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.

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.

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.

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.

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.

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).

[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.

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.

[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.

[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.

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.

Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to interact with the L-protein, which is also part of the l-ketoglutarate dehydrogenase complex present in fibroblasts.

Streamlined protein expression and purification using cleavable self-aggregating tags.

BACKGROUND: Recombinant protein expression and purification remains a fundamental issue for biotechnology. Recently we found that two short self-assembling amphipathic peptides 18A (EWLKAFYEKVLEKLKELF) and ELK16 (LELELKLKLELELKLK) can induce the formation of active protein aggregates in Escherichia coli (E. coli), in which the target proteins retain high enzymatic activities. Here we further explore this finding to develop a novel, facile, matrix-free protein expression and purification approach. RESULTS: In this paper, we describe a streamlined protein expression and purification approach by using cleavable self-aggregating tags comprising of one amphipathic peptide (18A or ELK16) and an intein molecule. In such a scheme, a target protein is first expressed as active protein aggregate, separated by simple centrifugation, and then released into solution by intein-mediated cleavage. Three target proteins including lipase A, amadoriase II and ß-xylosidase were used to demonstrate the feasibility of this approach. All the target proteins released after cleavage were highly active and pure (over 90% in the case of intein-ELK16 fusions). The yields were in the range of 1.6-10.4 µg/mg wet cell pellet at small laboratory scale, which is comparable with the typical yields from the classical his-tag purification, the IMPACT-CN system (New England Biolabs, Beverly, MA), and the ELP tag purification scheme. CONCLUSIONS: This tested single step purification is capable of producing proteins with high quantity and purity. It can greatly reduce the cost and time, and thus provides application potentials for both industrial scale up and laboratorial usage.

A novel L-aspartate dehydrogenase from the mesophilic bacterium Pseudomonas aeruginosa PAO1: molecular characterization and application for L-aspartate production.

L-aspartate dehydrogenase (EC 1.4.1.21; L: -AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative L-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very high specific activity for L-aspartate (L-Asp) and oxaloacetate (OAA) of 127 and 147 U mg(-1), respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T (m) value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent K (m) values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH. The L-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of L-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative production system C released 33 mM of L-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic AspDH and its potential applicability for efficient and attractive production of L-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production.

A non-NadB type L-aspartate dehydrogenase from Ralstonia eutropha strain JMP134: molecular characterization and physiological functions.

We report the molecular characterization and physiological function of a novel L-aspartate dehydrogenase (AspDH). The purified enzyme was a 28-kDa dimeric protein, exhibiting high catalytic activity for L-aspartate (L-Asp) oxidation using NAD and/or NADP as cofactors. Quantitative real-time PCR analysis indicated that the genes involved in the AspDH gene cluster, poly-3-hydroxyalkanoate (PHA) biosynthesis, and the TCA cycle were substantially induced by L-Asp in wild-type cells. In contrast, expression of the aspartase and aspartate aminotransferase genes was substantially induced in the AspDH gene knockout mutant (ΔB3576) but not in the wild type. GC-MS analyses revealed that the wild-type strain synthesized poly-3-hydroxybutyrate from fructose or L-Asp, whereas the ΔB3576 mutant did not synthesize PHA from L-Asp. AspDH gene cluster products might be involved in the biosynthesis of the PHA precursor, revealing that AspDH was a non-NadB type enzyme, and thus entirely different from the previously reported NadB type enzymes working in NAD biosynthesis.

[Investigation of antitumor substance from Trichoderma].

The effect of L-lysine-alpha-oxidase from Trichoderma harzianum Rifai on the functional activity of T-lymphocytes was investigated. It was shown that in a dose of 35 units/kg administered parentally the enzyme had no suppressive effect on the T-lymphocyte functional activity. An inhibitory effect of L-lysine-a-oxidase on some indices of the macrophages functional activity was observed. L-Lyzine-alpha-oxidase had a selective lymphotropic action and showed no mytostatic activity, which is in favour of the enzyme vs. other antitumor agents.

Steady-state kinetic mechanism and reductive half-reaction of D-arginine dehydrogenase from Pseudomonas aeruginosa.

D-arginine dehydrogenase from Pseudomonas aeruginosa catalyzes the oxidation of D-arginine to iminoarginine, which is hydrolyzed in solution to ketoarginine and ammonia. In the present study, we have genetically engineered an untagged form of the enzyme that was purified to high levels and characterized in its kinetic properties. The enzyme is a true dehydrogenase that does not react with molecular oxygen. Steady-state kinetic studies with D-arginine or D-histidine as substrate and PMS as the electron acceptor established a ping-pong bi-bi kinetic mechanism. With the fast substrate D-arginine a dead-end complex of the reduced enzyme and the substrate occurs at high concentrations of D-arginine yielding substrate inhibition, while the overall turnover is partially limited by the release of the iminoarginine product. With the slow substrate D-histidine the initial Michaelis complex undergoes an isomerization involving multiple conformations that are not all equally catalytically competent for the subsequent oxidation reaction, while the overall turnover is at least partially limited by flavin reduction. The kinetic data are interpreted in view of the high-resolution crystal structures of the iminoarginine--and iminohistidine--enzyme complexes.

Determination of plasma and serum L-lysine using L-lysine epsilon-oxidase from Marinomonas mediterranea NBRC 103028(T).

This article describes a successful application of l-lysine epsilon-oxidase (EC 1.4.3.20) for l-lysine determination. l-Lysine epsilon-oxidase was isolated from culture supernatant of Marinomonas mediterranea NBRC 103028(T) and was used for l-lysine determination. Comparison of the characteristics of l-lysine epsilon-oxidase with l-lysine alpha-oxidase, a commercial enzyme used for l-lysine determination, suggests that the use of l-lysine epsilon-oxidase would be more valuable for the determination of l-lysine because of its selectivity and sensitivity, especially in samples with low l-lysine concentration. The enzyme acted only on l-lysine and l-ornithine, to which the relative activity was only 3.4% of that on l-lysine. The value obtained by the colorimetric assay using l-lysine epsilon-oxidase and horseradish peroxidase was not affected by l-ornithine. The enzyme also shows a higher affinity for l-lysine (K(m)=0.0018mM). l-Lysine determination using l-lysine epsilon-oxidase in human plasma and serum was examined. The measured values were close to values determined by instrumental analyses using the precolumn AccQ.Tag Ultra Derivatization Kit. These results suggest that l-lysine epsilon-oxidase can be used for diagnosis based on plasma l-lysine concentration. This is the first report on the application of l-lysine epsilon-oxidase.