中文摘要
幾丁質水解酵素水解幾丁質所形成的產物之分析方法，目前大多利用高效能液相層析儀(HPLC)，來分析幾丁質水解產物的含量。然而高效能液相層析儀(HPLC)需於分析之前先將酵素活性破壞，經過透析膜過濾的處理步驟，才能進入高效能液相層析儀(HPLC)中進行分離，本篇論文利用核磁共振光譜儀(Nuclear Magnetic Resonance Spectroscopy, NMR)做為分析幾丁質水解產物的工具，此方法可以在不破壞酵素的活性前提下利用添加內部標準品定量幾丁質水解酵素水解幾丁質產物，而不需要標準品來製備檢量曲線，不僅具有極佳的濃度-積分值線性相關，也可以分析出水解產物α、β-from兩種型態的比例。依上述方法亦進行在不同pH值時酵素水解幾丁質的活性評估，除此之外，本篇論文還討論N-acetyl-D-glucosamine、N,N´-diacetylchitobiose，在pyridine中之動力行為研究。利用變溫的方式，發現乙醯基訊號會有明顯的變化，推測是因為乙醯基與anomeric位上的羥基會產生分子內的氫鍵，乙醯基的旋轉與分子內氫鍵有密切的關係，因此利用實驗數值計算出乙醯基旋轉的活化能，再利用理論計算的數據再加以證明。此外也發現N,N´-diacetylchitobiose在pyridine中會有開環-環合現象的發生，並推測其反應機構。

Chitinase (EC 3.2.1.14) hydrolyzes -1,4-glycosidic linkages of chitin .The most widely used method for the analysis of the chitinase-catalyzed hydrolysis products of - and -chitin is HPLC. However, this method associated with disadvantages such as the enzyme must be deactivated and filtered by dialytic membrane before going to separate by HPLC analysis and also needed the calibration curves to determine quantitatively the contents of chitin hydrolysis products. In the present study, a 1H NMR method was developed to analyze the chitinase-catalyzed chitin hydrolysis products. This method can be used for the analysis of chitin hydrolysis products without destroying of the enzyme activity, and to quantify the contents of products using internal standard without need of the calibration curves. Especially, this method has an excellent correlation between concentration of the products and integration of the peak, also the ratio’s of α,β conformers of products. The experiments were carried out in different pH phosphorous buffer solutions to estimate the activity of chitinase. In addation, this study also focused on the dynamic behavior of N-acetyl-D-glucosamine and N,N´-diacetylchitobiose in pyridine-d5. The temperature dependence of 1H NMR spectra has been studied in order to get activation parameters of the energetic barrier for the process (VT-NMR). The acetyl groups of products involved in intramolecular H-bonding with the OH group on anomeric site. The rotation of acetyl group is closely related to the intramolecular hydrogen bonding pattern, as suggested by experimental data which was well agreed with the theoretical data (molecular modeling). Moreover, a mechanism was also proposed to explain the mutarotation of N, N´-diacetylchitobiose through open-close ring system in pyridine-d5.

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