Reverse Reaction of Malic Enzyme for HCO3 − Fixation into Pyruvic Acid to Synthesize L-Malic Acid with Enzymatic Coenzyme Regeneration

Malic enzyme [L-malate: NAD(P)⁺ oxidoreductase (EC 1.1.1.39)] catalyzes the oxidative decarboxylation of L-malic acid to produce pyruvic acid using the oxidized form of NAD(P) (NAD(P)⁺). We used a reverse reaction of the malic enzyme of Pseudomonas diminuta IFO 13182 for HCO₃ ^? fixation into pyruvic acid to produce L-malic acid with coenzyme (NADH) generation. Glucose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuconostoc mesenteroides was suitable for coenzyme regeneration. Optimum conditions for the carboxylation of pyruvic acid were examined, including pyruvic acid, NAD⁺, and both malic enzyme and glucose-6-phosphate dehydrogenase concentrations. Under optimal conditions, the ratio of HCO₃ ^? and pyruvic acid to malic acid was about 38% after 24 h of incubation at 30 °C, and the concentration of the accumulated L-malic acid in the reaction mixture was 38 mM. The malic enzyme reverse reaction was also carried out by the conjugated redox enzyme reaction with water-soluble polymer-bound NAD⁺.     

Physicochemical and Enzymatic Properties of ATP: Nucleotide Pyrophosphotransferase

The molecular weight determined by the sedimentation equilibrium and SDS Polyacrylamide gel electrophoresis was 29,000 and 28,000, respectively. Isoelectric point of the enzyme was determined as pH 7.7. This enzyme contained large amounts of alanine, aspartic acid, glutamic acid and serine, and no cysteine residue was found. The enzyme was inhibited by SDS, KMnO₄, EDTA and tetracycline. GTP and GDP were the most active as pyrophosphate acceptor to the enzyme. The apparent Km for ATP was 2.2×10^?4 m and that for GTP was 2.1×10^?4m in the reaction of ATP+GTP→AMP+pppGpp. On the other hand, in the reaction of 2ATP→AMP+pppApp, the apparent Km for donor and acceptor ATP was 1.7×10^?3m. Effects of pH and metal ions on the enzymatic synthesis of pppGpp were also studied.     

Substrate Specificity of Alkaline Proteinases from Aspergillus sydowi and Aspergillus sulphureus

l-Fucose (l-galactose) dehydrogenase was isolated to homogeneity from a cell-free extract of Pseudomonas sp. No 1143 and purified about 380-fold with a yield of 23 %. The purification procedures were: treatment with polyethyleneimine, ammonium sulfate fractionation, chromatographies on phenyl-Sepharose and DEAE-Sephadex, preparative polyacrylamide gel electrophoresis, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of about 34,000. The optimum pH was at 9 — 10.5 and the isoelectric point was at pH 5.1. l-Fucose and l-galactose were effective substrates for the enzyme reaction, but d-arabinose was not so much. The anomeric requirement of the enzyme to l-fucose was the β-pyranose form, and the reaction product from l-fucose was l-fucono- lactone. The hydrogen acceptor for the enzyme reaction wasNADP⁺, and NAD ⁺ could be substituted for it to a very small degree. Km values were 1.9mm, 19mm, 0.016mm, and 5.6mm for l-fucose, l- galactose, NADP⁺, and NAD⁺, respectively. The enzyme activity was strongly inhibited by Hg^{2 +}, Cd^{2 +}, and PCMB, but metal-chelating reagents had almost no effect. In a preliminary experiment, it was indicated that the enzyme may be usable for the measurement of l-fucose.     

