Studies of human kidney gamma-glutamyl transpeptidase. Purification and structural, kinetic and immunological properties.

gamma-Glutamyl transpeptidase, present in various mammalian tissues, transfers the gamma-glutamyl moiety of glutathione to a variety of acceptor amino acids and peptides. This enzyme has been purified from human kidney cortex about 740-fold to a specific activity of 200 units/mg of protein. The purification steps involved incubation of the homogenate at 37 degrees followed by centrifugation and extraction of the sediment with 0.1 M Tris-HCl buffer, pH 8.0, containing 1% sodium deoxycholate; batchwise absorption on DEAE-cellulose; DEAE-cellulose (DE52) column chromatography; Sephadex G-200 gel filtration; and affinity chromatography using concanavalin A insolubilized on beaded Agarose. Detergents were used throughout the purification of the enzyme. The purified enzyme separated into three protein bands, all of which had enzyme activity, on polyacrylamide disc electrophoresis in the presence of Triton X-100. The enzyme has an apparent molecular weight of about 90,000 as shown by Sephadex G-200 gel filtration, and appears to be a tetramer with subunits of molecular weights of about 21,000. The Km for gamma-glutamyl transpeptidase using the artificial substrate, gamma-glutamyl-p-nitroanilide, with glycylglycine as the acceptor amino acid was found to be about 0.8 mM. The optimum pH for the enzyme activity is 8.2 and the isoelectric point is 4.5. Both GSH and GSSG competitively inhibited the activity of gamma-glutamyl transpeptidase when gamma-glutamyl-p-nitroanilide was used as the substrate. Treatment of the purified enzyme with papain has no effect on the enzyme activity or mobility on polyacrylamide disc electrophoresis. The purified gamma-glutamyl transpeptidase had no phosphate-independent glutaminase activity. The ratio of gamma-glutamyl transpeptidase to phosphate-independent glutaminase changed significantly through the initial steps of gamma-glutamyl transpeptidase purification. These studies indicate that the transpeptidase and phosphate-independent glutaminase activities are not exhibited by the same protein in human kidney.     

Identity of maleate-stimulated glutaminase with gamma-glutamyl transpeptidase in rat kidney.

Gamma-Glutamyl transpeptidase was purified from rat kidney by a procedure involving Lubrol extraction, acetone precipitation, ammonium sulfate fractionation, treatment with bromelain, and column chromatography on DEAE-cellulose and Sephadex G-100. The final preparation (enzyme III), which exhibits a specific activity about 8-fold higher than that of the purified rat kidney transpeptidase previously obtained in this laboratory (enzyme I), was apparently homogeneous on polyacrylamide gel electrophoresis. Enzyme III is a glycoprotein containing 10% hexose, 7% aminohexose, and 1.5% sialic acid; a tentative molecular weight value of about 70,000 was obtained by gel filtration. Enzyme III has a much lower molecular weight and a different amino acid and carbohydrate content than the less active rat kidney transpeptidase preparation previously obtained, but obtained, but the catalytic properties of these preparations are virtually identical. It is suggested that bromelain treatment may liberate the transpeptidase from a brush border complex that contains other proteins. An improved method is described for the isolation of the higher molecular weight form of the enzyme (enzyme I) in which affinity chromatography on concanavalin A-Sephrose is employed. The purified transpeptidase (enzyme III) is similar to the phosphate-independent maleate-stimulated glutaminase preparation obtained from rat kidney by Katunuma and colleagues with respect to amino acid and carbohydrate content, apparent molecular weight, and relative transpeptidase and maleate-stimulated "glutaminase" activities. Both of these enzyme preparations are much more active in transpeptidation reactions with glutathione and related gamma-glutamyl compounds than with glutamine. In the absence of maleate, the enzyme catalyzes the utilization of glutamine (by conversion to gamma-glutamylglutamine, glutamate, and ammonia) at about 2% of the rate observed for catalysis of transpeptidation between glutathione and glycylglycine; the utilization of glutamine occurs about 8 times more rapidly in the presence of 0.1 M maleate. The transpeptidation and maleate-stimulated glutaminase reactions catalyzed by both enzyme preprations are inhibited by 5 mM L-serine in the presence of 5 mM sodium borate. Studies on gamma-glutamyl transpeptidase and maleate-stimulated glutaminase in the kidneys of fetal rats, newborn rats, and rats after weaning showed parallel development of these activities. The evidence reported here and earlier work in this laboratory strongly support the conclusion that maleate-stimulated glutaminase activity is a catalytic function of gamma-glutamyl transpeptidase. The studies on the ontogeny of gamma-glutamyl transpeptidase and other data are considered in relation to the proposal that this enzyme is involved in amino acid and peptide transport. Its possible role in renal formation of ammonia is also discussed.     

