Properties of fibre-entrapped aspartase

Aspartase (l-aspartate ammonia lyase, EC 4.3.1.1) was extracted and purified from Escherichia intermedia cells. The enzyme was entrapped in cellulose triacetate porous fibres and the properties of the immobilized enzyme compared with those of the free enzyme. Similar behaviour was observed with regard to optimum pH, temperature, heat stability and kinetic constants. The stability of the entrapped enzyme was tested under operating conditions in a series of batch reactions. Good results were obtained for both the stability and the efficiency of the immobilized enzyme. The potential use of aspartase fibres for the production of l-aspartic acid is discussed.     

Identification of Flavone Compounds in Eighteen Gramineae Species

Conditions for tryptophan synthesis from pyruvic acid, indole and NH₄Cl by Enterobacter aerogenes AHU 1540 having a high tryptophanase activity, were investigated using a reaction mixture containing 1.7% of pyruvic acid. Under optimum conditions, 16.4g/liter of tryptophan was accumulated after 24 hr of incubation.

Agaricus campestris AHU 9382 produced pyruvic acid in amounts of 22 ~ 26.5 g/liter from 5% of glucose after 3-days shaking culture. When E. aerogenes was added to this fermentation broth together with indole and NH₄Cl, pyruvic acid produced was rapidly converted to tryptophan and yields of tryptophan as high as 15 g/liter were obtained after 12 hr of incubation. Furthermore, pyruvic acid fermentation by Saccharomyces exiguus AHU 3110 or Corynebacterium sp. 37-3A could also be used as a pyruvic acid source for subsequent tryptophan production.     

Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain

Glutamine synthetase (EC 6.3.1.2) has been purified from a collagenolytic Vibrio alginolyticus strain. The apparent molecular weight of the glutamine synthetase subunit was approximately 62,000. This indicates a particle weight for the undissociated enzyme of 744,000, assuming the enzyme is the typical dodecamer. The glutamine synthetase enzyme had a sedimentation coefficient of 25.9 S and seems to be regulated by a denylylation and deadenylylation. The pH profiles assayed by the -glutamyltransferase method were similar for NH₄-shocked and unshocked cell extracts and isoactivity point was not obtained from these eurves. The optimum pH for purified and crude cell extracts was 7.9. Cell-free glutamine synthetase was inhibited by some amino acids and AMP. The transferase activity of glutamine synthetase from mid-exponential phase cells varied greatly depending on the sources of nitrogen or carbon in the growth medium. Glutamine synthetase level was regulated by nitrogen catabolite repression by (NH₄)₂SO₄ and glutamine, but cells grown, in the presence of proline, leucine, isoleucine, tryptophan, histidine, glutamic acid, glycine and arginine had enhanced levels of transferase activity. Glutamine synthetase was not subject to glucose, sucrose, fructose, glycerol or maltose catabolite repression and these sugars had the opposite effect and markedly enhanced glutamine synthetase activity.Abbreviations GS glutamine synthetase - SMM succinate minimal medium - ASMM ammonium/succinate minimal medium - GT -glutamyl transferase - SVP snake venom phosphodiesterase     

Activities of Cysteine Synthetase and Cystathionase in Bacilhis subtilis during Sporulation

Cysteine synthetase (O-acetylserine sulfhydrylase) was partially purified from cells of Bacillus subtilis by the use of ammonium sulfate fractionation technique and DEAE-Sephadex A–50 chromatography. The cysteine synthetase preparation was compared with cystathionase (cystathionine β-cleavage enzyme) of the same organism in regard to biochemical properties and to changes in activity during sporulation.

The optimal pH and temperature for the cysteine synthetase were 8.5 and 25°C respectively. The enzyme was relatively stable at temperatures below 50°C and fairly resistant to proteases, in contrast to cystathionase. Production by B. subtilis of cysteine synthetase in sulfur-deficient synthetic medium was repressed by the addition of cysteine and derepressed by djenkolic acid. Activity of the enzyme was inhibited by methionine and increased by acetate. The cysteine synthetase activity was almost constant until the late sporulation stage commenced, but the specific activity of cystathionase (Fraction I) decreased rapidly in the course of sporulation and it could not be detected in the free spores.     

