| Summary: | 碩士 === 國立臺灣科技大學 === 化學工程系 === 97 === The objective of this thesis is to study the denaturation of recombinant N-carbamoyl-D-amino acid amidohydrolase C-terminally fused with a 6×His tag (DCaseH) and its two mutants, C250G and C193L/N197S/A273V. Urea and guanidine hydrochloride (GdnHCl) of different concentrations, respectively, were used to treat those three enzymes for various periods, and then the analyses of residual activities and fluorescence spectroscopy were employed to investigate the denaturation effects on the enzymatic activity and the protein tertiary structure.
The study on residual activities showed that C250G withstood urea at a higher concentration than the other two enzymes. When treated with urea for 120 min, C250G started to loose about 2% activity at 3 M urea, C193L/N197S/A273V at 1-2 M, and the wild-type (WT) enzyme at < 1 M. However, once the catalytic activity was affected, the inactivation of C250G went more dramatically than the others. Only 15% activity of C250G was observed after a 4-M urea treatment for 120 min, while the C193L/N197S/A273V and WT enzymes sustained 86% and 49% of the activities, respectively, under the same condition. The minimal GdnHCl concentrations to affect enzymatic activities were C250G > WT > C193L/N197S/A273V. As observed in the urea study, C250G was inactivated more significantly than the other two at GdnHCl concentrations higher than the minimal inactivation values. The fluorescence spectroscopy analyses yielded a similar result as the residual activity study. This study suggests C250G has a better stability than C193L/N197S/A273V and WT in the presence of denaturant.
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