Renal glutamine utilization: glycylglycine stimulation of gamma-glutamyltransferase

Glycylglycine stimulation of renal glutamine utilization was studied on the homogenate, subcellular and purified enzyme level. The results clearly establish the existence of two glutamine utilizing pathways, the mitochondrial dependent L-glutamine amidohydrolase (PDG) and a second, extramitochondrial pathway. In contrast to the mitochondrial pathway which produces stoichiometric amounts of ammonia and glutamate, this second pathway hydrolyzes glutamine to produce ammonia and transfers the gamma-glutamyl moiety, producing gamma-glutamyl peptides. In the crude systems, containing cyclotransferase, the gamma-glutamyl moiety appears mainly as 5-oxoproline; however, in the enzyme preparation, purified 112-fold, gamma-glutamyl peptides (transpeptidation) and a small amount of glutamate (hydrolysis) appear. D-Glutamine was also hydrolyzed, in contrast to the stereospecific PDG, but at less than one-half the rate of the L-isomer. The molecular weight of this extramitochondrial D- and L-glutamine utilizing enzyme was estimated by gel filtration on a Sephadex G-200 column and found to be approximately 70 000. Based on product formation, molecular weight estimation and copurification with the activity responsible for p-nitroanilide release from gamma-glutamyl-p-nitroanilide, we conclude that this reaction is catalyzed by gamma-glutamyltranspeptidase. Glycylglycine stimulated this enzyme to produce more ammonia while decreasing the appearance of glutamate; in contrast, the mitochondrial glutaminase was unaffected by glycylglycine. This extramitochondrial glutamine utilizing pathway can make a significant contribution to in vivo renal ammoniagenesis.     

An Enzyme Hydrolyzing l-Theanine in Tea Leaves

Theanine hydrolase activity in tea leaves was assayed by measuring enzymatically released ethylamine from l-theanine. The o-phthalaldehyde derivative of ethylamine was measured by reverse phase HPLC recorded with a spectrofluorometric detector.

Theanine hydrolase activity was purified about 4.6-fold by DEAE-cellulose column chromatography. Although this active fraction also had glutaminase activity, the yield of the glutaminase activity was about 50% of that of theanine hydrolytic activity. The theanine hydrolytic activity was inhibited by acidic amino acid and l-alanine, and stimulated by l-malic acid. The purified enzyme solution hydrolyzed not only theanine but also γ-glutamylmethylamide, γ-glutamyl-n-propylamide, γ-glutamyl-n-butylamide, γ-glutamyl-iso-butylamide, and γ-glutamyl-n-amylamide, which were synthesized from l-pyroglutamic acid and corresponding alkylamines. However, N-methylpropionamide and N-ethylpropionamide were not hydrolyzed. The theanine hydrolase activity and glutaminase in tea leaves showed the same pH optimum (8.5).

The activity of theanine hydrolase in tea leaves increased during the first lOhr after plucking but then decreased gradually, while that of glutaminase decreased constantly and was almost lost     

Phosphate-independent glutaminase from rat kidney. Partial purification and identity with gamma-glutamyltranspeptidase.