N-Carbamyl-L-Amino Acid Amidohydrolase of Pseudomonas sp. Strain NS671: Purification and Some Properties of the Enzyme Expressed in Escherichia coli

An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp. strain NS671 was expressed. The purified enzyme was homogeneous by the criterion of SDS–polyacrvlamide gel electrophoresis. The results of gel filtration chromatography and SDS–polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits. The enzyme required Mn²⁺ ion (above 1 mM) for the activity. The optimal pH and temperature were 7.5 and around 40°C, respectively, with N-carbamyl-L-methionine as the substrate. The enzyme activity was inhibited by ATP and was iost completely with p-chloromercuribenzoate (1 mM). The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-L-α-amino acids.     

Isolation and Identification of ( – ) - Quinic Acid as an Unidentified Major Tea-component

A new enzyme, N-acetyl- d-hexosamine dehydrogenase (N-acety 1-α-d-hexosamine: NAD⁺ 1-oxidoreductase), was purified to homogeneity on polyacrylamide gel electrophoresis from a strain of Pseudomonas sp. about 900-fold with a yield of 12 %. The molecular weight of the enzyme was about 124,000 on gel filtration and 30,000 on SD S-polyacrylamide gel electrophoresis, respectively. Its isoelectric point was 4.7. The optimum pH was about 10.0. The enzyme was most stable between pH 8.0 and pH 10.5. The highest enzyme activity was observed with N-acetyl-d-glucosamine (Km = 5.3mm) and N-acetyl-d-galactosamine (Km = 0.8mm) as the sugar substrate. But it was not so active on N-acetyl-d-mannosamine. NAD⁺ was used specifically as the hydrogen acceptor. The anomeric requirement of the enzyme for N-acetyl-d-glucosamine was the α-pyranose form, and the reaction product was N-acetyl-d-glucosaminic acid. The enzyme activity was inhibited by Hg and SDS, but many divalent cations, metal-chelating reagents, and sulfhydryl reagents had no effect.     

Isolation and amino acid sequence of a dehydratase acting on d-erythro-3-hydroxyaspartate from Pseudomonas sp. N99, and its application in the production of optically active 3-hydroxyaspartate

An enzyme catalyzing the ammonia-lyase reaction for the conversion of d-erythro-3-hydroxyaspartate to oxaloacetate was purified from the cell-free extract of a soil-isolated bacterium Pseudomonas sp. N99. The enzyme exhibited ammonia-lyase activity toward l-threo-3-hydroxyaspartate and d-erythro-3-hydroxyaspartate, but not toward other 3-hydroxyaspartate isomers. The deduced amino acid sequence of the enzyme, which belongs to the serine/threonine dehydratase family, shows similarity to the sequence of l-threo-3-hydroxyaspartate ammonia-lyase (EC 4.3.1.16) from Pseudomonas sp. T62 (74%) and Saccharomyces cerevisiae (64%) and serine racemase from Schizosaccharomyces pombe (65%). These results suggest that the enzyme is similar to l-threo-3-hydroxyaspartate ammonia-lyase from Pseudomonas sp. T62, which does not act on d-erythro-3-hydroxyaspartate. We also then used the recombinant enzyme expressed in Escherichia coli to produce optically pure l-erythro-3-hydroxyaspartate and d-threo-3-hydroxyaspartate from the corresponding dl-racemic mixtures. The enzymatic resolution reported here is one of the simplest and the first enzymatic method that can be used for obtaining optically pure l-erythro-3-hydroxyaspartate.     

