Starch-hydrolyzing Enzymes in Germinating Kidney Bean

Enzymatic production of D-Glu was investigated by the succesive reactions of a glutamate racemase (EC 5.1.1.3) and a glutamate decarboxylase (EC 4.1.1.15) on L-Glu.Lactobacillus brevis ATCC8287 was chosen as a source of glutamate racemase. This strain produced a glutamate decarboxylase simultaneously. The glutamate racemase activity in the cell free extracts was 0.035 units/mg protein. The enzyme kept its activity even at 500 Mm of L-Glu (74g/liter). The optimum pHs of the racemase and the decarboxylase were at around 8.5 and below 4.0, respectively. Both enzymes had no activity at the optimum pH for the other enzyme. L-Glu was racemized first by the glutamate racemase at pH 8.5, then the pH was shifted to 4.0 at which L-Glu was decarboxylated by the glutamate decarboxylase. Starting from 100 g/liter of L-Glu, 50 g/liter of D-Glu was produced and no L-Glu remained in the reaction mixture.     

Mutant of Escherichia coli Tolerant to the Lysis Induced by Cephalexin

2,3-Diaminopropionate ammonia-lyase (DAPAL), which catalyzes α,β-elimination of 2,3-diaminopropionate regardless of its stereochemistry, was purified from Salmonella typhimurium. We cloned the Escherichia coli ygeX gene encoding a putative DAPAL and purified the gene product to homogeneity. The protein obtained contained pyridoxal 5′-phosphate and was composed of two identical subunits with a calculated molecular weight of 43,327. It catalyzed the α,β-elimination of both D- and L-2,3-diaminopropionate. The results confirmed that ygeX encoded DAPAL. The enzyme acted on D-serine, but its catalytic efficiency was only 0.5% that with D-2,3-diaminopropionate. The enzymologic properties of E. coli DAPAL resembled those of Salmonella DAPAL, except that L-serine, D- and L-β-Cl-alanine were inert as substrates of the enzyme from E. coli. DAPAL had significant sequence similarity with the catalytic domain of L-threonine dehydratase, which is a member of the fold-type II group of pyridoxal phosphate enzymes, together with D-serine dehydratase and mammalian serine racemase.     

Purification and Characterization of γ-Glutamylmethylamide Synthetase from Methylophaga sp. AA-30

γ-Glutamylmethylamide synthetase [L-glutamate: methylamine ligase (ADP-forming), EC 6.3.4.12] was purified about 70-fold from a cell-free extract of Methylophaga sp. AA-30 by ammonium sulfate fractionation, Octyl-Sepharose column chromatography, and Sephacryl S-300 gel filtration. Only a single protein band was detected after SDS-polyacrylamide gel electrophoresis of the purified preparation; the band was at a position corresponding to a molecular weight of 56,000. The molecular weight of the enzyme was calculated to be 440,000 by Superose 6HR gel filtration, so we suggest that the enzyme is an octomer of identical subunits. The enzyme had maximum activity at pH 7.5 and 40°C. It could use ethylamine and propylamine instead of methylamine as the substrate, but it could not use D-glutamate or L-glutamine instead of L-glutamate.     

NADP-Specific Glutamate Dehydrogenase from Alkaliphilic Bacillus sp. KSM-635: Purification and Enzymatic Properties

An NADP-specific glutamate dehydrogenase [L-glutamate: NADP⁺ oxidoreductase (deaminating), EC 1.4.1.4] from alkaliphilic Bacillus sp. KSM-635 was purified 5840-fold to homogeneity by a several-step procedure involving Red-Toyopearl affinity chromatography. The native protein, with an isoelectric point of pH 4.87, had a molecular mass of approximately 315 kDa consisting of six identical summits each with a molecular mass of 52 kDa. The pH optima for the aminating and deaminating reactions were 7.5 and 8.5, respectively. The optimum temperature was around 60°C for both. The purified enzyme had a specific activity of 416units/mg protein for the aminating reaction, being over 20-fold greater than that for deaminating reaction, at the respective pH optima and at 30°C. The enzyme was specific for NADPH (K_m 44 μM), 2-oxoglutarate (K_m 3.13 mM), NADP⁺ (K_m 29 μM), and L-glutamate (K_m 6.06 mM). The K_m for NH₄Cl was 5.96 mM. The enzyme could be stored without appreciable loss of enzyme activity at 5°C for half a year in phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol, although the enzyme activity was abolished within 20 h by freezing at ?20°C.     

