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.

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.

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.

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.

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.

Alcohol Oxidase from the Methylotrophic Yeast Ogataea polymorpha: Isolation, Purification, and Bioanalytical Application.

Alcohol oxidase (EC 1.1.3.13; AOX) is a flavoprotein that catalyzes the oxidation of primary short-chain alcohols to corresponding carbonyl compounds with a concomitant release of hydrogen peroxide. It is a key enzyme of methanol metabolism in methylotrophic yeasts, catalyzing the first step of methanol oxidation to formaldehyde.Here we describe the isolation and purification of AOX from the thermotolerant methylotrophic yeast Ogataea (Hansenula) polymorpha, and using this enzyme in enzymatic assay of ethanol, simultaneous analysis of methanol and formaldehyde, and in construction of amperometric biosensors selective to primary alcohols and formaldehyde.

Creatinine Deiminase: Characterization, Using in Enzymatic Creatinine Assay, and Production of the Enzyme.

The goal of the review is description of the main characteristics of creatinine deiminase (CDI), an important bioanalytical tool for creatinine (Crn) assay. Crn is an essential metabolite for diagnostics of kidney disfunction and some other diseases, a biomarker to control the hemodialysis procedure, as well as an important analyte for sport medicine (estimation of general physiological status of athletes). We have described the important sources for CDI isolation, cloning of the corresponding gene, the construction of microbial recombinant strains, overproducing CDI, and characteristics of the enzyme from different microorganisms. There are reviewing also the new bioanalytical methods for quantitative determination of Crn, including enzymatic ones based on using CDI.

Amperometric biosensors based on oxidases and PtRu nanoparticles as artificial peroxidase.

Catalytically active nanomaterials have several advantages over their natural analogues when used as artificial enzymes (nanozymes), namely, higher stability and lower cost. Nanozymes with metallic nanocomposites are promising catalysts for biosensing applications. The aim of the current research is to construct oxidase-based bioelectrodes for food analysis using nanozymes as peroxidase mimetics. Bimetallic PtRu nanoparticles (nPtRu) coupled with alcohol oxidase (AO) and methylamine oxidase (AMO) were chosen to construct amperometric biosensors (ABSs) for primary alcohols and methylamine (MA). Both ABSs show high sensitivities (336 A·M-1·m-2 for the AO-ABS and 284 A·M-1·m-2 for the AMO-ABS), broad linear ranges (25-200 µM ethanol and 20-600 µM MA) and satisfactory storage stabilities. Practical feasibility of the constructed ABSs was demonstrated on food samples. High correlation between contents of MA and ethanol in foods determined by the ABSs and reference methods was observed.

Highly selective apo-arginase based method for sensitive enzymatic assay of manganese (II) and cobalt (II) ions.

A novel enzymatic method of manganese (II) and cobalt (II) ions assay, based on using apo-enzyme of Mn2+-dependent recombinant arginase I (arginase) and 2,3-butanedione monoxime (DMO) as a chemical reagent is proposed. The principle of the method is the evaluation of the activity of L-arginine-hydrolyzing of arginase holoenzyme after the specific binding of Mn2+ or Co2+ with apo-arginase. Urea, which is the product of enzymatic hydrolysis of L-arginine (Arg), reacts with DMO and the resulted compound is detected by both fluorometry and visual spectrophotometry. Thus, the content of metal ions in the tested samples can be determined by measuring the level of urea generated after enzymatic hydrolysis of Arg by reconstructed arginase holoenzyme in the presence of tested metal ions. The linearity range of the fluorometric apo-arginase-DMO method in the case of Mn2+ assay is from 4pM to 1.10nM with a limit of detection of 1pM Mn2+, whereas the linearity range of the present method in the case of Co2+ assay is from 8pM to 45nM with a limit of detection of 2.5pM Co2+. The proposed method being highly sensitive, selective, valid and low-cost, may be useful to monitor Mn2+ and Co2+ content in clinical laboratories, food industry and environmental control service.

Methylamine-sensitive amperometric biosensor based on (His)6-tagged Hansenula polymorpha methylamine oxidase immobilized on the gold nanoparticles.

A novel methylamine-selective amperometric bienzyme biosensor based on recombinant primary amine oxidase isolated from the recombinant yeast strain Saccharomyces cerevisiae and commercial horseradish peroxidase is described. Two amine oxidase preparations were used: free enzyme (AMO) and covalently immobilized on the surface of gold nanoparticles (AMO-nAu). Some bioanalytical parameters (sensitivity, selectivity, and storage stability) of the developed biosensors were investigated. The sensitivity for both sensors is high: 1450 ± 113 and 700 ± 30 A(-1) ·M(-1) ·m(-2) for AMO-nAu biosensor, respectively. The biosensors exhibit the linear range from 15 µM to 150 µM (AMO-nAu) and from 15 µM to 60 µM (AMO). The developed biosensor demonstrated a good selectivity toward methylamine (MA) (signal for dimethylamine and trimethylamine is less than 5% and for ethylamine 15% compared to MA output) and reveals a satisfactory storage stability. The constructed amperometric biosensor was used for MA assay in real samples of fish products in comparison with chemical method. The values obtained with both approaches different methods demonstrated a high correlation.

Structural and enzymatic properties of the AAA protein Drg1p from Saccharomyces cerevisiae. Decoupling of intracellular function from ATPase activity and hexamerization.

The AAA protein Drg1 from yeast was affinity-purified, and its ATPase activity and hexamerization properties were analyzed. The same parameters were also determined for several mutant proteins and compared in light of the growth characteristics of the corresponding cells. The protein from a thermosensitive mutant exhibited reduced ATPase activity and hexamerization. These defects were not reversed by an intragenic suppressor mutation, although this allele supported growth at the nonpermissive temperature. A different set of mutants was generated by site-specific mutagenesis intended to adjust the Walker A box of the D2 domain of Drg1p to that of the D1 domain. A S562G exchange in D2 produced a nonfunctional protein that did not hexamerize but showed above-normal ATPase activity. The C561T mutant protein, on the other hand, was functional but hexamerized less readily and had reduced ATPase activity. In contrast, the C561T/S562G protein hexamerized less than wild type but had much higher ATPase activity. We distinguished strong and weak ATP-binding sites in the wild type protein but two weak sites in the C561T/S562G protein, indicating that the stronger site resides in D2. These observations are discussed in terms of the inter-relationship of ATPase activity per se, oligomeric status, and intracellular function for AAA proteins.