Phosphate-independent glutaminase can be quantitatively solubilized from a microsomal preparation of rat kidney by treatment with papain. Subsequent gel filtration and chromatography on quaternary aminoethyl (QAE)-Sephadex and hydroxylapatite yield a 200-fold purified preparation of this glutaminase. The purified enzyme also hydrolyzes gamma-glutamylhydroxamate and exhibits substrate inhibition at high concentrations of either glutamine or gamma-glutamyhydroxamate, which is partially relieved by increasing concentrations of maleate. Rat kidney phosphate-independent glutaminase reaction is catalyzed by the same enzyme which catalyzes the gamma-glutamyltranspeptidase reaction. The ratio of glutaminase to transpeptidase activities remained constant throughout a 200-fold purification of this enzyme. The observation that the phosphate0independent glutaminase and gamma-glutamyltranspeptidase activities exhibit coincident mobilities during electrophoresis, both before and after extensive treatment with neuraminidase, strongly suggests that both reactions are catalyzed by the same enzyme. This conclusion is strengthened by the observation that maleate and various amino acids have reciprocal effects on the two activities. Maleate increases glutaminase activity and blocks transpeptidation, whereas amino acids activate the transpeptidase but inhibit glutaminase activity. In contrast, the addition of both maleate and alanine resulted in a strong inhibition of both activities. Both activities exhibit a similar distribution in the various regions of the kidney. Recovery of maximal activities in the outer stripe region of the medulla is consistent with previous quantitative microanalysis which indicated that this glutaminase activity is localized primarily in the proximal straight tubule cells. The glutaminase and transpeptidase activities have different pH optima. Examination of the product specificity suggests that decreasing pH also promotes glutaminase activity and that below pH 6.0, this enzyme functions strictly as a glutaminase. Because of the localization of this activity on the brush border membrane, these resuts are consistent with the possibility that the physiological conditions induced by metabolic acidosis could convert this enzyme from a broad specificity transpeptidase to a glutaminase. Therefore, this enzyme could contribute to the increased renal synthesis of ammonia from glutamine which is observed during metabolic acidosis.     

The intramitochondrial location of the glutaminase isoenzymes of pig kidney

1. The glutaminase activity of pig kidney is located almost entirely in the cortex. 2. Pig renal cortex contains two glutaminases, one phosphate-dependent and one phosphate-independent. Both isoenzymes are localized exclusively in the mitochondria. 3. After sonication of the mitochondria, the phosphate-dependent isoenzyme is entirely soluble, whereas approximately half the phosphate-independent isoenzyme is associated with the membranes. 4. In intact mitochondria, the activities of both isoenzymes respond to changes in the pH of the intramitochondrial compartment. 5. It is concluded that both glutaminase isoenzymes are situated in the intramitochondrial compartment, and that the phosphate-independent glutaminase may be bound to the inside of the inner mitochondrial membrane.     

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.     

Human kidney gamma-glutamyl transpeptidase. Catalytic properties, subunit structure, and localization of the gamma-glutamyl binding site on the light subunit.

Human kidney gamma-glutamyl transpeptidase has been purified by a procedure involving Lubrol extraction, acetone precipitation, treatment with bromelain, and column chromatography on DEAE-cellulose and Sephadex G-150. The final preparation is a glycoprotein (molecular weight of approximately 84,000) composed of two nonidentical glycopeptides (molecular weights of 62,000 and 22,000). The isozymic forms, separable by isoelectric focusing, have different contents of sialic acid. The utilization of L-glutamine (which is both a gamma-glutamyl donor and acceptor) is stimulated about 3-fold by maleate in contrast to 10-fold stimulation of glutamine utilization by the rat kidney enzyme. The gamma-glutamyl analogs, 6-diazo-5-oxo-L-norleucine (DON) and L-azaserine inactivate the human kidney enzyme with respect to its transpeptidase and hydrolase activities. Inactivation is prevented by gamma-glutamyl substrates (but not by acceptor substrates) and is accelerated by maleate. [14C]DON reacts covalently and stoichiometrically at the gamma-glutamyl site, which was localized to the light subunit of the enzyme. The light subunit of human transpeptidase closely resembles that of rat kidney enzyme in having the gamma-glutamyl binding site, and similar molecular weight and amino acid composition. The heavy subunits of the two enzymes are markedly different in both molecular weight and amino acid content; this may account for differences observed in acceptor amino acid specificity and in the magnitude of the maleate effect.     

