A Study on the Efficient Preparation of α-Ketoglutarate with L-Glutamate Oxidase.

Alpha-ketoglutaric acid (α-KG), as an intermediate product of the tricarboxylic acid cycle, plays a crucial role in peptide and amino acid synthesis. In order to reduce costs and improve efficiency in the oxidative production of α-ketoglutaric acid, this study successfully synthesized and expressed L-glutamate oxidase (LGOXStr) from Streptomyces viridosporus R111 and catalase (KatGEsc) from Escherichia coli H736. Two immobilization methods and the conditions for one-step whole-cell catalysis of α-ketoglutaric acid were investigated. α-Ketoglutaric acid has broad applications in the pharmaceutical, food, and chemical industries. The specific research results are as follows: (1) By fusing the sfGFP tag, L-glutamate oxidase (LGOXStr r) and catalase (KatGEsc) were successfully anchored to the outer membrane of Escherichia coli cells, achieving one-step whole-cell catalysis of α-ketoglutaric acid with a conversion efficiency of up to 75%. (2) Through the co-immobilization of LGOXStr and KatGEsc, optimization of the preparation parameters of immobilized cells, and exploration of the immobilization method using E.coli@ZIF-8, immobilized cells with conversion rates of over 60% were obtained even after 10 cycles of reuse. Under the optimal conditions, the production rate of α-ketoglutaric acid reached 96.7% in a 12 h reaction, which is 1.1 times that of E. coli@SA and 1.29 times that of free cells.

Reshaping the Substrate Binding Pocket of ß-Amino Acid Dehydrogenase for the Synthesis of Aromatic ß-Amino Acids.

By reshaping the substrate-binding pocket of ß-amino acid dehydrogenase (ß-AADH), some variants were obtained with up to 2560-fold enhanced activity toward the model substrates (S)-ß-homophenylalanine and (R)-ß-phenylalanine. A few aromatic ß-amino acids were prepared with >99% ee and high isolated yields via either kinetic resolution of racemates or reductive amination of the corresponding ß-keto acids. This work expands the catalytic capability of ß-AADHs and highlights their practical application in the synthesis of pharmaceutically relevant ß-amino acids.

Computational Insights on the Hydride and Proton Transfer Mechanisms of D-Arginine Dehydrogenase.

D-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) is an amine oxidase which catalyzes the conversion of D-arginine into iminoarginine. It contains a non-covalent FAD cofactor that is involved in the oxidation mechanism. Based on substrate, solvent, and multiple kinetic isotope effects studies, a stepwise hydride transfer mechanism is proposed. It was shown that D-arginine binds to the active site of enzyme as α-amino group protonated, and it is deprotonated before a hydride ion is transferred from its α-C to FAD. Based on a mutagenesis study, it was concluded that a water molecule is the most likely catalytic base responsible from the deprotonation of α-amino group. In this study, we formulated computational models based on ONIOM method to elucidate the oxidation mechanism of D-arginine into iminoarginine using the crystal structure of enzyme complexed with iminoarginine. The calculations showed that Arg222, Arg305, Tyr249, Glu87, His 48, and two active site water molecules play key roles in binding and catalysis. Model systems showed that the deprotonation step occurs prior to hydride transfer step, and active site water molecule(s) may have participated in the deprotonation process.

Directed evolution of glyphosate oxidase and a chemiluminescence system for glyphosate detection: A comprehensive practical laboratory experiment on biotechnology.

This article describes a comprehensive practical laboratory method for developing an enzyme to more easily measure glyphosate levels in solution. Through this article, undergraduate students of biology majors can conduct research experiments in critical fields by utilizing various techniques, such as chemiluminescence (CL) biosensors with engineered enzymes and are guided in molecular biology laboratories. A glyphosate oxidase mutant library was constructed by DNA shuffling, and a glyphosate oxidase variant with increased glyphosate degradation activity was selected by using a high-throughput screening assay. Following protein overexpression in Escherichia coli (DE3) and purification by affinity chromatography, the glyphosate oxidase variant protein combined with luminol-H2 O2 reaction was constructed as a new CL biosensor for detecting glyphosate in soils.

Non-active Site Residue in Loop L4 Alters Substrate Capture and Product Release in d-Arginine Dehydrogenase.

