Nanozyme can substitute a natural Ogataea polymorpha catalase enzyme in vivo.

Nanomaterials possessing artificial, enzyme-like catalytic activity (nanozymes, NZs) have a great potential for application in research, immunological assays, biosensors, in vivo imaging, and as therapeutic agents. Despite the obvious advances in construction and understanding of functional properties of NZs, there is still no clear evidence of whether they can complement the loss of corresponding enzymatic activity in vivo. Herein, we report the first, to the best to our knowledge, example of successful substitution of natural enzyme activity by catalase-like platinum (nPt) and platinum-gold (nPtAu) nanoparticles transferred to the cells of methylotrophic yeast Ogataea polymorpha. The nPt NZs were synthesized by the chemical reduction method and used as a seed to produce the nPt(core)Au(shell) particles. The produced nPt NZs were 68.1 and 91.3 nm in size, while the hydrids were of 531.2 and 615.1 nm. Both nPt and nPtAu demonstrated catalase activity in vitro. The catalase-deficient strain Ogataea polymorpha C-105 was shown to be able to grow on methanol and a mixture of glucose and methanol in the presence although not in the absence of NZs, this correlating with the decrease in intracellular hydrogen peroxide production. The results provide the first example of complementation of the natural enzyme function by synthetic NZs, the phenomenon which can further be used in a screening for new catalase-like nanozymes and as a fruitful tool to modify living cells by nanoparticles possessing catalytic activity and to use such modified cells as sensitive elements in cell-based biosensors.

Ammonium nanochelators in conjunction with arginine-specific enzymes in amperometric biosensors for arginine assay.

Amino acid L-arginine (Arg), usually presented in food products and biological liquids, can serve both as a useful indicator of food quality and an important biomarker in medicine. The biosensors based on Arg-selective enzymes are the most promising devices for Arg assay. In this research, three types of amperometric biosensors have been fabricated. They exploit arginine oxidase (ArgO), recombinant arginase I (ARG)/urease, and arginine deiminase (ADI) coupled with the ammonium-chelating redox-active nanoparticles. Cadmium-copper nanoparticles (nCdCu) as the most effective nanochelators were used for the development of ammonium chemosensors and enzyme-coupled Arg biosensors. The fabricated enzyme/nCdCu-containing bioelectrodes show wide linear ranges (up to 200 µM), satisfactory storage stabilities (14 days), and high sensitivities (A⋅M-1⋅m-2) to Arg: 1650, 1700, and 4500 for ADI-, ArgO- and ARG/urease-based sensors, respectively. All biosensors have been exploited to estimate Arg content in commercial juices. The obtained data correlate well with the values obtained by the reference method. A hypothetic scheme for mechanism of action of ammonium nanochelators in electron transfer reaction on the arginine-sensing electrodes has been proposed.

Amperometric biosensors based on alcohol oxidase and peroxidase-like nanozymes for ethanol determination.

The aim of the current research is to design alcohol oxidase-based amperometric biosensors (ABSs) using hybrid metallic nanoparticles as artificial peroxidases (PO) or PO-like nanozymes (NZs). A lot of metallic PO-like NZs were synthesized and tested with respect to their ability to substitute natural PO in solution and on amperometric electrode. The most effective PO mimetics were coupled with alcohol oxidase (AOX) on graphite electrodes (GEs) and characterized. Two types of modified GEs, namely, the AOX/nAuCePt/GE and the AOX/nFePtAu/GE show the highest sensitivities to ethanol (2600 A⋅M-1⋅m-2 and 1250 A⋅M-1⋅m-2, respectively), low limits of detection (1.5 µM and 2.2 µM), broad linear ranges (5 - 100 µM and 12 - 120 µM), as well as satisfactory storage stabilities. The most sensitive bioelectrode AOX/nAuCePt/GE was used as ABS for ethanol determination in real samples. The practical feasibility of the constructed ABS was demonstrated by determination of ethanol in beverages, human blood and saliva.

Extracellular laccase from Monilinia fructicola: isolation, primary characterization and application.