Developmental change of endogenous glutamate and gamma-glutamyl transferase in cultured cerebral cortical interneurons and cerebellar granule cells,and in mouse cerebral cortex and cerebellum in vivo

The developmental change of endogenous glutamate, as correlated to that of gamma-glutamyl transferase and other glutamate metabolizing enzymes such as phosphate activated glutaminase, glutamate dehydrogenase and aspartate, GABA and ornithine aminotransferases, has been investigated in cultured cerebral cortex interneurons and cerebellar granule cells. These cells are considered to be GABAergic and glutamatergic, respectively. Similar studies have also been performed in cerebral cortex and cerebellum in vivo. The developmental profiles of endogenous glutamate in cultured cerebral cortex interneurons and cerebellar granule cells corresponded rather closely with that of gamma-glutamyl transferase and not with other glutamate metabolizing enzymes. In cerebral cortex and cerebellum in vivo the developmental profiles of endogenous glutamate, gamma-glutamyl transferase and phosphate activated glutaminase corresponded with each other during the first 14 days in cerebellum, but this correspondence was less good in cerebral cortex. During the time period from 14 to 28 days post partum the endogenous glutamate concentration showed no close correspondence with any particular enzyme. It is suggested that gamma-glutamyltransferase regulates the endogenous glutamate concentration in culture neurons. The enzyme may also be important for regulation of endogenous glutamate in brain in vivo and particularly in cerebellum during the first 14 days post partum. Gamma-glutamyl transferase in cultured neurons and brain tissue in vivo appears to be devoid of maleate activated glutaminase.Abbreviations used Asp-T aspartate aminotransferase (EC 2.6.1.1) - GABA-T GABA aminotransferase (EC 2.6.1.19) - GAD glutamate decarboxylase (EC 4.1.1.15) - gamma-GT gamma-glutamyl transferase (gamma-glutamyl transpeptidase) (EC. 2.3.2.2) - Glu glutamate - GDH glutamate dehydrogenase (EC 1.4.1.3) - GS glutamine synthetase (EC 6.3.1.2) - MAG maleate activated glutaminase - Orn-T ornithine aminotransferase (EC 2.6.1.13) - PAG phosphate activated glutaminase (EC 3.5.1.1)     

Regulation of glutaminase B in Escherichia coli. I. Purification, properties, and cold lability.

Escherichia coli contains two glutaminases, A and B, with pH optima below pH 5 and above pH 7, respectively. Neither glutaminase A nor B is released from E. coli by osmotic shock. Glutaminase B has been purified 6,000-fold and the purified preparation is estimated to contain about 40% glutaminase B. The enzyme has a molecular weight of 90,000 and an isoelectric point of 5.4. Glutaminase B exhibits a broad pH optimum between 7.1 and 9.0. Only L-glutamine is deamidated by glutaminase B, L-asparagine and D-glutamine are not deamidated. The substrate saturation curve for glutaminase B shows an intermediary plateau region. Like many regulatory enzymes, glutaminase B is cold-labile. The enzyme is inactivated by cooling and activated by warming; both processes are first order with respect to time. The activation energy for activation by warming was calculated to be 5900 cal/mol. Activation by warming increased the Vmax and decreased the S0.5 for L-glutamine, but did not alter the molecular weight of the catalytically active enzyme. Borate and glutamate protected glutaminase B from inactivation by cold.     

gamma-Glutamyl cyclotransferase from rat kidney. Sulfhydryl groups and isolation of a stable form of the enzyme.

gamma-Glutamyl cyclotransferase, highly purified from rat kidney, contains several readily accessible sulfhydryl groups whose modification appears to be associated with the appearance of multiple enzyme forms as determined by isoelectric focusing and ion exchange chromatography. The enzyme was obtained in a 1000-fold purified and apparently homogeneous form by a procedures involving treatment with dithiothreitol followed by chromatography on thiol-Sepharose. The enzyme was also isolated in a highly active, apparently homogeneous, and stable form after reduction and treatment with iodoacetamide. The amino acid compositions and other properties of the two forms of the enzyme were very similar. Studies on the activity of the enzyme toward a variety of gamma-glutamyl amino acids and di-gamma-glutamyl amino acids showed that the enzyme is much more active toward certain di-gamma-glutamyl amino acids than toward the corresponding gamma-glutamyl amino acids; thus, the preferred substrates have the general structure gamma-Glu-gamma-Glu-NH-R in which the nature of the R moiety has relatively little effect on activity.     

Inhibition of the hydrolytic and transpeptidase activities of rat kidney gamma-glutamyl transpeptidase by specific monoclonal antibodies.

