L-lysine Α-oxidase Research Articles

糸状菌Trichoderma viride由来の抗腫瘍性酵素L-リシン α-オキシダーゼの組換え発現,分子特性及び結晶構造の解析(研究論文紹介)

糸状菌Trichoderma viride由来の抗腫瘍性酵素L-リシン α-オキシダーゼの組換え発現,分子特性及び結晶構造の解析(研究論文紹介)

Kinetic characteristics of L-lysine α- oxidase from Trichoderma cf. aureoviride Rifai VKM F-4268D: Substrate specificity and allosteric effects

The present work aims to investigate the kinetic characteristics of homodimer enzyme L-lysine α-oxidase from Trichoderma cf. aureoviride Rifai VKM F-4268D, taking into account allosteric effects. The enzyme was first shown to reveal positive cooperativeness, h=2.05±0.15. Using additional opportunities of Hill coefficient the value of the Michaelis–Menten constant has been estimated, Km=1.015∙10−5М, indicating high strength of substrate binding to the active site of each subunit. High selectivity and absolute L-stereospecificity of the enzyme were shown. The inhibition of L-lysine conversion by non-cleavable lysine analogs as well as the reaction product was found out to take place. These effects have been evaluated only as the inhibition coefficients (%). A more detailed study of these inhibition effects was complicated because of the cooperativeness of enzyme subunits mentioned above. The kinetic scheme of L-lysine α-oxidase was proposed involving parallel-subsequent action of each of two subunits in the catalytic act.We think that the results obtained will be useful for studying the kinetic properties of other multi-subunit enzymes and improve understanding of the mechanisms of their action.

Recombinant expression, molecular characterization and crystal structure of antitumor enzyme, L-lysine α-oxidase from Trichoderma viride.

L-Lysine α-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L-lysine to form α-keto-ε-aminocaproate. LysOX severely inhibited growth of cancer cells but showed relatively low cytotoxicity for normal cells. We have determined the cDNA nucleotide sequence encoding LysOX from T. viride. The full-length cDNA consists of 2,119 bp and encodes a possible signal peptide (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene have been cloned and heterologously expressed in Streptomyces lividans TK24 with the enzyme activity up to 9.8 U/ml. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity and thermal stability, are same as those of native LysOX. The crystal structure of LysOX at 1.9 Å resolution revealed that the overall structure is similar to that of snake venom L-amino acid oxidase (LAAO), and the residues involved in the interaction with the amino or carboxy group of the substrate are structurally conserved. However, the entrance and the inner surface structures of the funnel to the active site, as well as the residues involved in the substrate side-chain recognition, are distinct from LAAOs. These structural differences well explain the unique substrate specificity of LysOX.

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).

Heterologous expression of l-lysine α-oxidase from Scomber japonicus in Pichia pastoris and functional characterization of the recombinant enzyme.

Fish have a complex self-defense mechanism against microbial invasion. Recently, l-lysine α-oxidases have been identified from a number of fish species as a novel type of antibacterial protein in the integument. These enzymes exhibit strict substrate specificity for l-lysine, but the underlying mechanisms and details of their catalytic properties remain unknown. In this study, a synthetic gene coding for Scomber japonicus l-lysine α-oxidase, originally termed AIP (for apoptosis-inducing protein), was expressed in Pichia pastoris, and the recombinant enzyme (rAIP) was purified and characterized. rAIP exhibited essentially the same substrate specificity as the native enzyme, catalyzing the oxidative deamination of l-lysine as an exclusive substrate. rAIP was N-glycosylated and remained active over a wide range of pH, with an optimal pH of 7.5. The enzyme was stable in the pH range from 4.5 to 10.0 and was thermally stable up to 60°C. A molecular modelling of rAIP and a comparative structure/sequence analysis with homologous enzymes indicate that Asp(220) and Asp(320) are the substrate-binding residues that are likely to confer exclusive substrate specificity for l-lysine on the fish enzymes.

Enzymatic properties and anticancer activity of L-lysine α-oxidase from Trichoderma cf. aureoviride Rifai BKMF-4268D

L-Lysine α-oxidase (LO) from a novel Trichoderma strain: Trichoderma cf. aureoviride Rifai shows favorable biochemical and kinetic properties (Km for L-lysine of 17.9 µmol/l, optimum pH 8.0, high stability) and significant antiproliferative activity both in vitro and in vivo. The molecular weight of LO was determined to be 115-116 kDa; the active dimer consists of two identical 57-58 kDa subunits. LO shows considerable cytotoxicity against the following tumor cell lines: K562, LS174T, HT29, SCOV3, PC3, and MCF7, with the inhibition concentration (IC50) ranging from 3.0×10 to 7.8×10 U/ml (3.2×10 to 8.2×10 mg/ml). Two human colon cancer xenografts HCT116 and LS174T and breast adenocarcinoma T47D implanted subcutaneously into Balb/c nude mice showed high sensitivity to LO with a T/C of 12, 37, and 36%, respectively (P<0.05). The antitumor efficacy of LO was observed in the absence of pronounced morbidity or toxicity in vivo. Taken together, these data suggest that LO may be considered as an effective anticancer agent for the treatment of solid tumors in vivo. This study presents promising data on the possible application of LO in clinical oncology for patients with colorectal cancer.

