臺灣博碩士論文加值系統

本論文主要是將木瓜酵素固定於幾丁聚醣上，尋找最佳的幾丁質去醯化條
件，以及最佳的酵素固定化條件。另外探討固定化的木瓜酵素之一般性質;並
針對一些金屬離子探討它們在酵素催化反應上的影響。根據這些實驗研究結果
作為將來在工業應用上珍貴的參考資料。
結果發現，100℃、50%氫氧化鈉溶液反應55分鐘的操作條件，可製得約
79%去乙醯化程度的幾丁聚醣，以此作為固定化之擔體，分析得知若欲製得最
大的固定化酵素活性，則戊二醛最佳使用量約為1.8%，酵素最佳使用濃度約為
0.1mg/ml。經一系列實驗結果得知:溶性酵素最適反應pH值為pH7.0，固定
化酵素為pH8.0;兩者最適反應溫度皆為70℃。而活化劑半恍胺酸的最適使用
濃度為5mM。論文中也針對一些生物體內常見的離子(如:Na+、K+、Mg2+、
Ca2+或Mn2+等)及一些常見重金屬離子(Hg2+、Zn2+、Cd2+或Cu2+等)作
活性分析與探討。根據實驗結果大致上可將這些離子分類為三類:(1)離子活
化劑，如Na+、K+、Mg2+、Ca2+等;(2)離子去活劑，如Hg2+、Zn2+、Cd2+、
Cu2+等;(3)邊界(borderline)離子，如Mn2+。隨後由酵素催化中心的立體
構形(stereocatalytic conformation)、特異性囊袋(specificity pocket)、陽離子斥
離現象(the phenomenon of cation exclusion)、硬軟酸鹼系統(hard and soft acids/
bases sytem)與配位基效應(ligand effecy)、電荷傳遞系統(charge-relay system)
等方面去探討這些金屬離子的作用機制。另外，溶性木瓜酵素的pH安定度為pH
660，固定化木瓜酵素為pH8.0;兩者最適的溫度安定度為4℃。在操作安定度
方面，經過6次的重覆操作還保有68%的剩餘活性。最後透過動力學的實驗分
析，得知溶性酵素的Km(SE)=0.52(%)，Vmax(SE)=0.3(ΔA280/min．mgE);
固定化酵素的Km(IE)=0.34(%)，Vmax(IE)=0.21(ΔA280/min．mgE)。

Papain is immobilized on the chitosan to find out optimum conditions of deacetylation and immobilization. Besides the investigation made into the common properties of the immobilized papain. Research is made into the effect of the enzymatic catalysis reaction of some metal ions. Results based on these experimental studies will become precious reference for industrial application.
According to the experimental results, under 55 minutes of operational condition of 100℃, 50% NaOH, we can prepared to about 79% deacetylation, chitosan which is then used as a carrier for immobilizing papain in order to reach the best activity of immobilized papain, meanwhile the optimum concentrations of glutaraldehyde and papain are 1.8% and O.1 mg/ml respectively. As showed in the results of a series of experiments, they both have the soluble papain and immobilized papain give optimum reactive pH''s of 7.0 and 8.0 respectively, and the same optimum reactive temperature of 70℃ and 5mM concentration of cysteine activator. The relative activity of some common metal ions in the organism (for example: Na+、K+、Mg2+、Ca2+、Mn2+, etc.) and some usual heavy metal ions (for example: Hg2+、Zn2+、Cd2+、Cu2+, etc.) are researched in this thesis. According to the experimental results, we can divide thesemetal ions into three groups : (1) ionic activator: Na+、K+、Mg2+、Ca2+；(2) ionic deactivator : Hg2+、Zn2+、Cd2+、Cu2+; (3) borderline ion : Mn2+. The catalytic reactions is explored from the aspects of stereocatalytic conformation 、 specificity pocket 、 the phenomenon of cation exclusion 、 hard and soft acids / bases system 、 ligand effect and charge-relay system. On the other hand, the pH stability of soluble papain is pH 6.0, and the immobilized papain is pH 8.0. They have the same optimum temperature stability of 4℃. As for the operational stability, the residue activity is 68% after batch operations. Lastly, based on the results of kinetic analysis, soluble papain and immobilized papain are Km (SE) = 0.52 (%), Vmax (SE) = 0.33 (ΔA280/min ‧mgE) and Km (IE) = 0.34 (%), Vmax (IE)= 0.21 (ΔA280/min ‧ mgE) respectively.