Numerous studies demonstrate that enzymes undergo multiple conformational changes during catalysis. The malleability of enzymes forms the basis for allosteric regulation: residues located far from the active site can exert long-range dynamical effects on the active site residues to modulate catalysis. The structure of Pseudomonas aeruginosa d-arginine dehydrogenase (PaDADH) shows four loops (L1, L2, L3, and L4) that span the substrate and the FAD-binding domains. Loop L4 comprises residues 329-336, spanning over the flavin cofactor. The I335 residue on loop L4 is ∼10 Šaway from the active site and ∼3.8 Šfrom N(1)-C(2)═O atoms of the flavin. In this study, we used molecular dynamics and biochemical techniques to investigate the effect of the mutation of I335 to histidine on the catalytic function of PaDADH. Molecular dynamics showed that the conformational dynamics of PaDADH are shifted to a more closed conformation in the I335H variant. In agreement with an enzyme that samples more in a closed conformation, the kinetic data of the I335H variant showed a 40-fold decrease in the rate constant of substrate association (k1), a 340-fold reduction in the rate constant of substrate dissociation from the enzyme-substrate complex (k2), and a 24-fold decrease in the rate constant of product release (k5), compared to that of the wild-type. Surprisingly, the kinetic data are consistent with the mutation having a negligible effect on the reactivity of the flavin. Altogether, the data indicate that the residue at position 335 has a long-range dynamical effect on the catalytic function in PaDADH.

Development of oxidoreductases for amino acid quantification and mutagenesis techniques for heterologous soluble expression: screening and selection strategies.

The high stereo- and substrate specificities of enzymes have been utilized for microdetermination of amino acids. Here, I review the discovery of l-Arg oxidase from Pseudomonas sp. TPU 7192, l-Lys oxidase/decarboxylase from Burkholderia sp. AIU 395, and enzymes showing apparent l-His oxidase activity from Achromobacter sp. TPU 5009. I also discuss screening and uses of the selective enzymes for microdetermination of amino acids. In addition, functional modifications of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813, l-Trp dehydrogenase from Nostoc punctiforme ATCC 29133, and l-Lys ε-oxidase from Marinomonas mediterranea NBRC 103028 by directed evolution are reviewed. Finally, I review the rational identification of aggregation hotspots based on secondary structure and amino acid hydrophobicity-this process enables the wider use of natural enzymes.

A 3D-Predicted Structure of the Amine Oxidase Domain of Lysyl Oxidase-Like 2.

Lysyl oxidase-like 2 (LOXL2) has been recognized as an attractive drug target for anti-fibrotic and anti-tumor therapies. However, the structure-based drug design of LOXL2 has been very challenging due to the lack of structural information of the catalytically-competent LOXL2. In this study; we generated a 3D-predicted structure of the C-terminal amine oxidase domain of LOXL2 containing the lysine tyrosylquinone (LTQ) cofactor from the 2.4Å crystal structure of the Zn2+-bound precursor (lacking LTQ; PDB:5ZE3); this was achieved by molecular modeling and molecular dynamics simulation based on our solution studies of a mature LOXL2 that is inhibited by 2-hydrazinopyridine. The overall structures of the 3D-modeled mature LOXL2 and the Zn2+-bound precursor are very similar (RMSD = 1.070Å), and disulfide bonds are conserved. The major difference of the mature and the precursor LOXL2 is the secondary structure of the pentapeptide (His652-Lys653-Ala654-Ser655-Phe656) containing Lys653 (the precursor residue of the LTQ cofactor). We anticipate that this peptide is flexible in solution to accommodate the conformation that enables the LTQ cofactor formation as opposed to the ß-sheet observed in 5ZE3. We discuss the active site environment surrounding LTQ and Cu2+ of the 3D-predicted structure.

In silico characterization of fructosyl peptide oxidase properties from Eupenicillium terrenum.