Laccases are enzymes belonging to the family of blue copper oxidases. Due to their broad substrate specificity, they are widely used in many industrial processes and environmental bioremediations for removal of a large number of pollutants. During last decades, laccases attracted scientific interest also as highly promising enzymes to be used in bioanalytics. The aim of this study is to obtain a highly purified laccase from an efficient fungal producer and to demonstrate the applicability of this enzyme for analytics and bioremediation. To select the best microbial source of laccase, a screening of fungal strains was carried out and the fungus Monilinia fructicola was chosen as a producer of an extracellular enzyme. Optimal cultivation conditions for the highest yield of laccase were established; the enzyme was purified by a column chromatography and partially characterized. Molecular mass of the laccase subunit was determined to be near 35 kDa; the optimal pH ranges for the highest activity and stability are 4.5-5.0 and 3.0-5.0, respectively; the optimal temperature for laccase activity is 30°C. Laccase preparation was successfully used as a biocatalyst in the amperometric biosensor for bisphenol A assay and in the bioreactor for bioremediation of some xenobiotics.

Overexpression and one-step renaturation-purification of the tagged creatinine deiminase of Corynebacterium glutamicum in Escherichia coli cells.

The codA gene of Corynebacterium glutamicum PCM 1945 coding for a creatinine deiminase (CDI) (EC 3.5.4.21) has been amplified and cloned. The recombinant strain of Escherichia coli that overproduces the (His)6 -tagged inactive CDI of C. glutamicum as inclusion bodies has been constructed. After solubilization of inclusion bodies in the presence of 0.3% N-lauroylsarcosine, the enzyme was renaturated and purified by a single-step procedure using metal-affinity chromatography. The yield of the (His)6 -tagged CDI is ~30 mg from 1 L culture. The purified enzyme is sufficiently stable under the conditions designed and possesses an activity of 10-20 U/mg. The main characteristics of the tagged enzyme remained similar to that of the natural enzyme.

Synthesis, Catalytic Properties and Application in Biosensorics of Nanozymes and Electronanocatalysts: A Review.

The current review is devoted to nanozymes, i.e., nanostructured artificial enzymes which mimic the catalytic properties of natural enzymes. Use of the term "nanozyme" in the literature as indicating an enzyme is not always justified. For example, it is used inappropriately for nanomaterials bound with electrodes that possess catalytic activity only when applying an electric potential. If the enzyme-like activity of such a material is not proven in solution (without applying the potential), such a catalyst should be named an "electronanocatalyst", not a nanozyme. This paper presents a review of the classification of the nanozymes, their advantages vs. natural enzymes, and potential practical applications. Special attention is paid to nanozyme synthesis methods (hydrothermal and solvothermal, chemical reduction, sol-gel method, co-precipitation, polymerization/polycondensation, electrochemical deposition). The catalytic performance of nanozymes is characterized, a critical point of view on catalytic parameters of nanozymes described in scientific papers is presented and typical mistakes are analyzed. The central part of the review relates to characterization of nanozymes which mimic natural enzymes with analytical importance ("nanoperoxidase", "nanooxidases", "nanolaccase") and their use in the construction of electro-chemical (bio)sensors ("nanosensors").

D-lactate-selective amperometric biosensor based on the mitochondrial fraction of Ogataea polymorpha recombinant cells.

During the recent decades, a lot of data about the significance of D-lactate determination in food technology and quality control have been accumulated. Nowadays, the development of new methods for the determination of D-lactate is very relevant, especially with regard to biosensors. To construct a D-lactate-selective biosensor, we suggest using the mitochondria of recombinant yeast cells of Ogataea (Hansenula) polymorpha "tr6" (gcr1 catX/Δcyb2, prAOX_DLDH) overproducing D-lactate: cytochrome c-oxidoreductase (DLDH, EC 1.1.2.4) and lacking an L-lactate-specific enzyme (flavocytochrome b2 , E.C. 1.1.2.3). The usage of the pure enzyme is problematic due to the complexity of its isolation and stabilization because of the intramembranous localization of DLDH. The enzyme catalyzes D-lactate oxidation to pyruvate coupled with ferricytochrome c reduction to ferrocytochrome c. The constructed biosensor is characterized by high sensitivity (18.5 Ð·Ðœ-1 ·m-2 ), a low detection limit (3 µM of D-lactate), wide linear ranges, good selectivity, and sufficient stability. The real samples' analysis of D-lactate in dairy products was performed, and high correlation of the obtained results with the reference approach (0.7 < r < 1) and literature data was demonstrated.

