Biomedical subjects

I Yu Sakharov

Publications and source records attributed to I Yu Sakharov.

4 recordsLinked to original sources

PubMed2004

Palm tree peroxidases.

Over the years novel plant peroxidases have been isolated from palm trees leaves. Some molecular and catalytic properties of palm peroxidases have been studied. The substrate specificity of palm peroxidases is distinct from the specificity of other plant peroxidases. Palm peroxidases show extremely high stability under acidic and alkaline conditions and high thermal stability. Moreover, these enzymes are more stable with respect to hydrogen peroxide treatment than other peroxidases. Due to their extremely high stability, palm peroxidases have been used successfully in the development of new bioanalytical tests, the construction of improved biosensors, and in polymer synthesis.

Biotechnology↗

PubMed2003

Non-enzymatic interaction of reaction products and substrates in peroxidase catalysis.

A quantitative approach for estimation of the non-enzymatic interaction between ammonium 2,2;-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation product and a poorly oxidized substrate was developed using a system including tobacco peroxidase, a mediator substrate (ABTS), and a second substrate. The approach is based on the establishment of a pseudo-steady-state concentration of the ABTS oxidation product in the course of co-oxidation with a poor substrate. A mathematical description of the experimental curve shape has been proposed to linearize the kinetic data and estimate the rate constant for such non-enzymatic interaction. The rate constants calculated from the steady-state kinetics for the non-enzymatic interaction of ABTS oxidation product with phenol and resorcinol were 360 +/- 40 and 770 +/- 60 M(-1).sec(-1), respectively. The values obtained have the same order of magnitude as the rate constant for ABTS oxidation product interaction with veratryl alcohol, calculated from electrochemical measurements (170 M(-1).sec(-1)) by Donal Leech's group. However, the kinetic curves for co-oxidation of ABTS and veratryl alcohol catalyzed by tobacco peroxidase exhibit a pronounced lag-period, which either points to the high rate of the non-enzymatic interaction between ABTS oxidation product and veratryl alcohol and thus, contradicts the electrochemical calculations, or indicates an enzymatic nature of the co-oxidation phenomenon in this particular case.

Alcohols↗

PubMed2003

Expression and refolding of tobacco anionic peroxidase from E. coli inclusion bodies.

Coding DNA of the tobacco anionic peroxidase gene was cloned in pET40b vector. The problem of 11 arginine codons, rare in procaryotes, in the tobacco peroxidase gene was solved using E. coli BL21(DE3) Codon Plus strain. The expression level of the tobacco apo-peroxidase in the above strain was approximately 40% of the total E. coli protein. The tobacco peroxidase refolding was optimized based on the earlier developed protocol for horseradish peroxidase. The reactivation yield of recombinant tobacco enzyme was about 7% with the specific activity of 1100-1200 U/mg towards 2,2;-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was shown that the reaction of ABTS oxidation by hydrogen peroxide catalyzed by recombinant tobacco peroxidase proceeds via the ping-pong kinetic mechanism as for the native enzyme. In the presence of calcium ions, the recombinant peroxidase exhibits a 2.5-fold decrease in the second order rate constant for hydrogen peroxide and 1.5-fold decrease for ABTS. Thus, calcium ions have an inhibitory effect on the recombinant enzyme like that observed earlier for the native tobacco peroxidase. The data demonstrate that the oligosaccharide part of the enzyme has no effect on the kinetic properties and calcium inhibition of tobacco peroxidase.

Cloning, Molecular↗

PubMed2002

Substrate specificity of african oil palm tree peroxidase.

The optimal conditions for catalysis by the peroxidase isolated from leaves of African oil palm tree (AOPTP) have been determined. The pH optimum for oxidation of the majority of substrates studied in the presence of AOPTP is in the interval of 4.5-5.5. A feature of AOPTP is low pH value (3.0) at which the peroxidase shows its maximal activity toward 2,2;-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS). Increasing the buffer concentration changes the AOPTP activity, the degree of the effect depending upon the chemical structure of the substrate. Under optimal conditions of AOPTP catalysis, the values of second order rate constant characterizing efficiency of enzymatic oxidation of substrates have been calculated. It was shown that among 12 peroxidase substrates studied, ABTS and ferulic acid are the best substrates for AOPTP. The results show that substrate specificities of AOPTP and royal palm tree peroxidase are similar, but different from substrate specificity of other plant peroxidases.

Arecaceae↗