Improvement of activity and stability of chloroperoxidase by chemical modification.
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 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.


CONCLUSIONS
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.
Liu
Jian-Zhong
JZ
Key Laboratory of Gene Engineering of Ministry of Education and Biotechnology Research Center, State Key Laboratory of Biocontrol, Zhongshan University, Guangzhou, PR China. lssljz@mail.sysu.edu.cn
Wang
Min
M
D002384:Catalysis; D002711:Chloride Peroxidase / Q000737:chemistry*; D002949:Citraconic Anhydrides / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D008299:Maleic Anhydrides / Q000737:chemistry*; D010796:Phthalic Anhydrides / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry
D002949:Citraconic Anhydrides; D008299:Maleic Anhydrides; D010796:Phthalic Anhydrides; D012997:Solvents; D002711:Chloride Peroxidase; C043103:phthalic anhydride