DAHP Synthetase and Its Control in Corynebacterium glutamicum

Properties of 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthetase from Corynebacterium glutamicum were examined using the cell free extract. The optimum pH for the reaction was broad ranging from 5.5 to 7.0 and the optimum temperature was 37°C. Co²⁺ inhibited the enzyme activity at 20°C, whereas Co²⁺ apparently stimulated the enzyme activity at 37°C because the ion protected the enzyme from inactivation at 37°C. Co²⁺ reversed the inhibition of the enzyme activity by EDTA. The activity of DAHP synthetase was feedback inhibited only weakly by l-phenylalanine, l-tyrosine or l-tryptophan alone, but was strongly inhibited synergistically by l-phenylalanine and l-tyrosine. l-Tryptophan enhanced the inhibition by the pair of l-tyrosine and l-phenylalanine. Maximal inhibition was near 90 % in the simultaneous presence of the three amino acids. Sensitivity of the enzyme to the inhibitors was lost during the purification process of the enzyme or during the reaction at 37°C. Especially sensitivity to l-tryptophan was easily lost. Co²⁺ protected the enzyme from the desensitization. Mutants resistant to p-fluorophenylalanine plus l-tyrosine (or 3-aminotyrosine) had DAHP synthetase which was released from the feedback inhibition by the three amino acids. The formation of the enzyme was not affected by aromatic amino acids.     

Kinetic Studies on Crystalline Yeast Phosphoglyceric Acid Mutase

Effects of the substrate and the coenzyme on the crystalline yeast phosphoglyceric acid mutase activity have been investigated. Lineweaver-Burk plots at different concentrations of the substrate (d-3-phosphoglyceric acid: 3×10^?7 to 8×10^?3m) and the coenzyme (d-2, 3-diphosphoglyceric acid: 8×10^?7 to 10^?5m) change in such a way to indicate the involvement of an enzyme-substrate-coenzyme ternary complex as an active intermediate in the enzymic reaction process. It is concluded that the reaction catalyzed by the yeast enzyme follows the sequential pathway and that a phosphorylated enzyme does not participate as an obligatory intermediate in the reaction mechanism, if it occurs. Kinetic studies indicate Km values of 6×10^?4m for d-3-phosphoglyceric acid and 8×10^?7m for d-2, 3-diphosphoglyceric acid. The substrate is a competitive inhibitor of the coenzyme with a Ksi (inhibition constant) of 3.2×10^?3m. The coenzyme inhibition is not observed at concentration tested. A kinetic treatment to determine the mechanism of the enzyme reaction from the experimental data which are obtaind in the range of inhibitory substrate concentrations is presented.     

Metal-characterization of N-Acyl-D-glutamate Amidohydrolase from Pseudomonas sp. Strain 5f-l

N-Acyl-D-glutamate amidohydrolase (D-AGase) from Pseudomonas sp. 5f-1 was a zinc-metalloenzyme which contained 2.06–2.61 g. atom of Zn per mole of enzyme. The zinc atom was required for the catalytic activity and stability of the enzyme. The N-terminal amino acid sequence of Pseudomonas sp. 5f-l D-AGase showed 32% identity to that of Alcaligenes xylosoxydans subsp. xylosoxydans A-6.     

Cyclic (1→2)-β-d-Glucan-hydrolyzing Enzymes from Acremonium sp. 15 Purification and Some Properties of Endo-( 1 → 2)-α-d-glucanase and β-d-Glucosidase

Acremonium sp. 15 a fungus isolated from soil, produces an extracellular enzyme system degrading cyclic (1→2)-β-d-glucan. This enzyme was found to be a mixture of endo-(1→2)-β-d-glucanase and β-d-glucosidase. The (1→2)-β-d-glucanase was purified to homogeneity shown by disc-electrophoresis after SP-Sephadex column chromatography, Sephadex G-75 gel filtration, and rechromatography on SP-Sephadex. The molecular weight of the enzyme was 3.6 × 10⁴ by SDS-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was pH 9.6. The enzyme was most active at pH 4.0—4.5, and stable up to 40°C in 20 mm acetate buffer (pH 5.0) for 2 hr of incubation. This enzyme hydrolyzed only (l→2)-β-d-glucan and did not hydrolyze laminaran, curdlan, or CM-cellulose. The hydrolysis products from cyclic (1→2)-β-d-glucan were mainly sophorose.

The β-d-glucosidase was purified about 4000-fold. The rate of hydrolysis of the substrates by this β-d-glucosidase decreased in the following order: β-nitrophenyl-β-d-glucoside, sophorose, phenyl-β-d-glucoside, laminaribiose, and salicin. This enzyme has strong transfer action even at the low concentration of 0.75 mm substrate.     

