本文主要針對先前以SOEing定點隨機突變法，所挑選出C端帶有6×His tag之N-carbamoyl-D-amino acid amidohydrolase (DCaseH)突變酵素，其酵素活性或熱穩定性較原生種酵素稍佳的數個突變酵素進行分析，分別為C193L/N197S/A273V、C250G、M239L、M239K及M239C。為了探討這幾個突變酵素於熱失活下的非摺疊狀態，分別添加不同濃度且不同種類的還原劑，在不同溫度下進行熱反應，之後並利用HPLC進行活性分析，以求得最適溫度及條件來改善酵素的熱穩定性。而後以動態雷射光散射儀測量酵素經熱處理後因失活所造成的粒徑變化與粒徑分佈，並配合圓極偏光光譜儀了解酵素結構因熱處理被破壞所造成的變化。

活性分析結果顯示大部分的酵素在反應溫度42℃、添加5 mM DTT還原劑時會表現出最佳之比活性，而最適之TCEP濃度則隨不同的突變酵素有所變化。綜合活性、圓極偏光光譜及動態雷射光散射分析每一各會熱失活之酵素之結果，幾乎大部分的DCaseH酵素在40℃熱處理之數十秒內，其蛋白質二級結構會快速的形成部份非折疊態(20 ~ 30%)，緊接著開始有凝聚和隨之的聚集現象發生並導致酵素失活。在這些DCaseH酵素中，以C250G添加5 mM DTT具有最佳之比活性，而以C193L/N197S/A273V不含還原劑時及M239L添加2 mM TCEP時具有最高之熱失活活化能Ea，所以表現最為穩定。

We have previously screened several mutant 6×His-tagged N-carbamoyl-D-amino acid amidohydrolase (DCaseH), C193L/N197S/A273V, C250G, M239L, M239K, and M239C, with better activity and thermostability than the wild-type one using SOEing site-directed mutagenesis. This follow-on study focused on the thermostability and the inactivation mechanisms of wild-type and mutant DCaseHs under different types and concentrations of reducing agents. The activity assays showed most enzymes displayed the best activity at 42℃ and 5 mM DTT and the optimal TCEP concentrations were various for different enzymes. The combination study of the activity, circular dichroism, and dynamic light scattering analyses on each thermoinactivated enzyme concluded that nearly all DCaseHs rapidly underwent a partial unfolding (20~30%) of the secondary structure within the first ten seconds of heat treatment and the subsequent flocculation and then aggregation resulted in activity loss. Among the enzymes, C250G displayed the best activity, while C193L/N197S/A273V without adding reductant and M239L with adding 2 mM TCEP were the most thermostable.

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