Fructosyl peptide oxidase (FPOX) enzyme from Eupenicillium terrenum has a high potential to be applied as a diagnostic enzyme. The aim of the present study is the characterization of FPOX from E. terrenum using different bioinformatics tools. The computational prediction of the RNA and protein secondary structures of FPOX, solubility profile in Escherichia coli, stability, domains, and functional properties were performed. In the FPOX protein, six motifs were detected. The d-amino acid oxidase motif was found as the most important motif that is a FAD-dependent oxidoreductase. The cysteines including 97, 154, 234, 280, and 360 showed a lower score than -10 that have a low possibility for participitation in the formation of the SS bond. The 56.52% of FPOX amino acids are nonpolar. Random coils are dominant in the FPOX sequence, followed by alpha-helix and extended strand. The fpox gene is capable of generating a stable RNA secondary structure (-423.90 kcal/mol) in E. coli. FPOX has a large number of hydrophobic amino acids. FPOX showed a low solubility in E. coli which has several aggregation-prone sites in its 3-D structure. According to the scores, the best mutation candidate for increasing solubility was the conversion of methionine 302 to arginine. The melting temperature of FPOX based on its amino acid sequence was 55°C to 65°C. The amounts of thermodynamic parameters for the FPOX enzyme were -137.4 kcal/mol, -3.59 kcal/(mol K), and -6.8 kcal/mol for standard folding enthalpy, heat capacity, and folding free energy, respectively. In conclusion, the in silico study of proteins can provide a valuable method for better understanding the protein properties and functions for use in our purposes.

Recharacterization of the mammalian cytosolic type 2 (R)-ß-hydroxybutyrate dehydrogenase as 4-oxo-l-proline reductase (EC 1.1.1.104).

Early studies revealed that chicken embryos incubated with a rare analog of l-proline, 4-oxo-l-proline, showed increased levels of the metabolite 4-hydroxy-l-proline. In 1962, 4-oxo-l-proline reductase, an enzyme responsible for the reduction of 4-oxo-l-proline, was partially purified from rabbit kidneys and characterized biochemically. However, only recently was the molecular identity of this enzyme solved. Here, we report the purification from rat kidneys, identification, and biochemical characterization of 4-oxo-l-proline reductase. Following mass spectrometry analysis of the purified protein preparation, the previously annotated mammalian cytosolic type 2 (R)-ß-hydroxybutyrate dehydrogenase (BDH2) emerged as the only candidate for the reductase. We subsequently expressed rat and human BDH2 in Escherichia coli, then purified it, and showed that it catalyzed the reversible reduction of 4-oxo-l-proline to cis-4-hydroxy-l-proline via chromatographic and tandem mass spectrometry analysis. Specificity studies with an array of compounds carried out on both enzymes showed that 4-oxo-l-proline was the best substrate, and the human enzyme acted with 12,500-fold higher catalytic efficiency on 4-oxo-l-proline than on (R)-ß-hydroxybutyrate. In addition, human embryonic kidney 293T (HEK293T) cells efficiently metabolized 4-oxo-l-proline to cis-4-hydroxy-l-proline, whereas HEK293T BDH2 KO cells were incapable of producing cis-4-hydroxy-l-proline. Both WT and KO HEK293T cells also produced trans-4-hydroxy-l-proline in the presence of 4-oxo-l-proline, suggesting that the latter compound might interfere with the trans-4-hydroxy-l-proline breakdown in human cells. We conclude that BDH2 is a mammalian 4-oxo-l-proline reductase that converts 4-oxo-l-proline to cis-4-hydroxy-l-proline and not to trans-4-hydroxy-l-proline, as originally thought. We also hypothesize that this enzyme may be a potential source of cis-4-hydroxy-l-proline in mammalian tissues.

Combined active pocket and hinge region engineering to develop an NADPH-dependent phenylglycine dehydrogenase.

NADPH-dependent amino acid dehydrogenases (AADHs) are favorable enzymes to construct artificial biosynthetic pathways in whole-cell for high-value noncanonical amino acids (NcAAs) production. Glutamate dehydrogenases (GluDHs) represent attractive candidates for the development of novel NADPH-dependent AADHs. Here, we report the development of a novel NADPH-dependent phenylglycine dehydrogenase by combining active pocket engineering and hinge region engineering of a GluDH from Pseudomonas putida (PpGluDH). The active pocket of PpGluDH was firstly tailored to optimize its binding mode with bulky substrate α-oxobenzeneacetic acid (α-OA), and then, the hinge region was further engineered to tune the protein conformational dynamics, which finally resulted in a mutant M3 (T196A/T121I/L123D) with a 103-fold increase of catalytic efficiency (kcat/Km) toward α-OA. The M3 mutant exhibited high catalytic performance in both in vitro biocatalysis preparation and in vivo biosynthesis of l-phenylglycine, indicating its promising practical applications. Our results demonstrated that co-engineering of the active pocket and hinge region is an effective strategy for developing novel NADPH-dependent AADHs from GluDHs for NcAAs production.