Recombinant arginine-degrading enzymes in metabolic anticancer therapy and bioanalytics.

Tumor cells often exhibit specific metabolic defects due to the aberrations in oncogene-dependent regulatory and/or signaling pathways that distinguish them from normal cells. Among others, many malignant cells are deficient in biosynthesis of certain amino acids and concomitantly exhibit elevated sensitivity to deprivation of these amino acids. Although the underlying causes of such metabolic changes are still not fully understood, this feature of malignant cells is exploited in metabolic enzymotherapies based on single amino acid, e.g., arginine, deprivation. To achieve efficient arginine depletion in vivo, two recombinant enzymes, bacterial arginine deiminase and human arginase I have been evaluated and are undergoing further development. This review is aimed to summarize the current knowledge on the application of arginine-degrading enzymes as anticancer agents and as bioanalytical tools for arginine assays. The problems that have to be solved to optimize this therapy for clinical application are discussed.

Killing multiple myeloma cells with the small molecule 3-bromopyruvate: implications for therapy.

The small molecule 3-bromopyruvate (3-BP), which has emerged recently as the first member of a new class of potent anticancer agents, was tested for its capacity to kill multiple myeloma (MM) cancer cells. Human MM cells (RPMI 8226) begin to lose viability significantly within 8 h of incubation in the presence of 3-BP. The Km (0.3 mmol/l) for intracellular accumulation of 3-BP in MM cells is 24 times lower than that in control cells (7.2 mmol/l). Therefore, the uptake of 3-BP by MM cells is significantly higher than that by peripheral blood mononuclear cells. Further, the IC50 values for human MM cells and control peripheral blood mononuclear cells are 24 and 58 µmol/l, respectively. Therefore, specificity and selectivity of 3-BP toward MM cancer cells are evident on the basis of the above. In MM cells the transcription levels of the gene encoding the monocarboxylate transporter MCT1 is significantly amplified compared with control cells. The level of intracellular ATP in MM cells decreases by over 90% within 1 h after addition of 100 µmol/l 3-BP. The cytotoxicity of 3-BP, exemplified by a marked decrease in viability of MM cells, is potentiated by the inhibitor of glutathione synthesis buthionine sulfoximine. In addition, the lack of mutagenicity and its superior capacity relative to Glivec to kill MM cancer cells are presented in this study.

Bioconversion of airborne methylamine by immobilized recombinant amine oxidase from the thermotolerant yeast Hansenula polymorpha.

Aliphatic amines, including methylamine, are air-pollutants, due to their intensive use in industry and the natural degradation of proteins, amino acids, and other nitrogen-containing compounds in biological samples. It is necessary to develop systems for removal of methylamine from the air, since airborne methylamine has a negative effect on human health. The primary amine oxidase (primary amine : oxygen oxidoreductase (deaminating) or amine oxidase, AMO; EC 1.4.3.21), a copper-containing enzyme from the thermotolerant yeast Hansenula polymorpha which was overexpressed in baker's yeast Saccharomyces cerevisiae, was tested for its ability to oxidize airborne methylamine. A continuous fluidized bed bioreactor (CFBR) was designed to enable bioconversion of airborne methylamine by AMO immobilized in calcium alginate (CA) beads. The results demonstrated that the bioreactor with immobilized AMO eliminates nearly 97% of the airborne methylamine. However, the enzymatic activity of AMO causes formation of formaldehyde. A two-step bioconversion process was therefore proposed. In the first step, airborne methylamine was fed into a CFBR which contained immobilized AMO. In the second step, the gas flow was passed through another CFBR, with alcohol oxidase from the yeast H. polymorpha immobilized in CA, in order to decompose the formaldehyde formed in the first step. The proposed system provided almost total elimination of the airborne methylamine and the formaldehyde.

