Laboratory of Applied Microbiology, Department of Agrobioscience, Graduate School of Agricultural Science. Kobe University. Rokko. Kobe 657-8501. Japan

摘要

Kinetics of horseradish peroxidase (HRP) in the oxidation reaction of ortho-methoxy phenol (AH) by hydrogen peroxide was studied taking into account inactivation of HRP during reaction by its suicide substrate, H2O2. A new simple method was introduced to protect HRP against suicidal inactivation effect of peroxide. Histidine, tyrosine and cysteine were added to protein solution and peroxidatic activity of HRP was assayed (25 °C, pH 7.0) under suicide inactivation condition, [H2O2] >2 mM. Peroxidatic activity of HRP increased more than 1.5 times and HRP was protected against suicide inactivation, completely. A close match was observed for the proposed equations and experimental data. Surprisingly, inactivation rate constant (k_i) at 3.0 mM H2O2 was about zero in the presence of low-dose stabilizers of amino acids family ([amino acid]/HRP～0.2-10.0) and stabilized HRP solutions were stable more than 45 days keeping 80% of the initial catalytic activity. A very interesting observation was that peroxide-inactivated HRP could be recovered into a more active one in the presence of amino acids. Such improvements in the catalytic activity, long-term stability and preventing suicide inactivation of HRP are of paramount importance in clinical assays, biosensors preparation and biotechnological applications of peroxidase (biotransformation, chemical synthesis and phenol removal process).