Abstract
BackgroundEnzymes 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.ResultsIn 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 Km values and greater catalytic efficiencies kcat/Km 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 Km values and higher catalytic efficiencies kcat/Km 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.ConclusionChemical 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.
Highlights
Enzymes show relative instability in solvents or at elevated temperature and lower activity in organic solvent than in water
When 50 μl 50% (v/v) citraconic anhydride (CA), 100 μl 0.33 mM maleic anhydride (MA) or 150 μl 1 mM phthalic anhydride (PA) was added to the mixture containing 2 ml 4.5 μM CPO and 500 μl 0.38 mM thioanisole, the biggest activities of these modified CPOs were observed
The catalytic efficiencies of CA-CPO, MA-CPO and PA-CPO for sulfoxidation in aqueous buffer were increased by 26.2%, 22.6%, and 12.9%, respectively
Summary
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. Most other heme peroxidases use a histidine to fulfil the same function [3]. These structural features shared with both heme peroxidases and cytochromes P450 make CPO the most versatile of the known heme enzymes. In addition to catalyzing chlorination, bromination, and iodination reactions [9], CPO catalyses other reactions characteristic of heme peroxidases (dehydrogenation), catalases (H2O2 dismutation) and cytochromes P450 (monooxygenation) [1,10]. CPO is readily inactivated above 50°C [17], limiting its use in many fields
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