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Preparation and characterization of a temperature-sensitive sulfobetaine polymer–trypsin conjugate
Biochemical Engineering Journal 30(2006)48-54,-0001,():
A novel temperature-sensitive polymer-trypsin conjugate was prepared by the covalent linking of monodispersed carboxyl-terminated poly(3-dimethyl(methacryloyloxyethyl) ammonium propane sulfonate) (PDMAPS) to trypsin. The molar ratio of the polymer to trypsin was 2.4 and its upper critical solution temperature was 14◦C. Fluroscence emission spectroscopy and circular dichroic spectroscopy showed that the conjugated trypsin retained its native conformation. The hydrolysis ofN-R-benzoyl-d,l-arginine p-nitroanilidewas carried out at different pHs and temperatures using native trypsin and the conjugate, respectively. The optimal pH was 7.8 for native trypsin and 8.0 for the conjugate. Michaelis-Menten kinetics analysis showed that, as compared to the native trypsin, the conjugated trypsin has a smaller Km that decreases with temperature, while the Vm of the conjugate was the same. When casein was used to study the catalytic effect of the conjugated trypsin on a high molecular weight substrate, an increase in temperature from 40 to 60◦C gave a 2.6-fold increase in the enzyme activity of the conjugate. The half-life for the enzyme activity at 60◦C was 4.8 min for native trypsin and 315.9 min for the conjugate. The conjugate retained 85% of the initial enzyme activity after 10 cycles of temperature swinging from 4 to 40◦C.
【免责声明】以下全部内容由[刘铮]上传于[2006年07月10日 19时49分54秒],版权归原创者所有。本文仅代表作者本人观点,与本网站无关。本网站对文中陈述、观点判断保持中立,不对所包含内容的准确性、可靠性或完整性提供任何明示或暗示的保证。请读者仅作参考,并请自行承担全部责任。
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