Effect of pH and temperature on the substrate specificity of imidase

• Main Authors: Sheng-Wei Lin, 林聖偉
• Other Authors: Yuh-Shyong Yang
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/67394979751413362623

碩士 === 國立交通大學 === 生物科技研究所 === 88 === Due to its broad substrate specificity, thermophilic imidase is also known as dihydropyrimidinase (EC 3.5.2.2) and hydantoinase. The purpose of this research is to understand the effect of temperature and pH on the substrate specificity of imidase. At room temperature, hydrolytic activity of imidase decrease toward hydantoins as its size of side chain increase. Opposite results are observed at 60 ℃, in which condition hydantoins with large side chain become much better substrates. Three functional groups of substrate include six and five-member ring are used for this study. The optimal pHs of imidase are 8.0, 8.5, 9.5 for Vmax/Km of hydantoins, cyclic imides and dihydropyrimidines, respectively. Effect of pH on Vmax and Km is dependent on the substrate used. Km of a six-member ring cyclic imide and dihydropyrimidines are insensitive to the change of pHs from 7.5 to 9.5. The pH profile of Vmax of cyclic imides and hydantoins exhibit bell shapes profile, which is different from that of dihydropyrimidines. Substrate specificity of imidase for imides, hydantoins and dihydropyrimidines is temperature dependent. The shapes of pH profiles may remain similar (hydantoins and cyclic imides) or may differ (dihydropyrimidines) at different temperatures. Both Km and Vmax increase steadily with the elevation of reaction temperature up to 60 ℃ for dihydropyrimidines and glutarimide but not for hydantoins and succinimide. We conclude that ring structure and side chain are both important for the substrate selection of imidase at different temperature and pH.