Catalytic performance and thermostability of chloroperoxidase in reverse micelle: achievement of a catalytically favorable enzyme conformation.
The catalytic performance of chloroperoxidase (CPO) in peroxidation of 2, 2'-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) and oxidation of indole in a reverse micelle composed of surfactant-water-isooctane-pentanol was investigated and optimized in this work. Some positive results were obtained as follows: the peroxidation activity of CPO was enhanced 248% and 263%, while oxidation activity was enhanced 215% and 222% in cetyltrimethylammonium bromide (CTABr) reverse micelle medium and dodecyltrimethylammonium bromide (DTABr) medium, respectively. Thermostability was also greatly improved in reverse micelle: at 40 °C, CPO essentially lost all its activity after 5 h incubation, while 58-76% catalytic activity was retained for both reactions in the two reverse micelle media. At 50 °C, about 44-75% catalytic activity remained for both reactions in reverse micelle after 2 h compared with no observed activity in pure buffer under the same conditions. The enhancement of CPO activity was dependent mainly on the surfactant concentration and structure, organic solvent ratio (V(pentanol)/V(isooctane)), and water content in the reverse micelle. The obtained kinetic parameters showed that the catalytic turnover frequency (k(cat)) was increased in reverse micelle. Moreover, the lower K(m) and higher k(cat)/K(m) demonstrated that both the affinity and specificity of CPO to substrates were improved in reverse micelle media. Fluorescence, circular dichroism (CD) and UV-vis spectra assays indicated that a catalytically favorable conformation of enzyme was achieved in reverse micelle, including the strengthening of the protein α-helix structure, and greater exposure of the heme prosthetic group for easy access of the substrate in bulk solution. These results are promising in view of the industrial applications of this versatile biological catalyst.
Wang
Yali
Y
School of Chemistry and Materials Science, Shaanxi Normal University, Chang and South Road 199, 710062 Xi'an, Shaanxi, People's Republic of China.
Wu
Jinyue
J
Ru
Xuejiao
X
Jiang
Yucheng
Y
Hu
Mancheng
M
Li
Shuni
S
Zhai
Quanguo
Q
D052160:Benzothiazoles / Q000378:metabolism; D055162:Biocatalysis; D002711:Chloride Peroxidase / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004795:Enzyme Stability; D008823:Micelles; D009795:Octanes / Q000737:chemistry; D010084:Oxidation-Reduction; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D013451:Sulfonic Acids / Q000378:metabolism; D013501:Surface-Active Agents / Q000737:chemistry; D013696:Temperature
D052160:Benzothiazoles; D008823:Micelles; D009795:Octanes; D000644:Quaternary Ammonium Compounds; D013451:Sulfonic Acids; D013501:Surface-Active Agents; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D002711:Chloride Peroxidase; C022806:dodecyltrimethylammonium; C045798:2,2,4-trimethylpentane
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