Highly sensitive amperometric sensors based on laccase-mimetic nanozymes for the detection of dopamine.

The current research presents novel sensors based on laccase-like mimetics for the detection of dopamine (DA). The synthesized laccase-like nanozymes (nAuCu, nPtCu, nCuMnCo, and nCoCuCe) were prepared by a simple hydrothermal method and exhibited an attractive catalytic activity toward DA. The developed amperometric sensors based on laccase nanozymes (nAuCu and nPtCu) are more stable, selective, and revealed a higher sensitivity (6.5-fold than the biosensor based on the natural fungal laccase from Trametes zonata). The amperometric sensors were obtained by modification of the glassy carbon electrodes (GCEs) with AuPt nanoparticles. Functionalization of the electrode surface by AuPt NPs resulted in increased catalytic activity of the laccase-like layer and higher sensitivity. Among studied configurations, the sensor containing nAuCu and nAuPt possesses a wide linear range for dopamine detection (10-170 µM), the lowest limit of detection (20 nM), and the highest sensitivity (10 650 ± 8.3 A M-1 m-2) at a low applied potential (+0.2 V versus Ag/AgCl). The proposed simple and cost-effective sensor electrode was used for the determination of DA in pharmaceuticals.

Flavocytochrome b2-based enzymatic method of L-lactate assay in food products.

L-lactate, a key metabolite of the anaerobic glycolytic pathway, plays an important role as a biomarker in medicine, in the nutritional sector and food quality control. For these reasons, there is a need for very specific, sensitive, and simple analytical methods for the accurate L-lactate measuring. A new highly selective enzymatic method for L-lactate determination based on the use of flavocytochrome b 2 (EC 1.1.2.3; FC b 2) isolated from the recombinant strain of the yeast Hansenula polymorpha has been developed. A proposed enzymatic method exploits an enzymatic oxidation of L-lactate to pyruvate coupled with nitrotetrazolium blue (NTZB) reduction to a colored product, formazan. The maximal absorption peak of the colored product is near λ = 525 nm and the linear range is observed in the interval 0.005-0.14 mM of L-lactate. The main advantages of the proposed method when compared to the LDH-based routine approaches are a higher sensitivity (2.0 µM of L-lactate), simple procedure of analysis, usage of inexpensive, nontoxic reagents, and small amount of the enzyme. Enzymatic oxidation of L-lactate catalyzed by flavocytochrome b 2 and coupled with formazan production from nitrotetrazolium blue was shown to be used for L-lactate assay in food samples. A high correlation between results of the proposed method and reference ones proves the possibility to use flavocytochrome b 2-catalysed reaction for enzymatic measurement of L-lactate in biotechnology and food chemistry.

The Peroxidase-like Nanocomposites as Hydrogen Peroxide-Sensitive Elements in Cholesterol Oxidase-Based Biosensors for Cholesterol Assay.

Catalytically active nanomaterials, in particular, nanozymes, are promising candidates for applications in biosensors due to their excellent catalytic activity, stability and cost-effective preparation. Nanozymes with peroxidase-like activities are prospective candidates for applications in biosensors. The purpose of the current work is to develop cholesterol oxidase-based amperometric bionanosensors using novel nanocomposites as peroxidase (HRP) mimetics. To select the most electroactive chemosensor on hydrogen peroxide, a wide range of nanomaterials were synthesized and characterized using cyclic voltammetry (CV) and chronoamperometry. Pt NPs were deposited on the surface of a glassy carbon electrode (GCE) in order to improve the conductivity and sensitivity of the nanocomposites. The most HRP-like active bi-metallic CuFe nanoparticles (nCuFe) were placed on a previously nano-platinized electrode, followed by conjugation of cholesterol oxidase (ChOx) in a cross-linking film formed by cysteamine and glutaraldehyde. The constructed nanostructured bioelectrode ChOx/nCuFe/nPt/GCE was characterized by CV and chronoamperometry in the presence of cholesterol. The bionanosensor (ChOx/nCuFe/nPt/GCE) shows a high sensitivity (3960 A·M-1·m-2) for cholesterol, a wide linear range (2-50 µM) and good storage stability at a low working potential (-0.25 V vs. Ag/AgCl/3 M KCl). The constructed bionanosensor was tested on a real serum sample. A detailed comparative analysis of the bioanalytical characteristics of the developed cholesterol bionanosensor and the known analogs is presented.

