination of aspartic acid) reached the pyruvate pool.     

Characteristics of a transport-deficient mutant (nap) of Neurospora crassa

nap, previously characterized by Jacobson and Metzenberg as a neutral and acidic amino acid transport mutant, was found in this study to be reduced in transport activity for a variety of metabolites. Analysis of glycoprotein molecules involved in amino acid binding indicates that the deficiency is not at this level. The deficiency appears to be in some common component of all active transport systems.     

The estimation of l-phenylalanine ammonia-lyase shows phenylpropanoid biosynthesis to be regulated by l-phenylalanine supply and availability

l-Phenylalanine ammonia-lyase (PAL, E.C. 4.3.1.5) is the first committed enzyme in the pathway leading to phenylpropanoid biosynthesis in higher plants. PAL catalyses the conversion of l-phenylalanine to t-cinnamic acid with the elimination of ammonia. Standard methods for determination of PAL activity in both green and non-green tissues were found to lead to measurements of both l-phenylalanine amino-transferase (PAT, E.C. 2.6.1.1) and PAL activities together. The accurate estimation of PAL activity alone, necessitated the inhibition of PAT by a specific inhibitor of PAT activity, l-aspartic acid. The influence of PAT on the kinetics of PAL activity may explain (i) the diverse properties that have been attributed to PAL and (ii) the controversies regarding the control mechanism underlying the regulation of PAL activity. Evidence is presented for the regulation of phenylpropanoid biosynthesis via substrate supply and availability as opposed to feedback inhibition, during phaseollin production and hypersensitive necrosis in Phaseolus vulgaris.     

Synthesis of indole-3-acetylaspartic acid

A simple synthesis of indole-3-acetyl-d,l-aspartic acid and its l-isomer is described and their physical properties are listed.     

Ureidosuccinic acid permeation in Saccharomyces cerevisiae

Some strains of Saccharomyces cerevisiae exhibit a specific transport system for ureidosuccinic acid, which is regulated by nitrogen metabolism. Ureidosuccinic acid uptake occurs with proline but with ammonium sulfate as nitrogen source it is inhibited. The V for transport is 20–25 μmol/ml cell water per min. The apparent K_m is 3 · 10^-5. For the urep1 mutant (ureidosuccinic acid permease less) the internal concentration never exceeds the external one.In the permease plus strain ureidosuccinic acid can be concentrated up to 10 000 fold and the accumulated compound remains unchanged in the cells. Energy poisons such as dinitrophenol, carbonyl cyanide-m-chlorophenyl-drazone (CCCP) or NaN₃ inhibit the uptake. No significant efflux of the accumulated compound occurs even in the presence of these drugs.The specificity of the permease is very strict, only amino acids carrying an α-N-carbamyl group are strongly competitive inhibitors.The high concentration capacity of the cells and the lack of active exit of the accumulated compound support the hypothesis of a carrier mediated active transport system.     

Growth response of Nitrosomonas europaea to amino acids

Growth responses of Nitrosomonas europaea to individual amino acids or vitamins was observed in log-phase cultures, as was the incorporation of carbon-14 labeled amino acids. Nitrite formation and protein synthesis were increased by l-glutamic acid, l-aspartic acid, l-serine, and l-glutamine. l-Lysine, l-histidine, l-threonine, l-valine, l-methionine, and l-arginine were inhibitory. The other amino acids had no effect on growth. All of the uniformly labeled amino acids added at low concentrations were taken up by growing cells and distributed into cell fractions. From 1 to 12% of the added radioactivity was present in cells analyzed in late log phase, depending on the amino acid; glycine and l-serine caused accumulation of the label to the greatest extent, whereas l-aspartic and l-glutamic acids were among those incorporated to the least extent. Aspartic acid increased both cell protein and nitrite values, but did not alter the ratio of protein to nitrite from that found in controls.     

Determination of free aspartic acid enantiomers in rat brain by capillary electrophoresis with laser-induced fluorescence detection

Quantification of aspartic acid enantiomers in rat brain by using a chiral capillary electrophoresis procedure is described. Amino acids were pre-column derivatized with naphthalene-2,3-dialdehyde. Enantiomeric separation was achieved by micellar electrokinetic chromatography in the presence of methanol and β-cyclodextrin as chiral selector. The chiral separation was coupled with laser-induced fluorescence detection. Contents of d- and l-aspartic acids in rats at different stages of growth (from 1 day before birth to 90 days after birth) were determined. d-Aspartic acid was detected in all the brain tissue samples tested, but at different levels. In the cerebrum of rats 1 day before birth, d-aspartic acid was found to be at the highest concentration of 81 nmol/g wet tissue. The level of d-aspartic acid in rat brain falls rapidly after birth, while the l-aspartic acid level increases with age.     

α-Methyl-l-glutamic acid uptake by high affinity dicarboxylic amino acid transport system in Streptococcus faecalis

The transport of α-methyl-l-glutamic acid was studied in Streptococcus faecalis. Energy-dependent uptake against substantial concentration gradients was observed. Kinetic experiments indicated that, in contrast to l-glutamic acid, only a single catalytic component (high affinity) and a diffusion controlled process participated in α-methyl-l-glutamic acid uptake. At concentrations up to 10 mM, α-methylglutamate transport was almost completely abolished in a mutant strain lacking a high affinity dicarboxylic amino acid transport system. In competition experiments, α-methylglutamic acid antagonized glutamate uptake via the high affinity system, and only slightly via the low affinity system. Column chromatography of cell extracts showed that very little (approx. 5%) of the accumulated amino acid was converted to metabolites during short term incubations. These studies indicate that, at concentrations up to 3–5 mM, α-methyl-l-glutamic acid can be used as a specific, relatively metabolically inert substrate of the high affinity dicarboxylic amino acid transport system in S. faecalis.     

