A Novel Express Method to Determine Activity of Antitumor Enzyme L-Lysine-α-Oxidase of Trichoderma harzianum Rifai F-180.

A novel express method is developed to determine activity of antitumor enzyme L-lysine-α-oxidase obtained by culturing Trichoderma harzianum Rifai F-180 fungus. The carcinogenic reagent ortho-dianisidine-hydrochloride was replaced in the reaction medium with environmentally friendly reagents of the chromogenic mixture that included tetramethylbenzidine. This method improved precision and sensitivity of ELISA by 10 and 40 times, respectively. In addition, it could detect activity of L-lysine-α-oxidase not only in the producer strains with a pronounced activity of this enzyme, but also in the strains where this activity has not been previously determined.

Characterization of PlGoxB, a flavoprotein required for cysteine tryptophylquinone biosynthesis in glycine oxidase from Pseudoalteromonas luteoviolacea.

LodA-like proteins are oxidases with a protein-derived cysteine tryptophylquinone (CTQ) prosthetic group. In Pseudoalteromonas luteoviolacea glycine oxidase (PlGoxA), CTQ biosynthesis requires post-translational modifications catalyzed by a modifying enzyme encoded by PlgoxB. The PlGoxB protein was expressed and shown to possess a flavin cofactor. PlGoxB was unstable in solution as it readily lost the flavin and precipitated. PlGoxB precipitation was significantly reduced by incubation with either excess FAD or an equal concentration of prePlGoxA, the precursor protein that is its substrate. In contrast, the mature CTQ-bearing PlGoxA had no stabilizing effect. A homology model of PlGoxB was generated using the structure of Alkylhalidase CmIS. The FAD-binding site of PlGoxB in the model was nearly identical to that of the template structure. The bound FAD in PlGoxB had significant solvent exposure, consistent with the observed tendency to lose FAD. This also suggested that interaction of prePlGoxA with PlGoxB at the exposed FAD-binding site could prevent the observed loss of FAD and subsequent precipitation of PlGoxB. A docking model of the putative PlGoxB-prePlGoxA complex was consistent with these hypotheses. The experimental results and computational analysis implicate structural features of PlGoxB that contribute to its stability and function.

Cellulose binding domain fusion enhanced soluble expression of fructosyl peptide oxidase and its simultaneous purification and immobilization.

Hemoglobin A1c (HbA1c) is a hemoglobin molecule in which the N-terminal valine residue of the ß subunit has been grafted with the glucose in blood. Its detection has important implications for the diagnosis of diabetes. Enzymatic colorimetric method using fructosyl peptide oxidase (FPO) is simple and rapid for HbA1c detection. A FPO mutant with enhanced activity was constructed and produced by E. coli; however, most of expressed mutant FPO was insoluble. Significantly enhanced expression solubility was achieved when cellulose-binding domain (CBD) from Clostridium thermocellum was fused to the N-terminal of FPO mutant. Via the high affinity interaction between CBD and cellulose, the CBD fusion also facilitated the simultaneous purification and immobilization of FPO directly from E. coli cells lysate using bacterial cellulose (BC) nanofibrils as a matrix of very high specific area. A never-dried and water durable nanocellulose film with FPO activity could be easily obtained by collecting the FPO immobilized BC nanofibrils suspension on the surface of a microfiltration membrane. The activity of the ready-use FPO nanocellulose film was stable at least 7 days at room temperature for the detection of HbA1c level of 5.3-11% in blood samples.

L-Lysine-α-Oxidase: Acidovorax citrulli Bacterium Inhibitor.

Studies of the effects of Trichoderma harzianum Rifai F-180 culture fluid concentrate containing L-lysine-α-oxidase antitumor enzyme produced by the fungus and the homogenous enzyme, on ultrahazardous bacterium Acidovorax citrulli demonstrated the antibacterial activity of the concentrate. Trichoderma harzianum Rifai F-180 producing L-lysine-α-oxidase was cultured in a technological device at G. K. Skryabin Institute of Biochemistry and. Physiology of Microorganisms, Russian Academy of Sciences. Activity of L-lysine-α-oxidase in the resulted culture fluid concentrate was 0.54 U/ml, activity of the homogenous enzyme was 50 U/mg.

