Observation and Kinetic Analysis on the Reductive Denaturation of Lysozyme and Investigation on the Strategy of Lysozyme Refolding
Date Issued
2007
Date
2007
Author(s)
Lin, Jiun-Liang
DOI
zh-TW
Abstract
Many factors affect the extent of reductive denaturation of hen egg white lysozyme, e.g., the concentration of reducing agent such as dithiothreitol (DTT), the concentration of lysozyme and the time length of denaturation. It is thus crucial to identify the fully reductively denatured lysozyme. In this study, the reductive denaturation process of lysozyme was monitored with intrinsic fluorescence、hydrodynamic radius (size exclusion chromatography)、5-iodoacetamido fluroescein (IAF) labeling and activity recovery to characterize the fully reductively denatured lysozyme. It was found that lysozyme (5 mg/mL) was fully reductively denatured after being incubated in denaturing buffer which contained 0.2 M DTT for 30 hrs. Further, a reductively denatured intermediate of lysozyme was also identified as one-disulfide bond-left lysozyme. According to these findings, we proposed a “three-step reductive denaturation” kinetic model: N (native lysozyme) to I1 (intermediate 1) to I2 (intermediate 2) to U (fully unfolded lysozyme) to describe the existence of intermediates during reductive denaturation of lysozymes. The kinetic constants were estimated by fitting the shift in the wavelength of maximum emission intensity during denaturation with the three-step kinetic model. The rate constants for each steps were found to be 0.61 mM-0.467h-1, 0.20 h-1, and 0.0095 mM-0.333h-1, respectively, suggesting that I2 to U is the rate-limiting step. Additionally, the reductively denatured lysozyme intermediate I2 still has one uncleaved disulfide bond as revealed by IAF labeling. After that, by the method of dilution refolding, addition of 1.5 M urea in refolding buffer highly enhanced the refolding yield to 80 % and minimized the cost of GSSG. The denaturation of lysozyme in various extent could be generated by the addition of different concentrations of reducing agent (DTT) or different denaturing times under the influence of 1.5 M urea. The refolding of reductively denatured lysozyme was highly affected by the extent of denaturation. In general, a high DTT concentration and a long denaturing time resulted in slow refolding with low activity recovery. Finally, the beads of chitosan and its thiol derivative (MPA-GLA-chitosan) were prepared and used to facilitate the refolding of lysozyme. The use of MPA-GLA-chitosan beads as refolding additives increased the specific activity of lysozyme to 90 % after dialysis refolding.
Subjects
溶菌酶
還原變性
動力學模式
復性
雙硫鍵
Lysozyme
Reductive Denaturation
Kinetic Model
Renatutration
Disulfide bond
Type
thesis
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