On the asymmetric polymerization of aspartic acid enantiomers by kaolin

Summary Two recent reports in the literature claim thatl-aspartic acid polymerizes significantly faster thand-aspartic acid in the presence of kaolin in aqueous solution at 90°. The novelty of these observations and their potential significance for molecular evolution and the origin of optical activity in nature has prompted us to attempt a duplication of the experiments involved—using, however, analytical criteria which we felt would be more reliable than those previously employed. In our experimentsl- andd,l-aspartic acid in 0.01M solution were incubated with kaolin at 90° for 8 days. Careful examination of the aqueous residues from such experiments, however, failed to demonstrate any preferential polymerization ofl- overd-aspartic acid under the influence of kaolin, or indeed any significant gross polymerization of aspartic acid at all.     

Improvement of production of l-aspartic acid using immobilized microbial cells

Summary In our laboratory, EAPc-7 a strain having higher aspartase activity was derived from Escherichia coli ATCC 11303. For the improvement of l-aspartic acid productivity using EAPc-7 cells immobilized in -carrageenan, it was necessary to eliminate the fumarase activity which converts fumaric acid to l-malic acid. Several treatments for specifically eliminating fumarase activity from EAPc-7 cells were tested and it was found that when EAPc-7 cells were treated in a culture broth (pH 4.9) containing 50 mM l-aspartic acid at 45° C for 1 h, fumarase activity was almost completely eliminated without inactivation of the aspartase.The treated cells, immobilized in -carrageenan, were used for continuous production of l-aspartic acid from ammonium fumarate. The formation of l-malic acid was negligible and the half-life of the immobilized preparation was 126 days.Productivity of immobilized preparation of treated EAPc-7 cells in l-aspartic acid production was six times of that of the parent cell preparation.     

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.     

Removal of <Emphasis Type="SmallCaps">l</Emphasis>-alanine from the production of <Emphasis Type="SmallCaps">l</Emphasis>-2-aminobutyric acid by introduction of alanine racemase and <Emphasis Type="SmallCaps">d</Emphasis>-amino acid oxidase

l-2-Aminobutyric acid can be synthesized in a transamination reaction from l-threonine and l-aspartic acid as substrates by the action of threonine deaminase and aromatic aminotransferase, but the by-product l-alanine was produced simultaneously. A small amount of l-alanine increased the complexity of the l-2-aminobutyric acid recovery process because of their extreme similarity in physical and chemical properties. Acetolactate synthase has been introduced to remove the pyruvate intermediate for reducing the l-alanine concentration partially. To eliminate the remnant l-alanine, alanine racemase of Bacillus subtilis in combination with d-amino acid oxidase of Rhodotorula gracilis or Trigonopsis variabilis respectively was introduced into the reaction system for the l-2-aminobutyric acid synthesis. l-Alanine could be completely removed by the action of alanine racemase of B. subtilis and d-amino acid oxidase of R. gracilis; thereby, high-purity l-2-aminobutyric acid was achieved. The results revealed that alanine racemase could discriminate effectively between l-alanine and l-2-aminobutyric acid, and selectively catalyzed l-alanine to d-alanine reversibly. d-Amino acid oxidase then catalyzed d-alanine to pyruvate stereoselectively. Furthermore, this method was also successfully used to remove the by-product l-alanine in the production of other neutral amino acids such as l-tertiary leucine and l-valine, suggesting that multienzymatic whole-cell catalysis can be employed to provide high purity products.     

Optimization of aspartate ammonia lyase production by Bacillus cereus

In an attempt to clarify the function of l-aspartic acid and culture conditions in aspartate ammonia lyase induction, experiments were carried out on aspartase formation in Bacillus cereus cells. The enzyme was produced by microorganisms in response to l-aspartic acid, which is catabolized by direct deamination to fumarate. Enzyme synthesis by B. cereus was associated with physiological growth stages, which was confirmed by use of the protein synthesis inhibitor, chloramphenicol, whereas it did not influence synthesis when it was added directly to the reactor batch containing a biotransformation system. Aspartase activity was evaluated in a batch reactor by biotransformation of fumaric acid into l-aspartic acid catalyzed by whole B. cereus cells. The culture medium for the strain was optimized, which increased the initial aspartase activity threefold. B. cereus cells showed optimal aspartase activity at late log phase. Journal of Industrial Microbiology & Biotechnology (2000) 25, 225–228. Received 02 December 1999/ Accepted in revised form 09 August 2000     

Regulation of differentiation of the dimorphic fungusPaecilomyces viridis by nitrogen sources,antibiotics and metabolic inhibitors