Branched Chain Amino Acid Aminotransferase of Pseudomonas sp

Branched chain amino acid aminotransferase was partially purified from Pseudomonas sp. by ammonium sulfate fractionation, aminohexyl-agarose and Bio-Gel A-0.5 m column chromatography.

This enzyme showed different substrate specificity from those of other origins, namely lower reactivity for l-isoleucine and higher reactivity for l-methionine.

Km values at pH 8.0 were calculated to be 0.3 mm for l-leucine, 0.3 mm for α-ketoglutarate, 1.1 mm for α-ketoisocaproate and 3.2 mm for l-glutamate.

This enzyme was activated with β-mercaptoethanol, and this activated enzyme had different kinetic properties from unactivated enzyme, namely, Km values at pH 8.0 were calculated to be 1.2 mm for l-leucine, 0.3 mm for α-ketoglutarate.

Isocaproic acid which is the substrate analog of l-leucine was competitive inhibitor for pyridoxal form of unactivated and activated enzymes, and inhibitor constants were estimated to be 6 mm and 14 mm, respectively.     

Inactivation of Glutamate Racemase of Pediococcus pentosaceus with L-Serine O-Sulfate

Glutamate racemase of Pediococcus pentosaceus catalyzes the α,β-elimination of L-serine O-sulfate to produce a pyruvate concomitantly with an irreversible inactivation of the enzyme. α,β-Elimination and inactivation reactions proceed through a common intermediate. L-Serine O-sulfate serves as a suicide substrate of the enzyme.     

Cloning and Expression of the Pseudomonas Gene for Utilization of d-Leucine in Escherichia coli

Two genes of Pseudomonas putida (IFO 12996) which code for enzymes participating in amino acid metabolism, were cloned in Escherichia coli C600 using pBR322 as a vector. pST7549 is a 7.9 kb hybrid plasmid DNA which is composed of four SalI fragments (0.3, 1.4, 1.9 and 4.3 kb), and codes for β-isopropylmalate dehydrogenase (EC 1.1.1.85) in l-leucine biosynthesis. The enzyme activity in the crude extract from E. coli C600 bearing pST7549 was 80 ~ 90% lower than that of E. coli K12 or P. putida. When the foreign SalI fragments derived from P. putida were subcloned, a 1.9 kb SalI fragment was found to encode β-isopropylmalate dehydrogenase and it did not contain the promoter of P. putida DNA. Plasmid pST6961 has a 1.8 kb insert derived from the P. putida DNA in the SalI site of pBR322. E. coli cells carrying this recombinant plasmid show no leucine racemase activity and no d-leucine transaminase activity, but five-times higher d-leucine oxidation activity than the host strain, E. coli. Enzymological studies have suggested that plasmid pST6961 codes for d-amino acid dehydrogenase, a key enzyme in d-amino acid metabolism.     

Properties and Continuous Use of Microbial Fumarase Entrapped in Fibres

A new flavoprotein enzyme, l-glutamate oxidase, was purified to homogeneity from an aqueous extract of a wheat bran culture of Streptomyces sp. X-l 19–6. It showed absorption maxima at 273, 385 and 465 nm and a shoulder around 490 nm, and contained 2 mol of FAD per mol of enzyme. The enzyme had a molecular weight of approximately 140,000 and consisted of three sizes of subunits with molecular weights of 44,000, 16,000 and 9,000. Balance studies showed that 1 mol of l-glutamate was converted to 1 mol of α-ketoglutarate, ammonia and hydrogen peroxide with the consumption of 1 mol of oxygen. In addition to l-glutamate, l-aspartate was oxidized by the enzyme but only to an extent of 0.6% at pH 7.4; the Michaelis constants were as follows: 0.21 mM for l-glutamate and 29 mM for l-aspartate. The isoelectric point was pH 6.2, and the enzyme activity was optimal between pH 7.0 and 8.0. When the enzyme was heated at pH 5.5 for 15 min, the remaining activity was 100% of the original activity level at 65°C, 87% at 75°C and 47% at 85°C.     

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.     

Glutamate Metabolism in a Glutamate-producing Bacterium,Brevibacterium flavum

Brevibacterium flavum No. 2247 was found to grow with l-glutamate as the sole carbon and nitrogen source on an agar-plate medium when high concentrations of l-glutamate, FeSO₄ and biotin were added to the medium. It grew on l-glutamate in liquid medium only when yeast extract or high concentrations of FeSO₄ and glucose or organic acids of the tricarboxylic acid cycle were added to the medium. The growth on l-glutamate in liquid medium was also stimulated by high concentrations of l-glutamate, biotin and MgSO₄, and inhibited by a high concentration of (NH₄)₂SO₄.