Antibacterial Activity of L-Lysine-α-Oxidase from the Trichoderma.

We studied the effects of a concentrate of metabolites of Trichoderma harzianum Rifai F-180, an active producer of L-lysine-α-oxidase, and homogenous enzyme on a highly virulent bacteria Erwinia amylovora. The producer of antitumor and antiviral Trichoderma enzyme L-lysine-α-oxidase was cultured on a processing system of G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences (Pushchino). Activity of L-lysine-α-oxidase in the prepared concentrate of metabolic products of the producer was 5.4 U/ml, and activity of the homogenous enzyme was 50 U/ml. Antibacterial activity of the enzyme was shown in our experiments.

Escherichia coli-Based Expression and In Vitro Activity Assay of 1-Aminocyclopropane-1-Carboxylate (ACC) Synthase and ACC Oxidase.

1-Aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase are key enzymes in the ethylene biosynthetic pathway in plant tissues, and in vitro assay of their activities is indispensable for analysis, especially, for studying the action mechanism of inhibitors of ethylene biosynthesis. The enzymes can be obtained from plant tissues that are producing ethylene abundantly, such as ripening fruit- and senescing flower tissues, but it is necessary to separate the enzymes from co-extracted ACC by partial purification, making the procedure laborious and time-consuming. Here, we describe the production of the enzymes in Escherichia coli cells from corresponding cDNAs, and the procedures for assay of activities of the enzymes.

High-level expression of l-glutamate oxidase in Pichia pastoris using multi-copy expression strains and high cell density cultivation.

l-glutamate oxidase (GLOD), encoded by the gox gene, catalyses the transformation of l-glutamic acid into α-ketoglutaric acid (α-KG). In the present study, Pichia pastoris was used for heterologous production of GLOD following optimization of the gox coding sequence for expression in the yeast host. A series of constructs based on the pHBM905BDM plasmid were engineered and transformed into P. pastoris to increase the gox copy number. The results indicated that GLOD protein levels and enzyme activity increased with increasing gox copy number. Strain PGLOD4, which contained four copies of the target gene, was chosen for subsequent fermentation experiments, and a fermentation strategy involving two exponential feeding phases was developed. During the preinduction phase, glycerol was fed exponentially at µG = 0.15/h. When the cell density reached 300 g/l, methanol was fed exponentially at µM = 0.03/h to induce GLOD production. After 84 h of cultivation, the final cell density and total enzyme activity reached 420 g/L and 247.8 U/mL, respectively. The recombinant enzyme displayed an optimum temperature of 40 °C, which was higher than recombinant enzyme expressed in E. coli. This is important because increasing the temperature could accelerate enzymatic transformation of l-glutamic acid to α-KG. Experiments also demonstrated superior thermo-stability for the enzyme produced in yeast, which further enhances its potential for industrial applications.

Inhibition of Tobacco Ringspot Virus by the Culture Fluid of L-Lysine-α-Oxidase Producing Strain.

A producing strain of an anti-tumor and antiviral enzyme L-lysine-α-oxidase from Trichoderma was cultured using a technological device of G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences (Pushchino). L-lysine-α-oxidase activity in the obtained metabolite concentrate was 5.4 U/ml. We studied the effects of the concentrate of active L-lysine-α-oxidase producer on the highly infectious Tobacco ringspot virus and revealed anti-viral activity of it when enzyme concentration was at least 1.0 U/ml.

L-Arginine oxidase from Pseudomonas sp. TPU 7192: Characterization, gene cloning, heterologous expression, and application to L-arginine determination.