Vitamin B12-dependent Methionine Synthetase in Photosynthetic Bacteria Partial Purification and Properties

Vitamin B₁₂-dependent methionine synthetase (N⁵-methyItetrahydrofolate-homocysteine Bi2-methyltransferase; EC 2.1.1.13) was partially purified from two different types of photo-synthetic bacteria, Chromatium D and Rhodospirillum rubrum.

Chromatium D, which does not produce vitamin B₁₂, possessed apomethionine synthetase when grown in the absence of the vitamin. Partially purified apoenzyme was converted to holoenzyme efficiently with CH₃B₁₂ or OHB₁₂. Holo-methionine synthetase was purified 244 fold with 56.4 % recovery from Chromatium D cells grown with vitamin B₁₂ added. The partially purified enzyme required reductants but was only partially dependent on S-adenosylmethionine.

On the other hand, Rsp. rubrum methionine synthetase which was always present as holoenzyme, in contrast with that of Chromatium D, was purified 40 fold with 2.8% recovery. The obtained preparation required S-adenosylmethionine and reductants for the enzyme activity. The optimal pH of Chromatium D enzyme and of Rsp. rubrum enzyme was in the range of 7.5~7.8 and 6.5~6.75, respectively.     

Tryptophan Metabolism in Ochromonas malhamensis

Tryptophan enhanced the growth of Ochromonas malhamensis at concentrations up to 0.4 mg/ml; higher concentrations inhibited, the growth inhibition being reversible by tyrosine and adenine. The presence of a tryptophan synthetase system in vitro was demonstrated. Tyrosine and phenylalanine stimulated the activity of this enzyme. The uptake of exogenous tryptophan was accompanied by an increase in the free tryptophan pool which in turn suppressed the tryptophan synthetase system, thus pointing to a controlled mechanism. Incorporation of tryptophan in the growth medium enhanced the biosynthesis of folate-active compounds. An elucidation of the mode of action of tryptophan is attempted on the basis of known metabolic pathways.     

Biosynthesis and Characterization of 4-Fluorotryptophan-Labeled Escherichia coli Arginyl-tRNA Synthetase

Escherichia coli 4-fluorotryptophan-substituted arginyl-tRNA synthetase was biosynthetically prepared and purified from a tryptophan auxotroph which could overproduce this enzyme. A method was developed to separate 4-fluorotryptophan from tryptophan and to determine accurately their contents in the 4-fluorotryptophan-containing proteins. It was confirmed that more than 95% of the tryptophan residues in the purified 4-fluorotryptophan-substituted arginyl-tRNA synthetase were replaced by 4-fluorotryptophan. Studies on the effect of the 4-fluorotryptophan replacement on properties of the enzyme showed that, when compared with the native enzyme, both the specific activity and the first-order rate constant of the fluorinated enzyme decreased by approximately 20% with just slightly higher K _m values. CD studies, however, did not reveal any difference between the secondary structure of the native and fluorinated enzymes. In addition, thermal unfolding studies showed that the 4-fluorotryptophan replacement did not significantly affect the thermal stability of the enzyme. We may conclude that the substitution of 4-fluorotryptophan in arginyl-tRNA synthetase had no substantial effect on the structure and function of the enzyme. Finally, a preliminary study of ¹⁹F nuclear magnetic resonance spectroscopy of the fluorinated enzyme has shown promising prospect for further investigation of its structure and function with NMR.     

Evidence for Compartmentation of Tryptophan in Cultured Plant Tissues: Free Tryptophan Levels and Inhibition of Anthranilate Synthetase

1. Anthranilate synthetase activity in crude extracts from tissue cultures of Daucus carota L. (carrot), Nicotiana tabacum L. (tobacco; cv. Wisconsin 38 and xanthi), Glycine max Merr. (soybean) and Oryza sativa L. (rice) was completely inhibited by l -tryptophan (5 to 50 μM). Mutant carrot and tobacco lines, capable of growth in the presence of 5-methyltryptophan, required 500 to more than 1000 μM tryptophan for complete inhibition of enzyme activity, respectively. 2. Except for the mutant tobacco line, the concentrations of free tryptophan in all tissue cultures tested were greater than the levels necessary to completely inhibit the respective anthranilate synthetase activities in vitro. These findings would indicate that much of the free tryptophan is compartmentalized away from the regulatory enzyme, anthranilate synthetase. This could implicate compartmentalization of the inhibitor as a biosynthetic control mechanism. 3. During the growth of normal and mutant carrot tissues the anthranilate synthetase enzyme must be at least 7.8 and 10.8% active, respectively, in order to accumulate the amount of tryptophan found in the tissues. 4. Of the substrates and cofactors required for anthranilate synthetase activity in vitro, Mg²⁺ and glutamine were present at near optimal levels in the carrot and tobacco tissues, but chorismate was found to be significantly below the optimal concentrations.     