Monoclonal antibodies (mAb) against the native form of rat kidney gamma-glutamyl transpeptidase (GGT) were isolated by screening hybridomas with rat kidney brush-border membrane vesicles. They were directed against protein rather than sugar epitopes in that each recognized all GGT isoforms. All of them inhibited partially the enzyme activity of GGT. They were specific in that they inhibited the rat enzyme, but not the mouse or human enzyme. Kinetic analyses were carried out with free GGT and GGT-mAb complexes with d-gamma-glutamyl-p-nitroanilide in the presence or absence of maleate, or in the presence or absence of alanine, cysteine, cystine or glycylglycine as gamma-glutamyl acceptors. mAbs 2A10 and 2E9 inhibited the hydrolytic and glutaminase activities of GGT and had little effect on the transpeptidation activity of the enzyme, whereas mAbs 4D7 and 5F10 inhibited transpeptidation, but not hydrolytic or glutaminase activities. mAb 5F10 mimicked the effect of maleate on GGT, in that it inhibited transpeptidation, enhanced the glutaminase activity and increased the affinity of the donor site of GGT for acivicin. Such mAbs may be useful for long-term studies in tissue cultures and in vivo, and for the identification of GGT epitopes that are important for the hydrolytic and transpeptidase activities.     

Evidence that transpeptidation is a significant function of gamma-glutamyl transpeptidase

gamma-Glutamyl transpeptidase (purified from rat kidney) was incubated with glutathione and a mixture of amino acids that closely approximates the amino acid composition of blood plasma, and the relative extents of transpeptidation and hydrolysis were determined by quantitative measurement of the products formed (glutamate, cysteinylglycine, gamma-glutamyl amino acids). At pH 7.4, in the presence of 50 microM glutathione and the amino acid mixture, about 50% of the glutathione that was utilized participated in transpeptidation. Studies in which the formation of individual gamma-glutamyl amino acids was determined in the presence of glutathione and the amino acid mixture showed that L-cystine and L-glutamine are the most active amino acid acceptors, and that other neutral amino acids also participate in transpeptidation to a significant extent. These in vitro experiments are consistent with a number of other findings which indicate that transpeptidation is a significant physiological function of gamma-glutamyl transpeptidase.     

Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects

In Bacillus pasteurii glutamine is being taken up efficiently by a sodium-dependent uptake system and subsequently hydrolysed to ammonium and glutamate. Concerning the latter process, a catabolic L-glutamine amidohydrolase (glutaminase) was isolated from the cytoplasm of this alkaliphilic bacterium and purified to homogeneity using liquid chromatography. Biochemical and physical parameters of the pure enzyme were examined in detail. Interestingly, analysis of the glutaminase revealed a marked increase in catalytic activity in the presence of phosphate, a property yet restricted to animal glutaminases. This is the first report on the presence of a phosphate-activated glutaminase in bacteria.     

Phosphate-dependent glutaminase from rat kidney. Cause of increased activity in response to acidosis and identity with glutaminase from other tissues.

Immune serum was prepared against phosphate-dependent glutaminase purified from rat kidney and was used to investigate the cause of increased renal glutaminase activity in acidotic rats. Crude kidney homogenates from acidotic rats exhibited a fourfold greater specific activity for phosphate-dependent glutaminase. The glutaminase was solubilized initially by lyophilization of borate treated mitochondria with a 40–60% recovery and with maintenance of threefold difference in specific activity. Both preparations showed the same equivalence point in a quantitative precipitin experiment. To confirm these results, phosphate-dependent glutaminase was also solubilized by treatment of mitochondria isolated from normal and acidotic rat kidney cortex with 1% Triton X-100. The two preparations exhibited a fivefold difference in specific activity and again showed the same equivalence point in a quantitative precipitin experiment. These results indicate that the cause of increased phosphate-dependent glutaminase activity during acidosis is due to the presence of an increased amount of this enzyme. The antiserum prepared against the kidney phosphate-dependent glutaminase did not crossreact with glutaminase solubilized from rat liver mitochondria. But, rat brain mitochondria do contain a phosphate-dependent glutaminase that is immunologically identical to the enzyme from rat kidney.     

Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248

The use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme. To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties. We found that replacements of Asp248 affected glutamine turnover much more strongly than asparagine hydrolysis. In the wild-type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate. In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol(-1) for L-glutamine compared to 4 kJ mol(-1) for L-aspartic beta-hydroxamate and 7 kJ mol(-1) for L-asparagine. Smaller differences were seen with other N248 variants. Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active-site residues and catalytically relevant water molecules.     