L-Arginine oxidase (AROD, EC 1.4.3.-) was discovered in newly discovered Pseudomonas sp. TPU 7192 and its characteristics were described. The molecular mass (MS) of the enzyme was estimated to be 528 kDa, which was accounted for by eight identical subunits with MS of 66 kDa each. AROD was identified as a flavin adenine dinucleotide (FAD)-dependent enzyme with 1 mol of FAD being contained in each subunit. It catalyzed the oxidative deamination of L-arginine and converted L-arginine to 2-ketoarginine, which was non-enzymatically converted into 4-guanidinobutyric acid when the hydrogen peroxide (H2O2) formed by L-arginine oxidation was not removed. In contrast, 2-ketoarginine was present when H2O2was decomposed. AROD was specific to L-arginine with a Km value of 149 µM. It exhibited maximal activity at 55 °C and pH 5.5. AROD was stable in the pH range 5.5-7.5 and >95% of its original activity was below 60 °C at pH 7.0. Since these enzymatic properties are considered suitable for the determination of L-arginine, the gene was cloned and expressed in a heterologous expression system. We herein successfully developed a new simple enzymatic method for the determination of L-arginine using Pseudomonas AROD.

[L-Lysine-α-Oxidase in vitro Activity in Experiments on Models of Viruses Sindbis, Forest-Spring Encephalitis, Western Nile, Tyaginya and Dhori].

The antitumor effect of L-lysine-α-oxidase from the culture fluid of Trichoderma harzianum Rifai F-180 was investigated for the first time. The in vitro studies revealed its high activity on a model of the forest-spring encephalitis virus and no activity against the Sindbis, Western Nile, Tyaginya and Dhori viruses.

Functional expression of L-lysine α-oxidase from Scomber japonicus in Escherichia coli for one-pot synthesis of L-pipecolic acid from DL-lysine.

L-Pipecolic acid is a key component of biologically active molecules and a pharmaceutically important chiral building block. It can be stereoselectively produced from L-lysine by a two-step bioconversion involving L-lysine α-oxidase and ∆(1)-piperideine-2-carboxylae (Pip2C) reductase. In this study, we focused on an L-lysine α-oxidase from Scomber japonicus that was originally identified as an apoptosis-inducing protein (AIP) and applied the enzyme to one-pot fermentation of L-pipecolic acid in Escherichia coli. A synthetic gene coding for an AIP was expressed in E. coli, and the recombinant enzyme was purified and characterized. The purified enzyme was determined to be a homodimer with a molecular mass of 133.9 kDa. The enzyme essentially exhibited the same substrate specificity as the native enzyme. Optimal temperature and pH for the enzymatic reaction were 70 °C and 7.4, respectively. The enzyme was stable below 60 °C and at a pH range of 5.5-7.5 but was markedly inhibited by Co(2+). To establish a one-pot fermentation system for the synthesis of optically pure L-pipecolic acid from DL-lysine, an E. coli strain carrying a plasmid encoding AIP, Pip2C reductase from Pseudomonas putida, lysine racemase from P. putida, and glucose dehydrogenase from Bacillus subtilis was constructed. The one-pot process produced 45.1 g/L of L-pipecolic acid (87.4 % yield from DL-lysine) after a 46-h reaction with high optical purity (>99.9 % enantiomeric excess).

Purification and Properties of Glycine Oxidase from Pseudomonas putida KT2440.

Glycine oxidase, encoded by the thiO gene, participates in the biosynthesis of thiamin by providing glyoxyl imine to form the thiazole moiety of thiamin. We have purified and characterized ThiO from Pseudomonas putida KT2440. It has a monomeric structure that is distinct from the homotetrameric ThiOs from Bacillus subtilis and Geobacillus kaustophilus. The P. putida ThiO is unique in that glycine is its preferred substrate, which differs markedly from the B. subtilis and G. kaustophilus enzymes that use D-proline as the preferred substrate.

[Antitumor enzyme L-lyzine-alpha-oxidase from Trichoderma harzianum Rifai F-180 and investigation of its action on L-lysine oxidation by capillar electrophoresis].

Trichoderma harzianum Rifai F-180, an organism producing the antitumor enzyme L-lysine-alpha-oxidase was cultivated and the enzyme was isolated and purified under the manufacturing conditions. The effect of L-lysine-alpha-oxidase on oxidation of L-lysine was investigated for the first time by capillary electrophoresis and the procedure conditions were developed. The reaction of L-lysine oxidative deamination is described and location of the reaction components picks on the elecrophoregrams was identified. The average rate of the catalytic reaction of L-lysine oxidation equal to 0.46 RU/min (7.7 x 10(-3) RU/sec) was determined. The use of the antitumor enzyme L-lysine-alpha-oxidase is recommended as a drug for the treatment of superficial tumors and tissue relative oxygen excess.

