Background: Enzymes show relative instability in solvents or at elevated temperature and lower activity in organic solvent than in water. These limit the industrial applications of enzymes.
Results:
In order to improve the activity and stability of chloroperoxidase, chloroperoxidase was modified by citraconic anhydride, maleic anhydride or phthalic anhydride. The catalytic activities, thermostabilities and organic solvent tolerances of native and modified enzymes were compared. In aqueous buffer, modified chloroperoxidases showed similar K m values and greater catalytic efficiencies k cat /K m for both sulfoxidation and oxidation of phenol compared to native chloroperoxidase. Of these modified chloroperoxidases, citraconic anhydride-modified chloroperoxidase showed the greatest catalytic efficiency in aqueous buffer. These modifications of chloroperoxidase increased their catalytic efficiencies for sulfoxidation by 12%~26% and catalytic efficiencies for phenol oxidation by 7%~53% in aqueous buffer. However, in organic solvent (DMF), modified chloroperoxidases had lower K m values and higher catalytic efficiencies k cat /K m than native chloroperoxidase. These modifications also improved their thermostabilities by 1~2-fold and solvent tolerances of DMF. CD studies show that these modifications did not change the secondary structure of chloroperoxidase. Fluorescence spectra proved that these modifications changed the environment of tryptophan.
Conclusion:
Chemical modification of epsilon-amino groups of lysine residues of chloroperoxidase using citraconic anhydride, maleic anhydride or phthalic anhydride is a simple and powerful method to enhance catalytic properties of enzyme. The improvements of the activity and stability of chloroperoxidase are related to side chain reorientations of aromatics upon both modifications.
Background
Chloroperoxidase (CPO; EC 1.11.1.10) is a heavily glycocylated monomeric hemoprotein secreted from the filamentous fungus Caldariomyces fumago, with a sugar content of 18% of its molecular mass of 42 kDa [1,2]. Comparison of the X-ray crystallographic structure of CPO [3] with other known peroxidases, namely cytochrome C peroxidase [4], lignin peroxidase [5], peroxidases from Coprinus cinereus [6], peanut peroxidase [7], and horseradish peroxidase isoenzyme C [8], reveals dramatic structural differences between CPO and these traditional heme peroxidases. For example, the proximal axial