Thermostability enhancement of l-glutamate oxidase from Streptomyces sp. NT1 by full consensus protein design.

Thermostable l-glutamate oxidases (LGOXs) are desirable for use in l-glutamate (L-Glu) assay kits, enzymatic synthesis of α-ketoglutarate and for biosensor development. However, protein engineering efforts to improve thermostability often lead to a decrease in enzymatic activity. In this report, we aimed to enhance the thermostability (melting temperature, Tm) of a mesophilic LGOX from Streptomyces sp. NT1 (LGOXNT1) without a reduction in activity by a sequence-based protein design approach, termed full consensus (Fc) protein design. Among the 690 amino acids of LGOXNT1, 104 amino acids were substituted by the Fc protein design. The mutant gene was artificially synthesized and expressed in Escherichia coli BL21(DE3) cells. The Tm of the purified, recombinant LGOX mutant (FcLGOX) was determined to be ∼72 °C, which is an increase on the Tm of 65 °C for LGOXNT1 and the highest among known LGOXs. Importantly, purified FcLGOX showed no loss of specific activity or substrate specificity after a 30-min incubation at 70 °C. Our findings provide a new approach to improve the thermostability of enzymes.

Discovery of a new flavin N5-adduct in a tyrosine to phenylalanine variant of d-Arginine dehydrogenase.

d-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) catalyzes the flavin-dependent oxidation of d-arginine and other d-amino acids. Here, we report the crystal structure at 1.29 Å resolution for PaDADH-Y249F expressed and co-crystallized with d-arginine. The overall structure of PaDADH-Y249F resembled PaDADH-WT, but the electron density for the flavin cofactor was ambiguous, suggesting the presence of modified flavins. Electron density maps and mass spectrometric analysis confirmed the presence of both N5-(4-guanidino-oxobutyl)-FAD and 6-OH-FAD in a single crystal of PaDADH-Y249F and helped with the further refinement of the X-ray crystal structure. The versatility of the reduced flavin is apparent in the PaDADH-Y249F structure and is evidenced by the multiple functions it can perform in the same active site.

Oligomeric States and Hydrodynamic Properties of Lysyl Oxidase-Like 2.

Lysyl oxidase-like 2 (LOXL2) has emerged as a promising therapeutic target against metastatic/invasive tumors and organ and tissue fibrosis. LOXL2 catalyzes the oxidative deamination of lysine and hydroxylysine residues in extracellular matrix (ECM) proteins to promote crosslinking of these proteins, and thereby plays a major role in ECM remodeling. LOXL2 secretes as 100-kDa full-length protein (fl-LOXL2) and then undergoes proteolytic cleavage of the first two scavenger receptor cysteine-rich (SRCR) domains to yield 60-kDa protein (Δ1-2SRCR-LOXL2). This processing does not affect the amine oxidase activity of LOXL2 in vitro. However, the physiological importance of this cleavage still remains elusive. In this study, we focused on characterization of biophysical properties of fl- and Δ1-2SRCR-LOXL2s (e.g., oligomeric states, molecular weights, and hydrodynamic radii in solution) to gain insight into the structural role of the first two SRCR domains. Our study reveals that fl-LOXL2 exists predominantly as monomer but also dimer to the lesser extent when its concentration is <~1 mM. The hydrodynamic radius (Rh) determined by multi-angle light scattering coupled with size exclusion chromatography (SEC-MALS) indicates that fl-LOXL2 is a moderately asymmetric protein. In contrast, Δ1-2SRCR-LOXL2 exists solely as monomer and its Rh is in good agreement with the predicted value. The Rh values calculated from a 3D modeled structure of fl-LOXL2 and the crystal structure of the precursor Δ1-2SRCR-LOXL2 are within a reasonable margin of error of the values determined by SEC-MALS for fl- and Δ1-2SRCR-LOXL2s in mature forms in this study. Based on superimposition of the 3D model and the crystal structure of Δ1-2SRCR-LOXL2 (PDB:5ZE3), we propose a configuration of fl-LOXL2 that explains the difference observed in Rh between fl- and Δ1-2SRCR-LOXL2s in solution.