Purification and characterization of a new hydrolase for conjugated bile acids, chenodeoxycholyltaurine hydrolase, from Bacteroides vulgatus

A new hydrolase for conjugated bile acids, tentatively named chenodeoxycholyltaurine hydrolase, was purified to homogeneity from Bacteroides vulgatus. This enzyme hydrolyzed taurine-conjugated bile acids but showed no activity toward glycine conjugates. Among the taurine conjugates, taurochenodeoxycholic acid was most effectively hydrolyzed, tauro-beta-muricholic and ursodeoxycholic acids were moderately well hydrolyzed, and cholic and 7 beta-cholic acids were hardly hydrolyzed, suggesting that this enzyme has a specificity for not only the amino acid moiety but also the steroidal moiety. The molecular weight of the enzyme was estimated to be approximately 140,000 by Sephacryl S-300 gel filtration and the subunit molecular weight of the enzyme was 36,000 by SDS-polyacrylamide gel electrophoresis. The optimum pH was in the range of 5.6 to 6.4. The NH2-terminal amino acid sequence of the enzyme was Met-Glu-Arg-Thr-Ile-Thr-Ile-Gln-Gln-Ile-Lys-Asp-Ala-Ala-Gln. The enzyme was activated by dithiothreitol, but inhibited by sulfhydryl inhibitors, p-hydroxymercuribenzoate, N-ethylmaleimide, and dithiodipyridine.     

PRODUCTION AND OPTIMIZATION OF L-GLUTAMINASE ENZYME FROM Hypocrea jecorina PURE CULTURE

L-Glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) is the important enzyme that catalyzes the deamination of L-glutamine to L-glutamic acid and ammonium ions. Recently, L-glutaminase has received much attention with respect to its therapeutic and industrial applications. It acts as a potent antileukemic agent and shows flavor-enhancing capacity in the production of fermented foods. Glutaminase activity is widely distributed in plants, animal tissues, and microorganisms, including bacteria, yeasts, and fungi. This study presents microbial production of glutaminase enzyme from Hypocrea jecorina pure culture and determination of optimum conditions and calculation of kinetic parameters of the produced enzyme. The optimum values were determined by using sa Nesslerization reaction for our produced glutaminase enzyme. The optimum pH value was determined as 8.0 and optimum temperature as 50°C for the glutaminase enzyme. The Km and Vmax values, the kinetic parameters, of enzyme produced from Hypocrea jecorina, pure culture were determined as 0.491 mM for Km and 13.86 U/L for Vmax by plotted Lineweaver–Burk graphing, respectively. The glutaminase enzyme from H. jecorina microorganism has very high thermal and storage stability.     

Occurrence and some properties of a novel γ-glutamyltransferase responsible for the syntheis of γ-L-glutamul-D-alanine in pea seedlings

Occurrence of a novel γ-glutamyltransferase responsible for the formation of γ-L-glutamyl-D-alanine was demonstrated in pea seedlings, and the enzyme was purified 600-fold. The enzyme preparation catalyzed the transfer of the γ-glutamyl moiety of L-glutamine and other γ-glutamyl compounds to D-amino acids. In the formation of γ-L-glutamyl peptides of D-amino acids, L-glutamine served as the most effective γ-glutamyl donor and D-alanine acted as a highly-specific acceptor. The maximum activity of the γ-glutamyl transfer reaction between L-glutamine and D-alanine was observed at pH 9.5 and the apparent K_m values for these amino acids were estimated to be 2.0 and 2.9mM, respectively. This unique γ-glutamyltransferase activity was always accompanied by the catalytic activities of the known γ-glutamyltransferases during the purification procedure.     

Characterization of gamma-glutamyl hydrolase produced by Bacillus sp. isolated from Thai Thua-nao

Gamma-glutamyl hydrolase with a molecular mass of 28 kDa was purified from the culture broth of Bacillus sp. isolated from Thai Thua-nao, a natto-like fermented soybean food. The purified enzyme hydrolyzed chemically synthesized oligo-gamma-L-glutamates but not oligo-gamma-D-glutamates and degraded gamma-polyglutamic acid to a hydrolyzed product of only about 20 kDa (with D- and L-glutamic acid in a ratio of 70:30), suggesting that the enzyme is a gamma-glutamyl hydrolase that cleaves the gamma-glutamyl linkage between L- and L-glutamic acid of gamma-polyglutamic acid.