Glycine oxidase based high-throughput solid-phase assay for substrate profiling and directed evolution of (R)- and (S)-selective amine transaminases.

Transaminases represent one of the most important enzymes of the biocatalytic toolbox for chiral amine synthesis as they allow asymmetric synthesis with quantitative yields and high enantioselectivity. In order to enable substrate profiling of transaminases for acceptance of different amines, a glycine oxidase and horseradish peroxidase coupled assay was developed. Transaminase activity is detected upon transfer of an amine group from an amino donor substrate to glyoxylate, generating glycine, which is subsequently oxidized by glycine oxidase, releasing hydrogen peroxide in turn. Horseradish peroxidase uses the hydrogen peroxide to produce benzoquinone, which forms a red quinone imine dye by a subsequent condensation reaction. As glycine does not carry a chiral center, both (R)- and (S)-selective transaminases accepting glyoxylate as amino acceptor are amenable to screening. The principle has been transferred to establish a high-throughput solid-phase assay which dramatically decreases the screening effort in directed evolution of transaminases, as only active variants are selected for further analysis.

Characterization of a pyridoxal-5'-phosphate-dependent l-lysine decarboxylase/oxidase from Burkholderia sp. AIU 395.

A novel enzyme, which catalyzed decarboxylation of l-lysine into cadaverine with release of carbon dioxide and oxidative deamination of l-lysine into l-2-aminoadipic 5-semialdehyde with release of ammonia and hydrogen peroxide, was found from a newly isolated Burkholderia sp. AIU 395. The enzyme was specific to l-lysine and did not exhibit enzyme activities for other l-amino acids, l-lysine derivatives, d-amino acids, and amines. The apparent Km values for l-lysine in the oxidation and decarboxylation reactions were estimated to be 0.44 mM and 0.84 mM, respectively. The molecular mass was estimated to be 150 kDa, which was composed of two identical subunits with molecular mass of 76.5 kDa. The enzyme contained one mol of pyridoxal 5'-phosphate per subunit as a prosthetic group. The enzyme exhibiting decarboxylase and oxidase activities for l-lysine was first reported here, while the deduced amino acid sequence was homologous to that of putative lysine decarboxylases from the genus Burkholderia.

Characterization of a thermostable 2,4-diaminopentanoate dehydrogenase from Fervidobacterium nodosum Rt17-B1.

2,4-Diaminopentanoate dehydrogenase (2,4-DAPDH), which is involved in the oxidative ornithine degradation pathway, catalyzes the NAD(+)- or NADP(+)-dependent oxidative deamination of (2R,4S)-2,4-diaminopentanoate (2,4-DAP) to form 2-amino-4-oxopentanoate. A Fervidobacterium nodosum Rt17-B1 gene, Fnod_1646, which codes for a protein with sequence similarity to 2,4-DAPDH discovered in metagenomic DNA, was cloned and overexpressed in Escherichia coli, and the gene product was purified and characterized. The purified protein catalyzed the reduction of NAD(+) and NADP(+) in the presence of 2,4-DAP, indicating that the protein is a 2,4-DAPDH. The optimal pH and temperature were 9.5 and 85°C, respectively, and the half-denaturation time at 90°C was 38 min. Therefore, the 2,4-DAPDH from F. nodosum Rt17-B1 is an NAD(P)(+)-dependent thermophilic-alkaline amino acid dehydrogenase. This is the first thermophilic 2,4-DAPDH reported, and it is expected to be useful for structural and functional analyses of 2,4-DAPDH and for the enzymatic production of chiral amine compounds. Activity of 2,4-DAPDH from F. nodosum Rt17-B1 was suppressed by 2,4-DAP via uncompetitive substrate inhibition. In contrast, the enzyme showed typical Michaelis-Menten kinetics toward 2,5-diaminohexanoate. The enzyme was uncompetitively inhibited by d-ornithine with an apparent Ki value of 0.1 mM. These results suggest a regulatory role for this enzyme in the oxidative ornithine degradation pathway.