The effect of nitrogen and carbon sources, vitamins, antibiotics and metabolic inhibitors on growth and differentiation ofPaecilomyces viridis was investigated. Sodium nitrate,l-asparagine,l-proline and peptone were found to be suitable nitrogen sources for mycelial growth (M) in a synthetic medium with glucose.Paecilomyces viridis could also grow slowly in a synthetic medium containing benzylpenicillin or bacitracin as the only nitrogen sources and very slowly even in a medium with polymyxin as the nitrogen source. Ammonium salts, area,l-arginine,d, l-aspartic acid andl,-serine were found to support intensive sporulation. Partially yeast-like growth (Y) was facilitated by NaNO₂, (NH₄)₂SO₄, NH₄NO₃, urea,d, l-alanine,l-arginine,d, l-aspartic acid,l-cysteine,l-glutamic acid andl-serine. Partially yeastlike growth could be observed in a medium with peptone and at an initial pH of 2. The following compounds appear as suitable carbon sources for mycelial growth:d-glucose,d-galactose,d-mannose, maltose, sucrose, chitin andd-mannitol. No changes in morphology could be detected on any of the 25 used carbon sources in a synthetic medium with NaNO₃. Yeast-like growth was induced by the antibiotics azalomycin F, cyanein (brefeldin A), griseofulvin and monorden (radicicol). After removal of the antibiotics, mycelial growth was restored. Sporulation was stimulated by chloramphenicol, 2-deoxy-d-glucose, furancarboxylic acid and stipitatic acid. Deformation of phialides was observed after treatment with actinomycin D, amphotericin B, boromycin, citrinin, cycloheximide, cytochalasin D, fungicidin and scopathricin. Microcyclic conidiation or growth of phialides directly from conidia were induced by cycloheximide, desertomycin, ethidium bromide and 5-fluorouracil.     

Interaction of free indole-3-acetic acid and its amino acid conjugates in tomato hypocotyl cultures

The interaction of free IAA and its amino acid conjugates on growth and development of cultured tomato hypocotyl tissue (Lycopersicon esculentum Mill. cv. Marglobe) was studied. In a nutrient medium containing 10 mol/L of benzyladenine, free IAA stimulated shoot and root development with little callus proliferation. In contrast, all IAA-amino acid conjugates tested supported mostly callus growth. Simultaneous application of free IAA and its conjugates resulted in the expression of mixed morphogenetic responses (i.e., both vigorous callus growth and organogenesis resulted). Growth kinetics and the effect of temporal exposure of the tissues to the bound and the free auxin suggest that some IAA-amino acid conjugates may specifically influence plant morphogenesis in ways that cannot be easily explained as simply a function of their slow hydrolysis to release free IAA.Abbreviations IAA indole-3-acetic acid - IAA-Ala N-(indol-3-ylacetyl)-l-alanine - IAA-Asp N-(indol-3-ylacetyl)-dl-aspartic acid - IAA-Lys N -(indol-3-ylacetyl)-l-lysine - IAA-Orn N -(indol-3-ylacetyl)-l-ornithine - IAA-Thr N-(indol-3-ylaetyl)-l-threonine     

An ultrastructural and physiological investigation of the retractor muscles of Gené's organ in the cattle ticksBoophilus microplus andAmblyomma variegatum

The ultrastructures of the retractor muscles of Gené's organs in the cattle ticksBoophilus microplus andAmblyomma variegatum are described. The innervation, neuromuscular junctions, and insertions of the muscles are also described. The retractor muscles are important in controlling the actions of Gené's organ, the egg waxing organ in ticks, during oviposition in the female. The ultrastructural features of the muscles are typical of arthropod muscles, and the nerve terminals at the neuromuscular junctions contain small electron lucent synaptic vesicles with a diameter of 50 nm, and also larger dense core vesicles with a diameter of 100 nm. Evidence is presented implicatingl-glutamate andl-aspartate as putative excitatory transmitters at the tick neuromuscular junction. The excitatory post-synaptic potentials recorded in the muscles were abolished in the presence of low concentrations ofl-glutamic acid andl-aspartic acid, but were unaffected by acetyl-choline, 4-aminobutyric acid and octopamine, suggesting that glutamic acid and aspartic acid interact with receptors on the muscle membrane.     