Characteristics of a New Catechol 1,2-Oxygenase from Trichosporon cutaneum WY 2-2

Successive feeding of phenol at concentrations of less than 5.5 mM into a thick suspension of Trichosporon cutaneum WY 2-2 precultured in MPY-medium resulted in a high yield (approximately 28.7 g wet cells/liter) of intact cells capable of decomposing phenol actively (3.7 μmol/min/g of wet cells).

The effects of pH and additions of ethanol and 2-mercaptoethanol were tested on the stability of crude extracts from the strain grown on phenol. The crude extracts were stable at a pH range of 7.6 and 8.3, and were stable for 35 days when 10% ethanol and 5 mM 2-mercaptoethanol were added.

A highly purified preparation of catechol 1,2-oxygenase was obtained from strain WY 2-2 grown on phenol. The purified enzyme was homogeneous on polyacrylamide disc-gel electrophoresis. The enzyme had a molecular weight of about 105,000 and gave rise to subunits of molecular weight of 35,000 by SDS gel electrophoresis. Therefore, the enzyme appears to be a trimer of subunits with identical molecular weight. The Michaelis constants were 9.0 μM for catechol and 6.8 μM for 4-methylcatechol. The enzyme exhibited higher activities towards 4-methylcatechol and hydroxyquinol than towards catechol, and had essentially the same substrate specificity as the crude extracts. 4-Methylcatechol completely inhibited the enzyme activity towards catechol.     

Production of L-Tryptophan by 5-Fluorotryptophan Resistant Mutants of Bacillus subtilis

The growth of Bacillus subtilis TR–44, a prototrophic transductant from one of inosine producers, was completely inhibited by 200 µg/ml of 5-fiuorotryptophan, a tryptophan analogue, and the inhibition was reversed by the addition of L-tryptophan.

Several mutants resistant to 5FT* produced L-tryptophan in the growing cultures. The best producer, strain FT–39, which was selected on a medium containing 1500 µg/ml of 5FT, produced 2 g/liter of L-tryptophan, when cultured in a medium containing 8% of glucose but without any tryptophan precursors. In this mutant, anthranilate synthetase, a key enzyme of the tryptophan biosynthesis, had increased over 280-fold, presumably owing to a genetic derepression. From FT–39, mutants resistant to 7000 µg/ml of 5FT were derived. Among them, strain FF–25 produced 4 g/liter of L-tryptophan, twice as much as did the parental strain. Since this strain produced large amount of L-phenylalanine as well as L-tryptophan, the genetic alteration seemed to be involved in some metabolic regulation of common part of the aromatic amino acid biosynthetic pathway.

Further, some auxotrophs derived from these 5FT resistant mutants produced more L-tryptophan than did the parental strains.

Relationships between the accumulation of L-tryptophan and the regulation mechanisms of the L-tryptophan biosynthesis were discussed.     

Phytase from Aspergillus terreus

1) Aspergillus terreus No. 9A-1 was cultivated by a shaking method and the optimal cultural conditions for the phytase production were concluded as follows: Composition of medium; rice bran 30 g, ammonium sulfate 3 g, distilled water 1.0 liter; initial pH 5.5; shaking condition; 50 ml of medium/500 ml vol. flask; 120 oscil./min, 90 hr.

2) Phytase from Asp. terreus was purified by ammonium sulfate precipitation, acetone precipitation and chromatography on SE-Sephadex C-50 and Sephadex G-200 columns. The enzyme was purified about 520-folds with the yield of 20% from the broth. The purified enzyme was homogeneous by column chromatography, ultracentrifugation and electrophoresis.