[Antimycoplasmic Activity of Fermentation Broth of Trichoderma harzianum Rifai F-180, an Organism Producing L-Lysine-α-Oxidase, an Antitumor and Antiviral Enzyme].

A concentrate of the fermentation broth of Trichoderma harzianum Rifai F-180, an organism producing L-lysine-α-oxidase, an antitumor and antiviral enzyme, with the activity in the fermentation broth of 0.54-0.56 U/mI was recovered. The effect of the concentrate on the mycoplasmas growth was investigated for the first time. Two representatives of Mycoplasmafaceae, i.e. Mycoplasma hominis and Mycoplasma fermentans and one representative of Aholeplasmataceae. i. e. Aholeplasma laidlawii were used. It was shown that the fermentation broth inhibited the growth of Mycoplasma hominis after the preliminary exposure. The inhibition rate depended on the mycoplasma inoculation dose and the fermentation broth concentration.

Identification in Marinomonas mediterranea of a novel quinoprotein with glycine oxidase activity.

A novel enzyme with lysine-epsilon oxidase activity was previously described in the marine bacterium Marinomonas mediterranea. This enzyme differs from other l-amino acid oxidases in not being a flavoprotein but containing a quinone cofactor. It is encoded by an operon with two genes lodA and lodB. The first one codes for the oxidase, while the second one encodes a protein required for the expression of the former. Genome sequencing of M. mediterranea has revealed that it contains two additional operons encoding proteins with sequence similarity to LodA. In this study, it is shown that the product of one of such genes, Marme_1655, encodes a protein with glycine oxidase activity. This activity shows important differences in terms of substrate range and sensitivity to inhibitors to other glycine oxidases previously described which are flavoproteins synthesized by Bacillus. The results presented in this study indicate that the products of the genes with different degrees of similarity to lodA detected in bacterial genomes could constitute a reservoir of different oxidases.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of EtFPOX from Eupenicillium terrenum sp.

The flavoenzyme fructosyl peptide oxidase (FPOX) catalyses the oxidative deglycation of fructosyl amino acids or fructosyl dipeptides to produce amino acids, glucosone and hydrogen peroxide. In this study, FPOX protein from Eupenicillium terrenum sp. (EtFPOX) was expressed in Escherichia coli and purified by Ni-affinity and gel-filtration chromatography. EtFPOX crystals were obtained using the sitting-drop vapour-diffusion method with polyethylene glycol 3350 as precipitant. X-ray diffraction data were collected to 1.90 Å resolution using a synchrotron-radiation source. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 65.6, b = 80.0, c = 83.4 Å, and contained one molecule in the asymmetric unit. The calculated Matthews coefficient and solvent content were 2.22 Å(3) Da(-1) and 44.62%, respectively.

A thermostable L-aspartate oxidase: a new tool for biotechnological applications.

L-Amino acid oxidases (LAAOs) are homodimeric flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the stereospecific oxidative deamination of L-amino acids to α-keto acids, ammonia, and hydrogen peroxide. Unlike the D-selective counterpart, the biotechnological application of LAAOs has not been thoroughly advanced because of the difficulties in their expression as recombinant protein in prokaryotic hosts. In this work, L-aspartate oxidase from the thermophilic archea Sulfolobus tokodaii (StLASPO, specific for L-aspartate and L-asparagine only) was efficiently produced as recombinant protein in E. coli in the active form as holoenzyme. This recombinant flavoenzyme shows the classical properties of FAD-containing oxidases. Indeed, StLASPO shows distinctive features that makes it attractive for biotechnological applications: high thermal stability (it is fully stable up to 80 °C) and high temperature optimum, stable activity in a broad range of pH (7.0-10.0), weak inhibition by the product oxaloacetate and by D-aspartate, and tight binding of the FAD cofactor. This latter property significantly distinguishes StLASPO from the E. coli counterpart. StLASPO represents an appropriate novel biocatalyst for the production of D-aspartate and a well-suited protein scaffold to evolve a LAAO activity by protein engineering.