Flavocytochrome b2-Mediated Electroactive Nanoparticles for Developing Amperometric L-Lactate Biosensors.

L-Lactate is an indicator of food quality, so its monitoring is essential. Enzymes of L-Lactate metabolism are promising tools for this aim. We describe here some highly sensitive biosensors for L-Lactate determination which were developed using flavocytochrome b2 (Fcb2) as a bio-recognition element, and electroactive nanoparticles (NPs) for enzyme immobilization. The enzyme was isolated from cells of the thermotolerant yeast Ogataea polymorpha. The possibility of direct electron transfer from the reduced form of Fcb2 to graphite electrodes has been confirmed, and the amplification of the electrochemical communication between the immobilized Fcb2 and the electrode surface was demonstrated to be achieved using redox nanomediators, both bound and freely diffusing. The fabricated biosensors exhibited high sensitivity (up to 1436 A·M-1·m-2), fast responses, and low limits of detection. One of the most effective biosensors, which contained co-immobilized Fcb2 and the hexacyanoferrate of gold, having a sensitivity of 253 A·M-1·m-2 without freely diffusing redox mediators, was used for L-Lactate analysis in samples of yogurts. A high correlation was observed between the values of analyte content determined using the biosensor and referenced enzymatic-chemical photometric methods. The developed biosensors based on Fcb2-mediated electroactive nanoparticles can be promising for applications in laboratories of food control.

Overexpression of (His)6-tagged human arginase I in Saccharomyces cerevisiae and enzyme purification using metal affinity chromatography.

Arginase (EC 3.5.3.1; L-arginine amidinohydrolase) is a key enzyme of the urea cycle that catalyses the conversion of arginine to ornithine and urea, which is the final cytosolic reaction of urea formation in the mammalian liver. The recombinant strain of the yeast Saccharomyces cerevisiae that is capable of overproducing arginase I (rhARG1) from human liver under the control of the efficient copper-inducible promoter CUP1, was constructed. The (His)(6)-tagged rhARG1 was purified in one step from the cell-free extract of the recombinant strain by metal-affinity chromatography with Ni-NTA agarose. The maximal specific activity of the 40-fold purified enzyme was 1600 µmol min(-1) mg(-1) protein.

Nanozymes with reductase-like activities: antioxidant properties and electrochemical behavior.

Nanozymes (NZs) as stable cost-effective mimics of natural enzymes may be promising catalysts in food and environmental biotechnology, biosensors, alternative energy and medicine. The majority of known NZs are mimetics of oxidoreductases, although there are only limited data regarding mimetics of reductases. In the present research, a number of metal-based NZs were synthesized via chemical methods and screened for their antioxidant ability in solution. The most effective reductase-like Zn/Cd/Cu NZ was characterized in detail. Its antioxidant properties in comparison with several food products and Trolox, as well as substrate specificity, size and composition were studied. Zn/Cd/Cu NZ was shown to mimic preferentially selenite reductase. The amperometric sensor was constructed possessing a high sensitivity (1700 A M-1 m-2) and a broad linear range (16-1000 µM) for selenite ions. The possibility to apply the fabricated sensor for selenite determination in commercial mineral water has been demonstrated.

Highly Porous 3D Gold Enhances Sensitivity of Amperometric Biosensors Based on Oxidases and CuCe Nanoparticles.