A shared mechanistic pathway for pyridoxal phosphate-dependent arginine oxidases.

The mechanism by which molecular oxygen is activated by the organic cofactor pyridoxal phosphate (PLP) for oxidation reactions remains poorly understood. Recent work has identified arginine oxidases that catalyze desaturation or hydroxylation reactions. Here, we investigate a desaturase from the Pseudoalteromonas luteoviolacea indolmycin pathway. Our work, combining X-ray crystallographic, biochemical, spectroscopic, and computational studies, supports a shared mechanism with arginine hydroxylases, involving two rounds of single-electron transfer to oxygen and superoxide rebound at the 4' carbon of the PLP cofactor. The precise positioning of a water molecule in the active site is proposed to control the final reaction outcome. This proposed mechanism provides a unified framework to understand how oxygen can be activated by PLP-dependent enzymes for oxidation of arginine and elucidates a shared mechanistic pathway and intertwined evolutionary history for arginine desaturases and hydroxylases.

Catalytic mechanism of ancestral L-lysine oxidase assigned by sequence data mining.

A large number of protein sequences are registered in public databases such as PubMed. Functionally uncharacterized enzymes are included in these databases, some of which likely have potential for industrial applications. However, assignment of the enzymes remained difficult tasks for now. In this study, we assigned a total of 28 original sequences to uncharacterized enzymes in the FAD-dependent oxidase family expressed in some species of bacteria including Chryseobacterium, Flavobacterium, and Pedobactor. Progenitor sequence of the assigned 28 sequences was generated by ancestral sequence reconstruction, and the generated sequence exhibited L-lysine oxidase activity; thus, we named the enzyme AncLLysO. Crystal structures of ligand-free and ligand-bound forms of AncLLysO were determined, indicating that the enzyme recognizes L-Lys by hydrogen bond formation with R76 and E383. The binding of L-Lys to AncLLysO induced dynamic structural change at a plug loop formed by residues 251 to 254. Biochemical assays of AncLLysO variants revealed the functional importance of these substrate recognition residues and the plug loop. R76A and E383D variants were also observed to lose their activity, and the kcat/Km value of G251P and Y253A mutations were approximately 800- to 1800-fold lower than that of AncLLysO, despite the indirect interaction of the substrates with the mutated residues. Taken together, our data demonstrate that combinational approaches to sequence classification from database and ancestral sequence reconstruction may be effective not only to find new enzymes using databases of unknown sequences but also to elucidate their functions.

Monitoring Metabolites Using an NAD(P)H-sensitive Polymer Dot and a Metabolite-Specific Enzyme.

We introduce an NAD(P)H-sensitive polymer dot (Pdot) biosensor for point-of-care monitoring of metabolites. The Pdot is combined with a metabolite-specific NAD(P)H-dependent enzyme that catalyzes the oxidation of the metabolite, generating NAD(P)H. Upon UV illumination, the NAD(P)H quenches the fluorescence emission of Pdot at 627 nm via electron transfer, and also fluoresces at 458 nm, resulting in a shift from red to blue emission at higher NAD(P)H concentrations. Metabolite concentration is quantified ratiometrically-based on the ratio of blue-to-red channel emission intensities, with a digital camera-with high sensitivity and specificity. We demonstrate phenylalanine biosensing in human plasma for a phenylketonuria screening test, quantifying several other disease-related metabolites (lactate, glucose, glutamate, and ß-hydroxybutyrate), and a paper-based assay with smartphore imaging for point-of-care use.

A new l-arginine oxidase engineered from l-glutamate oxidase.

The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 catalyzes the oxidative deamination of l-glutamate to produce 2-ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l-glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l-glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l-arginine. The oxidative deamination activity of LGOX to l-arginine is higher than that of l-arginine oxidase form from Pseudomonas sp. TPU 7192. X-ray crystal structure analysis revealed that the guanidino group of l-arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild-type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l-arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l-arginine by LGOX R305E, is proportional to the concentration of l-arginine in a range from 0 to 100 µM. The linear relationship is maintained around 1 µM of l-arginine. Thus, LGOX R305E is suitable for the determination of l-arginine.