Production of extracellular H2O2 and L-lysine-α-oxidase during bulk growth of the fungus Trichoderma cf. aureoviride Rifai VKM F-4268D under salt stress

In this article, we report on Some physiological aspects of the synthesis of extracellular L-lysine-α-oxidase (LO) by the fungus Trichoderma cf. aureoviride Rifai VKM F-4268D under salt stress conditions and discuss the possible role of this enzyme for the producer. It has been shown that The synthesis of extracellular LO and proteolytic enzymes is induced in the fungus T. cf. aureoviride Rifai VKM F-4268D during submerged cultivation on wheat bran under salt stress. It has been shown that LO biosynthesis is accompanied by H2O2 accumulation in the growth medium. It seems that the extracellular LO synthesis followed by hydrogen peroxide production under stress conditions provides an adaptive advantage for the producer fungus in its competition with other organisms.

Isolation and properties of L-lysine-α-oxidase from the fungus Trichoderma cf. aureoviride RIFAI VKM F-4268D

L-lysine-α-oxidase (LO) synthesized by the fungus Trichoderma cf. aureoviride Rifai VKM F-4268D under salt stress conditions was isolated and characterized. The newly developed method for the isolation and purification of the enzyme was based on its precipitation from the culture liquid by copper sulfate. The subsequent LO purification by the methods of hydrophobic (Octyl Sepharose) and ion exchange (DEAE ToyoPearl) chromatography yielded a homogeneous enzyme preparation with a high degree of purification (310-fold) and high specific activity (90 U/mg protein). The molecular mass of the enzyme determined by gel filtration and native electrophoresis was 115–116 kDa. According to the data of SDS electrophoresis, LO was a dimer with identical subunits (57–58 kDa). The optical absorption spectrum of LO corresponded to the flavoprotein spectrum with maximums at 278, 390, and 465 (a shoulder at 490) nm. LO is a stereospecific enzyme oxidizing almost exclusively L-lysine (pH optimum 7.8–8.2). Insignificant activity was observed against L-ornithine and L-arginine. LO was shown to be stable at temperatures up to 50°C.

2PT109 L-リシン酸化酵素のX線結晶構造解析(日本生物物理学会第50回年会(2012年度))

2PT109 L-リシン酸化酵素のX線結晶構造解析(日本生物物理学会第50回年会(2012年度))

An efficient enzymatic synthesis of 5-aminovaleric acid

The title compound was prepared enzymatically from l-lysine in an excellent yield and under buffer-free conditions. l-Lysine was oxidized by the action of l-lysine α-oxidase from Trichoderma viride followed by spontaneous oxidative decarboxylation of the intermediate 6-amino-2-oxocaproic acid in the reaction medium. l-Lysine α-oxidase was immobilized on an epoxy-activated solid support (Sepabeads EC-EP) and the activity of both solution-based and immobilized enzyme in this reaction was determined.

Immobilization of l-lysine α-oxidase on gold-mercaptopropionic acid self-assembled monolayer: Preparation and electrochemical characterization

Immobilization of l-lysine α-oxidase on gold-mercaptopropionic acid self-assembled monolayer (Au-MPA-LOx SAM) electrode is verified experimentally in the present work. Fabrication steps and electrochemical interaction of Au-MPA-LOx with l-lysine were monitored by general electrochemical methods like cyclic voltammetry (CV) and chronoamperometry (CA), and by a more advanced method, electrochemical impedance spectroscopy (EIS) in the presence of parabenzoquinone (PBQ) redox probe. The data was analyzed from which quantitative kinetic parameters were extracted. The results confirmed successful immobilization of LOx, and thus, fabrication of Au-MPA-LOx SAM electrode. Our initial tests revealed a linear response for Au-MPA-LOx SAM electrode toward l-lysine concentration in solution at biological conditions, pH 7.4. The experimental data will be presented and discussed from which the Au-MPA-LOx SAM electrode is characterized, and the kinetic merits of the interface interactions are introduced.