Histidine uptake in mutant strains of Neurospora crassa via the general transport system for amino acids

A transport double mutant of Neurospora crassa has been isolated that has only one of the three transport systems capable of l-histidine uptake. The substrate specificity of the remaining transport system, termed the general transport system, has been fully characterized with regard to the contributions to binding of the side chain, the alpha-amino group, and the carboxylate group. The positively charged alpha-amino group is necessary for binding; the negatively charged carboxylate group is of less importance, since its replacement by a neutral carbonyl functional group does not completely abolish binding. The greatest structural latitude for binding was found in the side chain; affinity for alpha-amino acids was uniformly high except for l-aspartic and l-glutamic acids, l-asparagine, and l-proline. Thus, this transport system is "general" with these restrictions.     

Comparative transport activity of intact cells, membrane vesicles, and mesosomes of Bacillus licheniformis

Sodium ion was shown to stimulate strongly the transport of l-glutamic acid into cells of Bacillus licheniformis 6346 His(-). Lithium ion had a slight capacity to replace Na(+) in this capacity, but K(+) was without effect. Three of five amino acids tested. l-glutamic acid, l-aspartic acid, and l-alanine, were concentrated against a gradient in the cells. Intracellular pools of these amino acids were extractable with 5% trichloroacetic acid. Pools of l-histidine and l-lysine could not be detected. No evidence of active transport of lysine into cells could be detected, and histidine was taken up in the absence of chloramphenicol but not in its presence. The uptake of glutamic acid by membrane vesicle preparations was strongly stimulated by reduced nicotinamide adenine dinucleotide (NADH) and to a lesser extent by succinate. The presence of phenazine methosulfate increased uptake in the presence of succinate. Either l- or d-lactate and adenosine triphosphate were without effect. None of these compounds stimulated the uptake of glutamic acid by mesosomes, although some mesosome preparations contained separable membrane which was very active. NADH strongly stimulated the uptake of aspartic acid and alanine by membrane vesicles but had only a slight effect on the uptake of histidine and lysine. No evidence of active transport of any of the amino acids into mesosomes could be detected either in the presence or absence of NADH. NADH stimulation of the uptake of glutamic acid by membrane vesicles was destroyed by exposure to light of 360 nm; this inactivation was reversible by vitamin K(2(5)) or K(2(10)). Sodium ion stimulated transport of glutamic acid by membrane vesicles.     

Continuous production of l-aspartic acid from ammonium fumarate using immobilized cells by capture on the surface of nonwoven cloth coated with a pyridinium-type polymer

Cells of Escherichia coli possessing aspartase activity were immobilized by capture on the surface of nonwoven cloth coated with 10 mg/g of poly (N-benzyl-4-vinylpyridinium chloride-co-styrene), a pyridinium-type polymer. Continuous operation of a fixed-bed column reactor containing 21.7 g/l of the immobilized cells produced l-aspartic acid in 95% yield from ammonium fumarate in the case where influent solution contained 0.1 mol/l of the fumarate and space velocity was 1.36 h⁻¹ at 30°C and pH 8.9. Immobilization on the coated nonwoven cloth insignificantly affected optimal pH of the biochemical reaction. Stability of enzymic activity of the immobilized cells was much improved by use of the coated nonwoven cloth as the supporting material instead of beads of insoluble pyridinium-type resin. l-Aspartic acid was obtained in 77% yield after 160 d of continuous operation, and the initial yield was estimated to require about 500 d for halving.     

Control of mixed-substrate utilization in continuous cultures of Escherichia coli

The chemostat culture technique was used to study the control mechanisms which operate during utilization of mixtures of glucose and lactose and glucose and l-aspartic acid by populations of Escherichia coli B6. Constitutive mutants were rapidly selected during continuous culture on a mixture of glucose and lactose, and the beta-galactosidase level of the culture increased greatly. After mutant selection, the specific beta-galactosidase level of the culture was a decreasing function of growth rate. In cultures of both the inducible wild type and the constitutive mutant, glucose and lactose were simultaneously utilized at moderate growth rates, whereas only glucose was used in the inducible cultures at high growth rates. Catabolite repression was shown to be the primary mechanism of control of beta-galactosidase level and lactose utilization in continuous culture on mixed substrates. In batch culture, as in the chemostat, catabolite repression acting by itself on the lac enzymes was insufficient to prevent lactose utilization or cause diauxie. Interference with induction of the lac operon, as well as catabolite repression, was necessary to produce diauxic growth. Continuous cultures fed mixtures of glucose and l-aspartic acid utilized both substrates at moderate growth rates, even though the catabolic enzyme aspartase was linearly repressed with increasing growth rate. Although the repression of aspartase paralleled the catabolite repression of beta-galactosidase, l-aspartic acid could be utilized even at very low levels of the catabolic enzyme because of direct anabolic incorporation into protein.