Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/51827
標題: 幾丁聚醣-活性白土製備複合圓珠之吸附特性與酵素固定化作用
Adsorption Characteristics and Enzyme Immobilization on Complex Beads Prepared from Chitosan and Activated Clay
作者: 張敏雲
Chang, Min-Yun
關鍵字: chitosan;幾丁聚醣;complex beads;β- glucosidase;acid phosphatase;immobility;複合圓珠;β-葡萄苷;酶;酸性磷酸酶;固定化
出版社: 食品科學系
摘要: 
幾丁聚醣為酵素固定化廣泛應用之優良材質,幾丁聚醣圓珠能克服立體障礙展現更多活性基座和輸送空間。因此在酵素固定化和吸附作用能有優良表現,如果將其機械性質和比重改善,更能增進在酵素固定化工程上之操作和重覆使用性。
本研究以幾丁聚醣膠體加入活性白土製成複合圓珠,吸附兩種染料和兩種有機化合物之行為,以等溫平衡式和動力學模式解析,並以兩種酵素在複合圓珠固定化之活性探討,評估量產之可行性。
所得結果如下:
1. 由複合圓珠之等溫平衡吸附數據,顯示腐植酸、reactive red 222 (RR222)、丹寧酸和亞甲基藍等四種吸附質之等溫平衡皆以Freundlich等溫式描述較為適宜。
2. 在攪拌槽中完全混合,進行批次接觸實驗,以擬一階動力學(pseduo- first order kinetic)、擬二階動力學(pseduo-second order kinetic)及顆粒內部擴散模式(intraparticle diffusion model)等三種簡單動力學模式,比較單一材料活性白土、幾丁聚醣圓珠以及二者所製複合圓珠之吸附作用,顯示吸附較大分子量之RR222和丹寧酸時適用擬一階動力學式;並獲得各種吸附劑之顆粒內部擴散常數ki (g·kg-1·min-0.5)。
3. 複合圓珠經5.0g /L戊二醛(glutaraldehyde)交聯後,分別固定化酸性磷酸酶(acid phosphatase)和β-葡萄苷酶(β- glucosidase),利用可逆Michaelis-Menten機構描述複合圓珠固定化酵素之活性與基質濃度之關係,起始實驗數據以 Lineweaver-Burk畫圖法估算Michaelis常數(Km)和最大速率(Vm),酸性磷酸酶之Km為1.653mM,Vm為30.944 µmol·min-1;β-葡萄苷酶之Km為5.945mM,Vm為144.4µmol·min-1。進行50次反覆操作後活性均仍維持穩定。
4. 熱穩定性可依循一階熱失活公式描述,並且得到固定化酸性磷酸酶失活活化能Ea值為66.44kJ/ mol。探討溫度和pH 值等操作變數對酵素活性之影響,發現β-葡萄苷酶之溫度與活性,於15℃至75℃間,呈線性上升關係,酸性磷酸酶在57℃具有最高之活性,pH值在3.5至5.8範圍,兩種酵素活性變化皆不大,其穩定性高。
5. 本研究證實所製備的幾丁聚醣-活性白土複合圓珠,應用於酵素固定化具有開發潛力,此結果可供複合圓珠在酵素固定化工程應用之參考。

Chitosan is known as an ideal support material widely used for enzyme immobilization. It has been proved that chitosan beads could conquer steric hindrance and exhibit more activated seats and transported spaces. Though chitosan beads have excellent characterization for enzyme immobility and adsorption, if the beads can be modified to promote mechanical property and specific gravity, the operation and reuse of enzyme immobility engineering will be increased.
In this study, activated clay was added in chitosan colloid to prepare complex beads. The adsorption action of the complex beads were analyzed by isotherm equations and kinetic models for two dyes and two organic compounds. The activity of two enzymes immobilized on the complex beads were also determined to evaluate the availability of the product.
The following results were obtained.
1.All adsorption isotherms of humic acid, RR222, tannic acid and methylene blue from water onto complex beads could be well fitted by the Freundich equation.
2.Three simplified kinetic models including pseudo-first order equation, pseudo-second order equation and intraparticle diffusion model were selected to follow the adsorption processes. It was shown that the adsorption of larger molecules including RR222 and tannic acid could be best described by the pseudo-first order equation. A comparison of the kinetic parameter of intraparticle diffusion ki (g·kg-1 ·min-0.5) among such adsorbents was also obtained.
3.The immobilization of acid phosphatase andβ-glucosidase onto the complex beads was finished by cross-linking with 5.0 g / L glutaraldehyde. The activity of both immobilized enzymes could be described by reversible Michaelis-Menten mechanism applying the pseudo- steady- state hypothesis. The Michaelis constant (Km) and maximum velocity (Vm) were estimated according to the Lineweaver- Burk plot. The values of Km and Vm of acid phosphatase were obtained to be 1.65 mM and 30.9 µmol·min-1, respectively, and those of Km and Vm ofβ-glucosidase were 5.95 mM and 144.4 µmol·min-1, respectively. After 50 times reuse, the immobilized enzyme can keep stability of its activity.
4.The activated energy of thermal inactivation of the immobilized acid phosphatase, Ea, could be obtained if the inactivation was assumed to the first order, which was equal to 66.4 kJ/mol. It was show that the activity of the immobilized β-glucosidase increased with increasing temperature in the range 15-75 oC. The activity of the immobilized acid phosphatase at 57 oC reached maximum. The activities of both immobilized enzymes remained nearly constant at pH 3.5-5.8.
5.This work provided some basic data for solute adsorption and enzyme immobilization onto complex beads for further design of production process.
URI: http://hdl.handle.net/11455/51827
Appears in Collections:食品暨應用生物科技學系

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