Recombinant human arginase I immobilized on gold and silver nanoparticles: preparation and properties

Abstract

Metal nanoparticles (NPs), such as gold (Au) and silver (Ag), are important for chemistry, physics, and biology due to their unique optical, electrical, and photothermal properties. Such NPs are widely used for immobilization of various bioactive substances, including peptides, enzymes, antibodies and DNA. The synthesis of silver and gold nanoparticles was carried out by reduction of silver nitrate by glucose and reduction of tetrachloroauric acid by sodium citrate, respectively. The size and structure of the AgNPs and AuNPs were characterized using TEM, AFM and XRD methods. The average size of the AgNPs and AuNPs was between 8 and 15 nm. Recombinant arginase I was immobilized using the carbodiimidepentafluorophenol method on the surface of NPs functionalized with ω-mercaptohexadecanoic acid. It was shown that recombinant human liver arginase I isolated from the yeast Hansenula polymorpha maintains satisfactory stability after immobilization on both NPs. The immobilized arginase retained 40% of its activity on the surface of AuNPs and 25% on AgNPs compared to the free arginase after storage at +4 ºC during 25 days. The immobilized enzyme can be used for assay of arginine in pharmaceuticals, in food products and in blood

Surface enhanced Raman scattering as a probe of the cholesterol oxidase enzyme

“…In the next step, the MHDA linker was immobilized on the surface of nanoparticles, 19 and the ChOX enzyme was covalently bonded with them. The immobilization was performed by the following method: the AuNPs were incubated overnight with 5 mM MHDA solubilized in ethanol at temperature 4 C. The conjugate was rinsed with DMF (N,Ndimethylformamide) and incubated with 20 mM CMC (N-cyclohexyl-N 0 -(2-morpholinoethyl)carbodiimidemethop-toluenesulfonate), 20 mM PFP (pentafluorophenyl-4-vinylbenzoate), and 20 mM DIPEA (N,N-diisopropylethylamine) in DMF for 30 min at 25 C, under constant mixing.…”

L-arginine assay with the use of arginase I

“…The enzyme was isolated from a cell free extract of the producer strain and purified by chromatography on an affinity sorbent Arg macroporous glass [12,13]. The enzyme preparation with a specific activity of 600 μmol/min per milligram of protein (600 U/mg) was used to develop a method of enzymatic assay of Arg.…”

“…Arginase preparations were successfully used for immobilization on gold and silver nanoparticles for constructing Arg biosensors [13][14][15][16] and for develop ing an enzymatic method of Arg assay with the spec trophotometric detection of the reaction product [17].…”

Gold Nanoparticles Like A Matrix For Covalent Immobilization Of Cholesterol Oxidase – Application For Biosensing

“…The reaction mixtures were cooled, precipitated (by centrifugation at 24000g force) and washed twice with distilled water or toluene. In the next steps, the 16-mercaptohexadecanoic acid MHDA linker is bounded to the surface of nanoparticles [10] and ChOX enzyme is covalently immobilized with them as it shown in Fig. 1.…”

“…The AuNPs have an excellent conductivity and they are suitable for acting as "electronic wires" to enhance the transfer of electrons (from biological element to electrode) generated during the chemical reactions catalyzed by enzymes [9]. Simultaneously, the bioactivity of the enzyme is improved as well as stability also: the enzyme conjugated with the AuNPs is more stable in various environmental conditions and it is more stable during the time in comparison with a free enzyme [10].…”