Aspartate aminotransferase (TA)- and α-ketoglutarate dehydrogenase (KD)-defective mutants did not grow on l-glutamate, and glutamate-utilizing revertants derived from these mutants recovered TA and KD activity, respectively, whereas glutamate dehydrogenase (GD)-defective mutants grew on l-glutamate. Washed cells of strain No. 2247 grown on glutamate decomposed the amino acid, whereas those grown on glucose did not. The degradation was observed only under aerobic conditions. The former cells showed higher KD, succinate dehydrogenase and fumarase activities than the latter cells. Of 75 mutants which did not grow on glutamate but grew on succinate, three strains lacked KD but showed the same glutamate productivity as the parent strain. Four other strains with normal KD levels showed higher glutamate productivity than the parent.     

Automated Measurement of L-Glutamate in Soy Sauce Using L-Glutamate Dehydrogenase

An automated method for rapid and convenient measurement of L-glutamate has been developed by using a discrete analyzer, EEL Auto Chemist. It is based on the colorimetric measurement of NADH produced on a mole-mole basis by enzymatic dehydrogenation of L-glutamate using L-glutamate dehydrogenase from bovine liver. The values of L-glutamate obtained by this method were well agreed with those obtained by the routine Waruburg mano-metric method using L-glutamate decarboxylase from Escherichia coli.     

γ-Glutamylcysteine Synthetase from Proteus mirabilis

The distribution of γ-glutamylcysteine synthetase (l-glutamate: L-cysteine γ-ligase, EC 6.3.2.2) was investigated in bacteria, and the enzyme was purified from Proteus mirabilis approximately 9,000-fold with an over-all yield of 10%. The purification procedure included ammonium sulfate fractionation, protamine treatment, DEAE-cellulose and hydroxylapatite column chromatographies and Sephadex gel filtrations. The purified enzyme was homogeneous by the criteria of ultracentrifugation. It showed multiple bands on disc-polyacrylamide gel electrophoresis and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band with a molecular weight of 62,000 was obtained on SDS-polyacrylamide gel electrophoresis after cross-linking of the enzyme with dimethylsuberimidate. The molecular weight was determined from the sedimentation and diffusion coefficients to be 64,000 and by Sephadex G-150 gel filtration to be 62,000. The purified enzyme catalyzed the stoichiometric formation of γ-glutamylcysteine and the reaction showed a sigmoidal dependence upon l-cysteine concentration. The enzyme also catalyzed γ-glutamyl amino acid formation from l-α-aminobutyrate, l-homoserine, glycine, l-serine, dl-norvaline or dl-homocysteine, but at lower rates than from l-cysteine. The γ-glutamyl-α-aminobutyrate formation by the enzyme did not show a sigmoidal but a hyperbolic dependence upon l-α-aminobutyrate concentration.     

Production and Purification of Alkaline Protease Inhibitors of Streptomyces sp.

N-Acetyl-d-glutamate deacetylase and N-acetyl-d-aspartate deacetylase were found in cell extracts from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. N-Acetyl-d-glutamate deacetylase was produced inducibly by N-acetyl-d-glutamate and was highly specific to N-acetyl-d-glutamate. N-Acetyl-d-aspartate deacetylase was produced inducibly by N-acetyl-d-aspartate and was highly specific to N-acetyl-d-aspartate.     

Insect Ecdysis Inhibitors from the East African Medicinal Plant Ajuga remota (Labiatae)

γ-Glutamy Icy steine synthetase was purified from E. coli B. The enzyme had a molecular weight of 5.5 × 10⁴ and required only magnesium ion for activity. The optimal pH and temperature for reaction were 8.5 and 45°C, respectively. The Km values for l-glutamate, l-cysteine, and ATP were 0.50, 0.09, and 0.01 mm, respectively. GTP and UTP were also used as energy sources. The enzyme activity was inhibited by phosphate anions and by various sulfhydryl reagents. Unlike the enzyme from mammalian tissues, the E. coli B enzyme was not inhibited by α-alkyl analogues of methionine. The enzyme was feedback inhibited by reduced glutathione, although oxidized glutathione had no inhibitory effect.     