Biotechnological production of polyamines by bacteria: recent achievements and future perspectives

In Bacteria, the pathways of polyamine biosynthesis start with the amino acids l-lysine, l-ornithine, l-arginine, or l-aspartic acid. Some of these polyamines are of special interest due to their use in the production of engineering plastics (e.g., polyamides) or as curing agents in polymer applications. At present, the polyamines for industrial use are mainly synthesized on chemical routes. However, since a commercial market for polyamines as well as an industry for the fermentative production of amino acid exist, and since bacterial strains overproducing the polyamine precursors l-lysine, l-ornithine, and l-arginine are known, it was envisioned to engineer these amino acid-producing strains for polyamine production. Only recently, researchers have investigated the potential of amino acid-producing strains of Corynebacterium glutamicum and Escherichia coli for polyamine production. This mini-review illustrates the current knowledge of polyamine metabolism in Bacteria, including anabolism, catabolism, uptake, and excretion. The recent advances in engineering the industrial model bacteria C. glutamicum and E. coli for efficient production of the most promising polyamines, putrescine (1,4-diaminobutane), and cadaverine (1,5-diaminopentane), are discussed in more detail.     

Uptake of amino acids by actidione-treated yeast cells

Uptake of glyeine,l-cysteine,l-leucine,l-methionine,l-aspartic acid andl-lysine was investigated in resting cells ofSaccharomyces cerevisiae treated with 0.3mm actidione for blocking protein synthesis. The amino acids were taken up against substantial concentration gradients (up to nearly 1,000∶1 for μm l-cysteine and glycine). They were present in the free form inside the cells. Their unidirectional transmembrane fluxes were under a negative feedback control by the intracellular concentration of the amino acid involved. The amino acids tested apparently employed more than one transport agéncies for their membrane passage, the half-saturation constants being 6.2–7.7×10⁻⁴ m for glycine, 2.5×10⁻⁴ m forl-cysteine, 6×10⁻⁵ and 4×10⁻⁴ m forl-lysine, 3×10⁻⁵ and 6×10⁻⁴ m forl-methionine, 7–18×10⁻⁵ and 1.6×10⁻³ m forl-aspartic acid and 6×10⁻⁵ and 2×10⁻³ m forl-leucine. The specificities of the transport systems are overlapping but there emerges a wide-affinity transport system for glycine, alanine, leucine, methionine, serine, cysteine, phenylalanine, aspartic acid, asparagine, glutamic acid and tryptophan (and possibly for other amino acids), and more specific systems for each of the following: glycine, lysine, methionine, histidine, arginine, and aspartic and glutamic acids. Proline had the peculiar effect of stimulating the transport of all the amino acids tested. The amino acids apparently interacted in the uptake not only by competition for the binding site but also by allotopic inhibition (e.g.l-cysteine) and possibly stimulation (l-proline). The initial rate of uptake of amino acids and their steady-state level of distribution were characterized by identical activation energies: 7.5 kcal/mole forl-lysine, 6.9 kcal/mole forl-aspartic acid, and 13.2 kcal/mole for glycine.     

ThenadB gene ofSalmonella typhimurium complements the nicotinic acid auxotrophy ofShigella flexneri

Shigella species are characteristically nicotinic acid (NA) auxotrophs. The invasiveS. flexneri strain M90T, transformed with the multicopy plasmid pZT349 encoding thenadB gene ofSalmonella typhimurium, can grow in minimal glucose medium without exogenous NA, whereas, M90T containing the control vector, pUC18 does not, suggesting that this species lacksl-aspartic acid oxidase, the first enzyme in the de novo NAD biosynthetic pathway. The estimated growth rate of strain M90T (pZT349) in HeLa cells was identical to that of M90T (pUC18), indicating the available intracellular concentration of NA is not limiting for bacterial growth.     

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.     

Biotransformation of alkyl and aryl carbonates: enantioselective hydrolysis

Summary N-(Benzyloxycarbonyl)-l-asparty-l-phenylalanine methyl ester, the precursor of the synthetic sweetener aspartame, was continuously synthesized in an immobilized thermolysin plug-flow type reactor at 25° C with the substrates (N-benzyloxycarbonyl-l-aspartic acid and l-phenylalanine methyl ester) dissolved in ethyl acetate. The immobilized enzyme was quite stable in ethyl acetate containing 2.5% 0.01 M 2-(N-morpholino)ethanesulphonic acid-NaOH buffer, pH 6.0, and 20 mM CaCl₂ with or without the substrate at 25° C. By periodically washing the column, we could conduct a continuous reaction for over 500 h with an average yield of 95% and a space velocity of 1.85 h ^–1.Offprint requests to: K. Nakanishi     

A chemically defined medium for cephalosporin C production by Paecilomyces persicinus