Partially disposable biosensors for the quick assessment of damage in foodstuff after thermal treatment

A novel approach towards a quick, easy and reliable solution to the quite complex problems connected with the Amadori's compounds and furosine formation in several foodstuff is here proposed and based on enzymatic reactions, either as far as L-lysine release after partial hydrolysis from proteic material of the food matrix (bioavailable L-lysine), or as far as the determination of total and free L-lysine is concerned. The contribution proposes the use of specific L-lysine sensitive bioelectrodes for the evaluation of the possible heat damage through the rapid measure of L-lysine consumption. The L-lysine biosensor has been realized by immobilizing the enzyme L-lysine-α-oxidase (LOx) on an amperometric H 2O 2 sensitive electrode. The bioelectrode-enzymatic reaction arrangement allows to perform determinations of L-lysine and bioavailable L-lysine in foodstuff from different sources and after different thermal treatments in a very rapid inexpensive and reliable way in the range 1.0–33 ppm, with a limit of detection LOD = 0.5 ppm, so that a considerable number of samples can be easily monitored quickly and with very low costs.

Flow Injection Analysis System for l-Lysine Estimation in Foodstuffs Using a Biosensor Based on Lysine Oxidase Immobilization on a Gold-Poly(m-Phenylenediamine) Electrode

The development and the evaluation of an amperometric FIA biosensing system for l-lysine determination in foodstuffs is described. The system employs a home-made flow-cell with an amperometric biosensor, prepared by immobilization of lysine oxidase on a gold-poly(m-phenylenediamine) electrode. A peristaltic pump is connected to the flow-cell via a 2-position 6-port sample injector/switching valve for the on-line transfer of the samples. The analytical procedure is based on the oxidation of l-lysine by l-lysine α-oxidase. The produced H2O2 is monitored amperometrically at 650 mV in FIA set-up. The lysine biosensor construction procedure comprises: a) the electropolymerization of m-phenylenediamine on a Si-gold strip electrode and b) the immobilization of l-lysine α-oxidase on the poly(m-phenylenediamine) membrane with glutaraldehyde in the presence of bovine serum albumin. Parameters, such as, flow rate, enzyme amount, monomer concentration, matrix effect, effect of interference's, etc., were studied. The flow-cell biosensor system provides a linear response from 1 × 10−3 to 5 × 10−5 M of lysine, with excellent characteristics of reproducibility, stability, and life-time. The system was further applied and evaluated for the estimation of lysine in various hydrolysate food samples (e.g., pasta, milk, wheat flour, etc.) without any pretreatment with an average recovery of 97.3%.

Spectrophotometric Assays for l-Lysine α-Oxidase and γ-Glutamylamine Cyclotransferase

A new assay for l-lysine α-oxidase is described. In this assay, the oxidized product generated from l-lysine is reacted with semicarbazide to form α-keto-ϵ-aminocaproate semicarbazone. Formation of the α-keto acid semicarbazone is continuously monitored spectrophotometrically at 248 nm (ϵ 10,160 ± 240 M−1 cm−1). The method was adapted to provide a new assay for γ-glutamylamine cyclotransferase. This enzyme catalyzes the conversion of many l-γ-glutamylamines to 5-oxo-l-proline and free amine. A biologically important substrate is Nϵ-(γ-l-glutamyl)-l-lysine, which is converted to 5-oxo-l-proline and l-lysine by the action of γ-glutamylamine cyclotransferase. The l-lysine generated from Nϵ-(γ-l-glutamyl)-l-lysine in an endpoint assay is converted to α-keto ϵ-aminocaproate semicarbazone in the presence of semicarbazide, excess l-lysine α-oxidase, and catalase. The methods were applied to the determination of γ-glutamylamine cyclotransferase activity of partially purified preparations of the bovine kidney enzyme and to detect γ-glutamylamine cyclotransferase activity in rat kidney and liver homogenates.

Development of an interferent free amperometric biosensor for determination of l-lysine in food

A highly selective, fast responding amperometric biosensor is described, useful for the determination of l-lysine in food. Common electrochemical interferences, like acetoaminophen and ascorbic acid have zero response at +100 mV applied onto a ruthenium/rhodium coated glassy carbon electrode covered with 1,2-diaminobenzene polymer. This novel transducer was coupled with l-lysine α-oxidase purified from Trichoderma viride and at the appropriate pH, classic substrate interferences from l-ornithine, l-arginine and l-phenylalanine are reduced to 3.4, 1.1 and 0.7% of the response to l-lysine (taken as 100%). No other amino acids respond. The sensor is inexpensive to produce, has excellent repeatability and very good reproducibility. Thus, the l-lysine (protein) content of foods can be almost specifically determined following rapid microwave digestion of the product.