3) This purified preparation of phytase showed following properties, a) Optimal pH for the reaction was 4.5; b) optimal temperature for the reaction was about 70°C; c) the enzyme was stable in the range of pH from 1.2 to 9.0     

Measurement of the Five Enzymes which Convert Chorismate to Tryptophan in Wheat Plants (Triticum aestivum cv. Kalyansona)

Conditions are described for measuring anthranilate synthetase, anthranilate-PRPP-phosphoribosyl transferase, N-5′-phosphoribosyl anthranilate isomerase, indole-3-glycerol phosphate synthetase and tryptophan synthetase in crude extracts from Triticum aestivum (wheat) plants. Only the last enzyme has been measured before in extracts from green plants. The extractable quantities of each enzyme in all plant parts at all stages of growth were sufficient to synthesize the amount of tryptophan present within the same tissue in 48 h. Anthranilate synthetase activity was the lowest of the five enzyme activities and was the only one inhibited by tryptophan in vitro, indicating that this enzyme may be the control point in tryptophan biosynthesis in wheat plants.     

Control of Tryptophan Biosynthesis in Plant Tissue Cultures: Lack of Repression of Anthranilate and Tryptophan Synthetases by Tryptophan

Tobacco (cv. Xanthi and cv. Wisconsin 38), rice, carrot, tomato, and soybean tissue cultures were grown in liquid media containing L-tryptophan. The addition of tryptophan increased the cellular tryptophan levels greatly (12–2500 fold), but did not lower appreciably the levels of two tryptophan biosynthetic enzymes, anthranilate synthetase and tryptophan synthetase. However, the addition of 50 μM tryptophan to the crude enzyme extract completely inhibited the anthranilate synthetase activity while 1 mM tryptophan inhibited the tryptophan synthetase activity by only 10–20°/o. This information indicates that tryptophan biosynthesis is controlled by the feedback inhibition of anthranilate synthetase by tryptophan and not by repression of enzyme synthesis. All of the species had significant enzyme levels. Anthranilate synthetase activity could not be detected in extracts from cells grown on tryptophan unless the extracts were first passed through two G-25 Sephadex columns with a short 30 °C warming step in between, a procedure shown to remove an inhibitor of the enzyme.     

Inactivation of glutamine synthetase by adenylylation in intact cells of E. coli

Summary The adenine pool of a purineless mutant of E. coli was radioactively labelled by short incubation with ¹⁴C-adenine.The glutamine synthetase was inactivated in vivo by incubation of the cell suspension with 2x10^-3 M NH₄ ⁺ for 2 min. The inactivated glutamine synthetase was extracted from the cells and purified 20-fold.Incubation of the purified glutamine synthetase with phosphodiesterase regenerated the biosynthetic activity of the enzyme paralleled by the liberation of ¹⁴C-adenine and ¹⁴C-adenosine. ¹⁴C-adenine and ¹⁴C-adenosine were also obtained when inactivated glutamine synthetase, prepared in vitro by use of ¹⁴C-ATP and purified adenylylating enzyme, was incubated with phosphodiesterase under the same conditions.The similar liberation of adenine derivatives by phosphodiesterase from glutamine synthetase inactivated in a cell-free system as well as in intact cells, demonstrates that in both cases the inactivation consists in an adenylylation of the enzyme.     

Extracellular Accumulation of DNA by Hydrocarbon-utilizing Bacteria

Some hydrocarbon-utilizing bacteria grown on n-paraffin as the sole source of carbon accumulated extracellularly a considerable amount of DNA (0.1 g to 0.6 g per liter) which was free from intact cells and slime materials. This was particularly noted when the strains belonging to Pseudomonad were employed. n-Paraffin was a preferable carbon source for the accumulation of DNA by Pseudomonad, while a strain of Arthrobacter accumulated DNA by growing it on glucose.

The DNA thus accumulated was easily isolated and purified free from other cellular components. The purified DNA was highly polymerized (above 4 × l0⁶ daltons), having the same base composition as the cellular DNA.     

