Amperometric biosensors for L-arginine and creatinine assay based on recombinant deiminases and ammonium-sensitive Cu/Zn(Hg)S nanoparticles.

There are limited data on amperometric biosensors (ABSs) based on deiminases that produce ammonium as a byproduct of enzymatic reaction. The most frequently proposed biosensors utilizing such a mode are based on potentiometric transducers, which contain at least two enzymes in the bioselective layer; this complicates the procedure and increases the cost of analysis. Thus, the construction of a one-enzyme ABS is a practical problem. In our manuscript ABSs for the direct measurement of creatinine (Crn) and l-arginine (Arg), based on the recombinant bacterial creatinine deiminase (CDI) and arginine deiminase (ADI), are described. To choose the best chemosensor on ammonium ions, a number of nanoparticles (NPs) were synthesized and characterized using cyclic voltammetry. Hybrid Cu/Zn(Hg)S-NPs, having a good selectivity and an extremely high sensitivities towards ammonium ions (5660 A M-1 m-2 at +170 mV and 1870 A M-1 m-2 at -300 mV, respectively), was selected for the development of deiminase-based ABSs. The novel biosensors exhibited very high sensitivities (2660 A M-1 m-2 to Crn for CDI-ABS; 1570 A M-1 m-2 to Arg for ADI-ABS), broad linear ranges, low limits of detection, satisfactory storage stabilities and good selectivities towards natural substrates. The constructed CDI-ABS and ADI-ABS were tested on real samples of biological fluids and juices for Crn and Arg assay, respectively. High correlations of the obtained results with the reference methods were demonstrated for the target analytes.

Novel arginine deiminase-based method to assay L-arginine in beverages.

A highly selective and sensitive enzymatic method for the quantitative determination of L-arginine (Arg) has been developed. The method is based on the use of recombinant bacterial arginine deiminase (ADI) isolated from the cells of a recombinant strain Escherichia coli and o-phthalaldehyde (OPA) as a chemical reagent. Ammonia, the product of the enzymatic digestion of Arg by ADI, reacts with OPA and forms in the presence of sulfite a product, which can be detected by spectrophotometry (S) and fluorometry (F). The linear concentration range for Arg assay in the final reaction mixture varies for ADI-OPA-F variant of the method from 0.35 µM to 24 µM with the detection limit of 0.25 µM. For ADI-OPA-S variant of the assay, the linearity varies from 0.7 µM to 50 µM with the detection limit of 0.55 µM. The new method was tested on real samples of wines and juices. A high correlation (R=0.978) was shown for the results obtained with the proposed and the reference enzymatic method.

Purification and characterization of alcohol oxidase from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha.

Alcohol oxidase (AOX) has been purified 8-fold from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha C-105 (gcr1 catX) with impaired glucose-induced catabolite repression and completely devoid of catalase. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and HPLC. Some physicochemical and biochemical properties of AOX were studied in detail: molecular weight (approximately 620 kD), isoelectric point (pI 6.1), and UV-VIS, circular dichroism (CD), and fluorescence spectra. The content of different secondary structure motifs of the enzyme has been calculated from the CD spectra using a computer program. It was found that the native protein contains about 50% alpha-helix, 25% beta-sheet, and about 20% random structures. The kinetic parameters for different substrates, such as methanol, ethanol, and formaldehyde, were measured using a Clark oxygen electrode. The rate of enzymatic oxidation of formaldehyde by alcohol oxidase from H. polymorpha is only twice lower compared to the best substrate of the enzyme, methanol.