A chemically defined medium was developed for the biosynthesis of cephalosporin C by Paecilomyces persicinus Nicot strain P-10. Glucose served as the major carbon source and nitrogen was supplied by five amino acids, l-arginine, l-aspartic acid, l-glutamic acid, glycine and dl-methionine. Omission of any of the first four diminished or prevented production of cephalosporin C; omission of methionine did not. Methionine is not critical for the production of cephalosporin C in this defined medium. Production of the antibiotic was affected by the concentrations of inorganic salts employed. Biotin was required for growth and cephalosporin C synthesis. The addition of l-lysine precursors to the medium did not influence cephalosporin C levels and l-lysine itself inhibited antibiotic production. Known precursors of -lactam antibiotics as well as oleic acid did not affect biosynthesis of cephalosporin C. Chemical changes occurring in the defined medium revealed that glucose was efficiently utilized after 96 hours incubation whereas total soluble nitrogen levels increased following an initial sharp decrease. Mycelial weight and cephalosporin C production were both maximal after 96 hours incubation. Mycelial nitrogen was highest after 48 hours incubation whereas mycelial lipid levels were greatest after 72 hours.     

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.     

Modification of cell membrane lipids inMicrococcus lysodeikticus induced by pantoyl lactone

Summary Growth ofMicroccoccus lysodeikticus in the presence of pantoyl lactone brings about both qualitative and quantitative changes in cell membrane lipids. Significant amounts of the two major phospholipids (phosphatidylglycerol and diphosphatidylglycerol) are converted to lyso forms; the largest conversion occurs in the phosphatidylglycerol. In addition, amounts of several phospholipid fatty acids are changed. Physical alteration of the cell membrane can be demonstrated using differential scanning calorimetry. Although growth and transport are significantly inhibited when pantoyl lactone is present, cells possessing altered cell membrane phospholipids and phospholipid fatty acids, brought about by growth in the presence of pantoyl lactone, transportd-alanine,l-glutamic andl-aspartic acid normally when washed free of the pantoyl lactone.     

The cytosolic pathway of l-malic acid synthesis in Saccharomyces cerevisiae: the role of fumarase

Saccharomyces cerevisiae accumulates l-malic acid but only minute amounts of fumaric acid. A ¹³C-nuclear magnetic resonance study following the label from glucose to l-malic acid indicates that the l-malic acid is synthesized from pyruvic acid via oxaloacetic acid. From this, and from previously published studies, we conclude that a cytosolic reductive pathway leading from pyruvic acid via oxaloacetic acid to l-malic acid is responsible for the l-malic acid production in yeast. The non-production of fumaric acid can be explained by the conclusion that, in the cell, cytosolic fumarase catalyzes the conversion of fumaric acid to l-malic acid but not the reverse. This conclusion is based on the following findings. (a) The cytosolic enzyme exhibits a 17-fold higher affinity towards fumaric acid than towards l-malic acid; the K _m for l-malic acid is very high indicating that l-malic acid is not an in vivo substrate of the enzyme. (b) Overexpression of cytosolic fumarase does not cause accumulation of fumaric acid (but rather more l-malic acid). (c) According to ¹³C NMR studies there is no interconversion of cytosolic l-malic and fumaric acids.     

Selection ofRhizopus strains forl(+)-lactic acid andγ-linolenic acid production

The production ofl(+)-lactic acid and formation ofγ-linolenic acid by 50Rhizopus strains growing on saccharidic substrates were investigated. Formation of acids was observed on solid cultivation media but mainly during submerged fermentation. Strains with the highest selectivity of bothl(+)-lactic acid production andγ-linolenic acid formation were tested in a laboratory fermenter. The best producer was treated by UV irradiation to increase the fatty acid content in the biomass, especially that ofγ-linolenic acid. The conversion of 10% saccharidic substrate by this newly prepared strainRhizopus arrhizus CCM 8109 results in more than 95% of theoretical yield ofl(+)-lactic acid and permits a volume productivity of 0.4 gγ-linolenic acid per liter.     

Purification of brain microtubule-associated protein MAP1A

Brain microtubule-associated protein MAP1A has been purified until homogeneity by using a novel procedure involving copolymerization with microtubules, treatment with poly-l-aspartic acid and FPLC. The purified protein retains its capacity to facilitate microtubule assembly.     

Malic acid accumulation by Aspergillus flavus

Summary Scanning electron microscopy revealed that Aspergillus flavus produced unusual crystals and hair-like processes during its l-malic acid production phase. Crystallinic dendritic aggregates were formed on the hyphae growing as pellets. The size and number of crystal aggregates increased during the fermentation in parallel with l-malic acid accumulation. The crystals (composed of calcium malate as well as small amounts of calcium succinate and calcium fumarate) were removed from the hyphae, after incubation with 6N HCl. On day 5 of the fermentation, about 9% of the total amount of l-malic acid produced was accounted for by the attached crystals. In addition to crystal formation we observed the appearance of hair-like processes during the early phase (2 days) of malic acid production only.