Improvement of Gene Targeting in Aspergillus nidulans with Excess Non-Homologous Fragments

An α-glucosidase was purified in an electrophoretically pure state from an extract of koji culture of Aspergillus sp. KT-11. This enzyme was found to have a transferring activity when the reaction was done in a high concentration of leucrose at pH 4.5. Two kinds of transfer products, fractions I and II, were obtained from leucrose by the enzyme and they were identified as [(α-D-glucopyranosyl-(1 →6)-α-D-glucopyranosyl-(1 →6)- α -D-glucopyranosyl-(1→5)-D-fructopyranose] and [α-D-glucopyranosyl-(1 →6)-α-D-glucopyranosyl-(1→5)-D- fructopyranose], respectively. These are considered to be novel oligosaccharides     

Structure of Oligosaccharides Obtained by Controlled Degradation of Mung Bean Xyloglucan with Acid and Aspergillus oryzae Enzyme Preparation

A xyloglucan (MBXG) from the cell walls of etiolated mung bean hypocotyls was characterized by analyzing the fragment oligosaccharides from controlled degradation products of the polymer with acid and enzyme.

Cellobiose, cellotriose and cellotetraose were isolated from the partial acid hydrolyzate of MBXG. Isoprimeverose (6-O-α-d-xylopyranosyl-d-glucopyranose) and a pentasaccharide, α-l-fucosyl-(1 → 2)-β-d-galactosyl-(1 → 2)-α-d-xylosyl-(1 → 6)-β-d-glucosyl-(1 → 4)-d-glucose, were isolated from the hydrolyzate of MBXG with an Asp. oryzae enzyme preparation.     

Functional Characterization of D-Galacturonic Acid Reductase,a Key Enzyme of the Ascorbate Biosynthesis Pathway,from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38–39 kDa, as judged by SDS–PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5±4.5 μM and uronic acids, such as D-galacturonic acid (Km=3.79±0.5 mM) and D-glucuronic acid (Km=4.67±0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP⁺. The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H₂O₂, suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

A Novel Glucanotransferase from a Bacillus circulans Strain That Produces a Cyclomaltopentaose Cyclized by an α-1,6-Linkage

A novel glucanotransferase, involved in the synthesis of a cyclomaltopentaose cyclized by an α-1,6-linkage [ICG5; cyclo-{→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→}], from starch, was purified to homogeneity from the culture supernatant of Bacillus circulans AM7. The pI was estimated to be 7.5. The molecular mass of the enzyme was estimated to be 184 kDa by gel filtration and 106 kDa by SDS–PAGE. These results suggest that the enzyme forms a dimer structure. It was most active at pH 4.5 to 8.0 at 50 °C, and stable from pH 4.5 to 9.0 at up to 35 °C. The addition of 1 mM Ca²⁺ enhanced the thermal stability of the enzyme up to 40 °C. It acted on maltooligosaccharides that have degrees of polymerization of 3 or more, amylose, and soluble starch, to produce ICG5 by an intramolecular α-1,6-glycosyl transfer reaction. It also catalyzed the transfer of part of a linear oligosaccharide to another oligosaccharide by an intermolecular α-1,4-glycosyl transfer reaction. Thus the ICG5-forming enzyme was found to be a novel glucanotransferase. We propose isocyclomaltooligosaccharide glucanotransferase (IGTase) as the trivial name of this enzyme.     