Anticancer Enzyme L-Lysine α-Oxidase: Properties and Application Perspectives

Fungal L-lysine α-oxidase (1.4.3.14) (LO) from Trichoderma harzianum Rifai presents an oxidoreductase with a firmly attached coenzyme-FAD. This stable enzyme catalyzes an oxidative deamination of L-lysine yielding hydrogen peroxide, ammonia, and α-keto acid. LO exhibits antitumor activity toward 5 of 12 tested transplantable tumors. The sensitive tumors were ascitic hepatoma 22 (T/C = 201%, CR = 66%); mammary adenocarcinoma Ca-755 (TGI = 96%); melanoma B-16 (TGI = 81%); AKATOL (TGI = 75%); RSHM 5 (TGI = 79%). LO therapeutic activity was observed within a wide range of doses, 35-350 U/kg, by intraperitoneal daily injections for 5 d. Contrary to Escherichia coli L-asparaginase, LO demonstrates its antitumor activity by the low therapeutic doses in vivo within a wide range of optimal doses and through another antitumor spectrum. Fisher lymphadenosis L-5178y highly sensitive toward L-asparaginase appeared to be LO resistant. The possible mechanisms of LO antitumor activity through the key biochemical processes are discussed.

Isoforms of L-lysine α-oxidase fromTrichoderma sp.

Several purification methods were tested and the optimal procedure for obtaining L-lysine α-oxidase from fungiTrichoderma sp. and its isoform (minor L-lysine α-oxidase) was worked out. The enzyme and its isoform were obtained in a homogeneous state, the most important physicochemical properties were studied, and a number of differences between them were found. The most marked differences between L-lysine α-oxidase and its isoform were observed in the molecular weight (120 and 100 kD, respectively), in the isoelectric point (pI 4.4 and 5.6, respectively), and in the specific activity (90–95 and 17–20 U/mg) in experiments where L-lysine where used as substrate.

L‐Lysine α‐oxidase from Trichoderma viride i4. Purification and characterization

AbstractA L‐lysine α‐oxidase (LOD) has been purified to homogeneity in a two‐step procedure with 300‐fold enrichment and 60% recovery from the culture extract of Trichoderma viride i4. The enzyme catalyzes the reaction between L‐lysine and molecular oxygen forming 2‐oxo‐6‐aminocaproate, ammonia and hydrogen peroxide. Numerous substrates have been tested. The Km value for L‐lysine was found to be 0.026 mM. Its apparent molecular mass is 110000 Da when determined by gel filtration on Sephadex G‐200, the estimated molecular weight of the subunits being 55000 Da in the SDS‐PAGE. The enzyme is a glycoprotein and shows absorption maxima at 276, 386 and 463 nm. It was found to contain 1 mol of FAD per subunit. The coenzyme is bound non‐covalently. Its isoelectric point is at pH 4.3. The enzyme is stable at extreme pH values, at relatively high temperatures and in diluted hydrogen peroxide.The enzyme described here differs from two other known L‐lysine oxidases previously characterized with regard to its amino acid composition and its substrate specificity.

The use of an enzyme single fiber reactor in the study of leukemic cell proliferation: In vitro experiments and computer simulation

This paper describes the use of an immobilized enzyme reactor in the study of the in vitro effects of lysine deprivation on leukemic blood. l-lysine α-oxidase is immobilized in a single hollow fiber reactor to remove lysine from the blood of sheep infected by BLV. The treatment relies on the higher sensitivity of leukemic cells to nutrient depletion than that of normal cells. A population balance model is used to describe the changes in the leukocyte proliferative capacity after treatment. Additionally, preliminary data from in vitro tests with human blood demonstrate the potential of l-lysine α-oxidase and the enzymatic reactor in treating leukemia.

Evaluation of intrinsic immobilized kinetics in hollow fiber reactor systems

Immobilized cell and enzyme hollow fiber reactors have been developed for a variety of biochemical and biomedical applications. Reported mathematical models for predicting substrate conversion in these reactors have been limited in accuracy because of the use of free-solution kinetic parameters. This paper describes a method for determining the intrinsic kinetics of enzymes immobilized in hollow fiber reactor systems using a mathematical model for diffusion and reaction in porous media and an optimization procedure to fit intrinsic kinetic parameters to experimental data. Two enzymes, a thermophilic β-galactosidase that exhibits product inhibition and L-lysine α-oxidase, were used in the analysis. The intrinsic kinetic parameters show that immobilization enhanced the activity of the β-galactosidase while decreasing the activity of L-lysine α-oxidase. Both immobilized enzymes had higher K m values than did the soluble enzyme, indicating less affinity for the substrate. These results are used to illustrate the significant improvement in the ability to predict substrate conversion in hollow fiber reactors.