Metallic nanoparticles potentially have wide practical applications in various fields of science and industry. In biosensorics, they usually act as catalysts or nanozymes (NZs) and as mediators of electron transfer. We describe here the development of amperometric biosensors (ABSs) based on purified oxidases, synthesized nanoparticles of CuCe (nCuCe), and micro/nanoporous gold (pAu), which were electro-deposited on a graphite electrode (GE). As an effective peroxidase (PO)-like NZ, nCuCe was used here as a hydrogen-peroxide-sensing platform in ABSs that were based on glucose oxidase, alcohol oxidase, methylamine oxidase, and L-arginine oxidase. At the same time, nCuCe is an electroactive mediator and has been used in laccase-based ABSs. As a result, the ABSs we constructed and characterized were based on glucose, methanol, methyl amine, L-arginine, and catechol, respectively. The developed nCuCe-based ABSs exhibited improved analytical characteristics in comparison with the corresponding PO-based ABSs. Additionally, the presence of pAu, with its extremely advanced chemo-sensing surface layer, was shown to significantly increase the sensitivities of all constructed ABSs. As an example, the bioelectrodes containing laccase/GE, laccase/nCuCe/GE, and laccase/nCuCe/pAu/GE exhibited sensitivities to catechol at 2300, 5055, and 9280 A·M-1·m-2, respectively. We demonstrate here that pAu is an effective carrier of electroactive nanomaterials coupled with oxidases, which may be promising in biosensors.

Nanomaterials as Redox Mediators in Laccase-Based Amperometric Biosensors for Catechol Assay.

Laccase is a copper-containing enzyme that does not require hydrogen peroxide as a co-substrate or additional cofactors for an enzymatic reaction. Nanomaterials of various chemical structures are usually applied to the construction of enzyme-based biosensors. Metals, metal oxides, semiconductors, and composite NPs perform various functions in electrochemical transformation schemes as a platform for the enzyme immobilization, a mediator of an electron transfer, and a signal amplifier. We describe here the development of amperometric biosensors (ABSs) based on laccase and redox-active micro/nanoparticles (hereafter-NPs), which were immobilized on a graphite electrode (GE). For this purpose, we isolated a highly purified enzyme from the fungus Trametes zonatus, and then synthesized bi- and trimetallic NPs of noble and transition metals, as well as hexacyanoferrates (HCF) of noble metals; these were layered onto the surfaces of GEs. The electroactivity of many of the NPs immobilized on the GEs was characterized by cyclic voltammetry (CV) experiments. The most effective mediators of electron transfer were selected as the platform for the development of laccase-based ABSs. As a result, a number of catechol-sensitive ABSs were constructed and characterized. The laccase/CuCo/GE was demonstrated to possess the highest sensitivity to catechol (4523 A·M-1·m-2) among the tested ABSs. The proposed ABSs may be promising for the analysis of phenolic derivatives in real samples of drinking water, wastewater, and food products.

Reusable alcohol oxidase-nPtCu/alginate beads for highly sensitive ethanol assay in beverages.

Nanozymes (NZs) are nanoparticles that mimic the catalytic properties of natural enzymes. The present work aimed to obtain effective peroxidase mimetics (PO-like NZs), to characterize their morphological properties, estimate the kinetic parameters of NZs and evaluate the prospects of their application in analysis of ethanol. Herein, we have proposed a convenient spectrophotometric method for ethanol assay using reusable alginate beads enriched with alcohol oxidase (AO) and nanoparticles of PtCu (nPtCu) as PO-like NZs, and 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogen. The linear range for the proposed nPtCu-AO/alginate beads/TMB-based method is from 0.01 mM to 0.15 mM with a limit of detection of 3.3 µM ethanol. The method is used for the quantitative determination of ethanol in alcoholic beverages. The obtained results proved to be in a good correlation with the enzymatic reference method. These results highlight the potential of the nPtCu with PO-like activity in bioanalytical applications. The proposed method, being sensitive, economical and suitable for routine and micro-volume formats, can be used in clinical diagnostics for the detection of ethanol.