Metabolism of Aromatic Amino Acid in Microorganisms

Phenylalanine ammonia-lyase, which catalyzes the conversion of l-phenylalanine to trans-cinnamic acid and ammonia, has been partially purified from the cells of Rhodotorula. Some of the properties of this phenylalanine ammoyia-lyase were investigated. The enzyme was stable in phosphate buffer of pH over the range of 6.0 to 7.0 On heating, the enzyme was stable up to 50°C, but above 60°C, it was destroyed. The enzyme activity was strongly inhibited by p-chloromercuribenzoate at 10^?5 m and almost recovered by the addition of glutathione or mercaptoethanol at 10^?3 m. The present enzyme preparation of Rhodotorula also catalyzed the deamination of l-tyrosine to trans-p-coumaric acid. trans-p-Coumaric acid was isolated from the reaction mixture and identified by its absorption spectra. The rates of deamination showed optima at pH 9.0 and 9.5 for l-phenylalanine and l-tyrosine, respectively.     

Inhibition of l-Alanine Adding Enzyme by Glycine

l-Alanine adding enzymes from Bacillus subtilis and Bacillus cereus which catalyzed l-alanine incorporation into UDPMurNAc were partially purified and the properties of the enzymes were examined. The enzyme from B. subtilis was markedly stimulated by reducing agents including 2-mercaptoethanol, dithiothreitol, glutathione and cysteine. Mn²⁺ and Mg²⁺ activated l-alanine adding activity and their optimal concentrations were 2 to 5 mm and 10 mm, respectively. The optimum pH was 9.5 and the Km for l-alanine was 1.8×10^?4m. l-Alanine adding reaction was strongly inhibited by p-chloromercuribenzoate and N-ethyl-maleimide. Among glycine, l- and d-amino acids and glycine derivatives, glycine was the most effective inhibitor of the l-alanine adding reaction. The enzyme from B. cereus was more resistant to glycine than that from B. subtilis. Glycine was incorporated into UDPMurNAc in place of l-alanine, and the Ki for glycine was 4.2×l0^?3m with the enzyme from B. subtilis. From these data, the growth inhibition of bacteria by glycine is discussed.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

l-Glutamic acid was formed from d-, l-, and dl-PCA with cell-free extract of Pseudomonas alcaligenes ATCC-12815 grown in the medium containing dl-PCA as a sole source of carbon and nitrogen. The enzyme(s) involved in this conversion reaction was distributed in the soluble fraction within the cell and in 0.5 saturated fraction at the fractionation procedure with the saturation of ammonium sulfate. Optimum pH of this enzyme(s) lied at pH 8.5 and optimum temperature was 30°C. Cu (5 × 10^?3 m) inhibited the reaction considerably while Ca or Fe accelerated it. PALP (1×10^?3 m) also gave an enhanced activity to some extent. The enzyme preparation converted dextro-rotatory enan-thiomorph of PCA to its laevo-rotatory one which in turn was not converted to the opposite rotation direction by this enzyme. Furthermore, the preparation did not, if any, show d-glutamic acid racemase activity. Isotopic experiments with using dl-PCA-1-¹⁴C revealed that l-glutamic acid-1-¹⁴C was formed by the cleavage of –CO–NH– bond of pyrrolidone ring of PCA. It was concluded that dl-PCA when assimilated by the present bacterium is at first transformed to l-PCA by the optically isomerizing enzyme and subsequently is cleaved to l-glutamic acid probably by the PCA hydrolysing enzyme.     

Metabolism of l-Theanine,d-Theanine and the Related Compounds in Bacteria

Some strains of Pseudomonas was found capable of utilizing l-theanine or d-theanine as a sole nitrogen and carbon source. The cell-free extract catalyzes the hydrolysis of the amide group of the compounds and the hydrolase activity was influenced remarkably by the nitrogen source in the medium. l-Theanine and d-theanine were hydrolyzed to yield stoichiometrically l-glutamic acid and d-glutamic acid, respectively, and ethylamine, which were isolated from the reaction mixture and identified.