Extracellular Lytic Enzymes of Myxococcus virescens

An easy and rapid method for the purification of a bacteriolytic endopeptidase produced by Myxococcus virescens is described. The bacteria were grown in casitone media and the cells were sedimented by centrifugation. About 1.2 g of montmorillonite were added per liter of cell-free culture solution. The clay was sedimented by centrifugation and the enzyme was then eluted by 0.05 M Na-phosphate buffer pH 6.0, containing 0.4 M NaCl. The enzyme was diluted with water and chromatographed on carboxymethyl-cellulose columns. The purified enzyme liberated free amino groups but no reducing sugars or N-acetylhexosamines when acting on purified N-acetylated cell walls of Micrococcus lysodeikticus. Analysis of N- and C-terminal amino acids in the digestion products showed that the enzyme had liberated about 110 nmoles of lysine ε-amino groups and 60 nmoles of alanine carboxyl groups per mg of cell wall. When it acted on a bisdisaccharide pentapeptide dimer isolated from M. lysodeikticus cell walls, it cleaved about 30% of the alanyl-lysine linkages. Consequently the enzyme was an alanyl-lysine endopeptidase. It had no muramyl-alanine amidase activity.     

Purification of Inulin Fructotransferase (DFA I-producing) from Arthrobacter MCI2493 and Production of DFA I from Inulin by the Enzyme

An extracellular enzyme that produces di-d-fructofuranose-2′, 1;2, 1′ dianhydride from inulin was purified from the culture broth cf Arthrobacter sp. MCI2493. The molecular weight of the enzyme was 40,000 by gel filtration and SDS polyacrylamide gel electrophoresis. The enzyme had maximum activity at pH 6.0 and 50°C. Using this purified enzyme, 100g/liter inulin was converted into 60 g/liter of DFA I, nystose, and 1-f-fructofuranosyl-nystose after incubation for 30 h.     

Studies on tryptophan synthetases from various strains of Bacillus subtilis

1. Tryptophan synthetase B of three strains of Bacillus subtilis was prepared from ;exo-protoplastic' and ;endo-protoplastic' fractions; the enzyme from ;exo-protoplastic' fraction was purified 30- to 120-fold by ammonium sulphate precipitation and DEAE-cellulose column chromatography; the latter step separated this enzyme from tryptophan synthetase A, tryptophanase and proteolytic enzymes, but the purified preparations were not stable. 2. The activity of tryptophan synthetase B did not depend on the presence of tryptophan synthetase A. 3. Tryptophan synthetases B of the strains tested differed in their utilization of 2- and 7-methylindole as compared with indole; this suggests that these tryptophan synthetases B are not identical.     

Biosynthesis and Characterization of 4-Fluorotryptophan-Labeled Escherichia coli Arginyl-tRNA Synthetase

Escherichia coli 4-fluorotryptophan-substituted arginyl-tRNA synthetase was biosynthetically prepared and purified from a tryptophan auxotroph which could overproduce this enzyme. A method was developed to separate 4-fluorotryptophan from tryptophan and to determine accurately their contents in the 4-fluorotryptophan-containing proteins. It was confirmed that more than 95% of the tryptophan residues in the purified 4-fluorotryptophan-substituted arginyl-tRNA synthetase were replaced by 4-fluorotryptophan. Studies on the effect of the 4-fluorotryptophan replacement on properties of the enzyme showed that, when compared with the native enzyme, both the specific activity and the first-order rate constant of the fluorinated enzyme decreased by approximately 20% with just slightly higher K _m values. CD studies, however, did not reveal any difference between the secondary structure of the native and fluorinated enzymes. In addition, thermal unfolding studies showed that the 4-fluorotryptophan replacement did not significantly affect the thermal stability of the enzyme. We may conclude that the substitution of 4-fluorotryptophan in arginyl-tRNA synthetase had no substantial effect on the structure and function of the enzyme. Finally, a preliminary study of ¹⁹F nuclear magnetic resonance spectroscopy of the fluorinated enzyme has shown promising prospect for further investigation of its structure and function with NMR.     

Nachweis und Lokalisation von Tryptophan-Synthetase in Samen von Juglans regia L.

Summary Cell-free extracts from seeds of Juglans regia synthesize tryptophan from L-serine and indole. Tryptophan synthetase has maximal activity in the range between pH 7 and 8. The enzyme is associated with a particulate fraction (density 1,210 g/ml) which is separated from the mitochondria (density 1,191 g/ml) after isopycnic density centrifugation on a continuous sucrose gradient.