Purification and Characterization of Glucokinase in Escherichia coli B

Glucokinase was purified from Escherichia coli B cells dosed with a hybrid plasmid carrying the gene for glucokinase. The enzyme was purified about 170-fold and was homogeneous on polyacrylamide gel electrophoresis. The enzyme was 49,000 in molecular weight and consisted of two subunits having a molecular weight of 24,500. The glucokinase catalyzed phosphorylation of D-glucose, D-mannose, D-glucosamine, and 2-deoxy-D-glucose, consuming ATP as a phosphoryl donor. Besides ATP, other nucleoside triphosphates such as ITP, GTP and UTP were also utilized as phosphoryl donors. The enzyme required free sulfhydryl groups and Mg²⁺ for activity. Other properties of the glucokinase were characterized and compared with those of glucokinases from various sources.     

Cloning of the d-Xylose Uptake Gene Linked to the xyl A Gene in Escherichia coli

An Escherichia coli mutant (MX-5) deficient in d-xylose utilization was isolated. The d-xylose uptake and d-xylose isomerase activities of the mutant were much lower than those of the parental strain (C600). The genes responsible for the d-xylose uptake by E. coli were cloned onto vector plasmid pBR322, and the resultant hybrid plasmid was designated as pXP5. Hybrid plasmid pXP5 improved the growth rate of the mutant (MX-5) on d-xylose, and also both the d-xylose uptake and d-xylose isomerase activities of the mutant were recovered when pXP5 was introduced into the mutant cells. Based on these results, it was suggested that one (xyl T) of the d-xylose transport genes could be closely linked to the d-xylose isomerase gene (xylA) known to be present at 80 min on E. coli chromosomal DNA.     

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.     

Properties of α-Amino-ε-caprolactam Racemase from Achromobacter obae

α-Amino-ε-caprolactam racemase, which occurs in the cytoplasmic fraction of Achromobacter obae, has been purified to homogeneity. It has a monomeric structure with a molecular weight of approximately 50,000. The absorption spectrum of the enzyme exhibits maxima at 280 and 412 nm at pH 7.3, and is independent of pH from 6.0 to 8.0. One mole of pyridoxal 5′-phosphate is bound per mol of the enzyme. Incubation of the enzyme with hydroxylamine resulted in the formation of the apoenzyme. d- and l-α-Amino-ε-caprolactams are the only substrates. The maximum activity is found at pH 8.8 for both the isomers. Michaelis constants are as follows: 8 mm for d-α-amino-ε-caprolactam, 6mm for l-α-amino-ε-caprolactam and 2.1 × 10^?7 m for pyridoxal 5′-phosphate. The enzyme is inhibited significantly by CuSO₄, HgCl₂, thiol reagents such as N-ethylmaleimide and p-chloromercuribenzoate, and carbonyl reagents (e.g., phenylhydrazine and hydroxylamine). α-Amino-ε-caprolactam racemase catalyzes the α-proton exchange of the substrate with deuteron during racemization in deuterium oxide.     

Inhibitors of alanine racemase enzyme: a review

Alanine racemase is a fold type III PLP-dependent amino acid racemase enzyme catalysing the conversion of l-alanine to d-alanine utilised by bacterial cell wall for peptidoglycan synthesis. As there are no known homologs in humans, it is considered as an excellent antibacterial drug target. The standard inhibitors of this enzyme include O-carbamyl-d-serine, d-cycloserine, chlorovinyl glycine, alafosfalin, etc. d-Cycloserine is indicated for pulmonary and extra pulmonary tuberculosis but therapeutic use of drug is limited due to its severe toxic effects. Toxic effects due to off-target affinities of cycloserine and other substrate analogs have prompted new research efforts to identify alanine racemase inhibitors that are not substrate analogs. In this review, an updated status of known inhibitors of alanine racemase enzyme has been provided which will serve as a rich source of structural information and will be helpful in generating selective and potent inhibitor of alanine racemase.     

Preparation of optically active 4-chlorophenylalanine from its racemate by deracemization technique using transformant Escherichia coli cells

We have developed a method for the preparation of l-4-chlorophenylalanine from its racemate with Escherichia coli cells expressing a single foreign gene. l-4-Chlorophenylalanine was obtained in a high optical yield by the inversion of configuration of its d-form via the tandem reactions catalyzed by d-amino acid dehydrogenase (DadA) and branched-chain amino acid aminotransferase (BCAAT). While we constructed a plasmid for BCAAT utilizing the gene from Sinorhizobium meliloti ATCC 51124, the first enzyme DadA was the dadA-gene product from E. coli host cell itself, which was activated by the addition of l-alanine in the growth medium.