The theanine hydrolase of Pseudomonas aeruginosa was purified approximately 200-fold. It was shown that the activities of l-theanine hydrolase, d-theanine hydrolase and the heat-stable l-glutamine hydrolase and d-glutamine hydrolase are ascribed to a single enzyme, which may be regarded as a γ-glutamyltransferase from the point of view of the substrate specificity and the properties. This theanine hydrolase catalyzed the transfer of γ-glutamyl moiety of the substrates and glutathione to hydroxylamine. l-Glutamine and d-glutamine were hydrolyzed by the theanine hydrolase and also by the heat-labile enzyme of the same strain of Pseudomonas aeruginosa, whose properties resembled the common glutaminase.     

Efficient Production and Purification of Extracellular 1,2-α-L-Fucosidase of Bacillus sp. K40T

When Bacillus sp. K40T was cultured in the presence of L-fucose, 1,2-α-L-fucosidase was found to be produced specifically in the culture fluid. The enzyme was purified to homogeneity from a culture containing only L-fucose by chromatography on hydroxylapatite and chromatofocusing. The molecular weight of the enzyme was estimated to be 200,000 by gel filtration on Sephadex G-200. The enzyme was optimal at pH 5.5–7.0 and was stable at pH 6.0–9.0. The enzyme hydrolyzed the α(1 → 2)-L-fucosidic linkages in various oligosaccharides and glycoproteins such as lacto-N-fucopentaose (LNF)-I 〈O-α-L-fucose-(1 → 2)-O-β-D-galactose-(1 → 3)-N-acetyl-O-β-D-glucosamine-(1 → 3)-O-β-D-galactose-(1 → 4)-D-glucose〉, porcine gastric mucin, and porcine submaxillary mucin. The enzyme also acted on human erythrocytes, which was confirmed by the hemagglutination test using Ulex anti-H lectin. The enzyme did not hydrolyze α(1 → 3)-, α-(1 → 4)- and α-(1 → 6)-L-fucosidic linkages in LNF-III 〈O-β-D-galactose-(1 → 4)[O-α-L-fucose-(1 → 3)-]-N-acetyl-O-β-D-glucosamine-(1 → 3)-O-β-D-galactose-(1 → 4)-D-glucose〉, LNF-II 〈O-β-D-galactose-(1 → 3)[O-α-L-fucose-(1 → 4)-]-N-acetyl-O-β-D-galactose-(1 → 3)-O-β-D-galactose-(1 → 4)-D-glucose〉 or 6-O-α-L-fucopyranosyl-N-acetylglucosamine.     

Antitumor Activities and Immunochemical Properties of the Cell-wall Polysaccharides from Aureobasidium pullulans

Delipidated cell walls from Aureobasidium pullulans were fractionated systematically.

The cell surface heteropolysaccharide contains D-mannose, D-galactose, D-glucose, and D-glucuronic acid (ratio, 8.5:3.9:1.0:1.0). It consists of a backbone of (1→6)-α-linked D-mannose residues, some of which are substituted at O-3 with single or β-(1→6)-linked D-galactofuranosyl side chains, some terminated with a D-glucuronic acid residue, and also with single residues of D-glucopyranose, D-galactopyranose, and D-mannopyranose.

This glucurono-gluco-galactomannan interacted with antiserum against Elsinoe leucospila, which also reacted with its galactomannan, indicating that both polysaccharides contain a common epitope, i.e., at least terminal β-galactofuranosyl groups and also possibly internal β-(1→6)-linked galactofuranose residues.

It was further separated by DEAE-Sephacel column chromatography to gluco-galactomannan and glucurono-gluco-galactomannan.

The alkali-extracted β-D-glucan was purified by DEAE-cellulose chromatography to afford two antitumor-active (1→3)-β-D-glucans. One of the glucans (M_r, 1–2 × 10⁵) was a O-6-branched (1→3)-β-D-glucan with a single β-D-glucosyl residue, d.b., 1/7, and the other (M_r, 3.5–4.5 × 10⁵) had similar branched structure, but having d.b., 1/5. Side chains of both glucans contain small proportions of β-(1→6)-and β-(1→4)-D-glucosidic linkages.