Crystal Structures and Catalytic Mechanism of l-erythro-3,5-Diaminohexanoate Dehydrogenase and Rational Engineering for Asymmetric Synthesis of ß-Amino Acids.

Amino acid dehydrogenases (AADHs) have shown considerable potential as biocatalysts in the asymmetric synthesis of chiral amino acids. However, compared to the widely studied α-AADHs, limited knowledge is available about ß-AADHs that enable the synthesis of ß-amino acids. Herein, we report the crystal structures of a l-erythro-3,5-diaminohexanoate dehydrogenase and its variants, the only known member of ß-AADH family. Crystal structure analysis, site-directed mutagenesis studies and quantum chemical calculations revealed the differences in the substrate binding and catalytic mechanism from α-AADHs. A number of rationally engineered variants were then obtained with improved activity (by 110-800 times) toward various aliphatic ß-amino acids without an enantioselectivity trade-off. Two ß-amino acids were prepared by using the outstanding variants with excellent enantioselectivity (>99 % ee) and high isolated yields (86-87 %). These results provide important insights into the molecular mechanism of 3,5-DAHDH, and establish a solid foundation for further design of ß-AADHs for the asymmetric synthesis of ß-amino acids.

A Single-Point Mutation in d-Arginine Dehydrogenase Unlocks a Transient Conformational State Resulting in Altered Cofactor Reactivity.

Proteins are inherently dynamic, and proper enzyme function relies on conformational flexibility. In this study, we demonstrated how an active site residue changes an enzyme's reactivity by modulating fluctuations between conformational states. Replacement of tyrosine 249 (Y249) with phenylalanine in the active site of the flavin-dependent d-arginine dehydrogenase yielded an enzyme with both an active yellow FAD (Y249F-y) and an inactive chemically modified green FAD, identified as 6-OH-FAD (Y249F-g) through various spectroscopic techniques. Structural investigation of Y249F-g and Y249F-y variants by comparison to the wild-type enzyme showed no differences in the overall protein structure and fold. A closer observation of the active site of the Y249F-y enzyme revealed an alternative conformation for some active site residues and the flavin cofactor. Molecular dynamics simulations probed the alternate conformations observed in the Y249F-y enzyme structure and showed that the enzyme variant with FAD samples a metastable conformational state, not available to the wild-type enzyme. Hybrid quantum/molecular mechanical calculations identified differences in flavin electronics between the wild type and the alternate conformation of the Y249F-y enzyme. The computational studies further indicated that the alternate conformation in the Y249F-y enzyme is responsible for the higher spin density at the C6 atom of flavin, which is consistent with the formation of 6-OH-FAD in the variant enzyme. The observations in this study are consistent with an alternate conformational space that results in fine-tuning the microenvironment around a versatile cofactor playing a critical role in enzyme function.

Extraction and Purification of Extracellular L-Glutamate Oxidase from Streptomyces.

The bacterial isolates Streptomyces were obtained from the soil and cultivated in a wheat bran medium, which was used to produce the L-glutamate oxidase enzyme. The extracellular enzyme was then extracted using a cooling centrifugation process to obtain the filtrate that represents the crude enzyme. Afterward, the enzyme purification processes were carried out which included precipitation with ammonium sulfate as a preliminary purification step followed by dialysis to remove the salts. Next, ion-exchange chromatography and gel filtration were used to finish the purification process, and the enzyme activity was determined for each purification step. The results of purification of L-glutamate oxidase enzyme from streptomyces using ammonium sulfate showed that the specific activity was 8.25 units/mg protein with a saturation ratio of 60%. Moreover, the results of purification using a dialysis tube indicated that the specific activity was 9.5 units/mg protein. In addition, the result of purification using diethylaminoethyl cellulose ion column revealed that the specific activity was 25 unit/mg protein and the results of purification using gel filtration showed that the specific activity was 56 units/mg protein which was the best step in the purification process due to high specific activity of the enzyme. The optimum temperature and pH for the activity and stability of the enzyme were tested. Based on the findings, the optimum temperature for the activity of the enzyme was 37 °C. In addition, it was found that the optimum temperature range for the stability of the enzyme was 30-50 °C. Besides, the optimum pH for the activity was 7.0 and the optimum pH range for the enzyme stability was 5.0-7.0.