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標題: 固定化酵素N-carbamoyl-D-amino acid amidohydrolase於再生纖維膜上之效率探討
Study on Immobilization of N-carbamoyl-D-amino acid amidohydrolase with regenerated cellulose membrane
作者: 余焜乾
Yu, Kun-Cian
關鍵字: 固定化;N-carbamoyl-D-amino acid amidohydrolase;Cp-HPG;D-HGP;Glutaraldehyde
出版社: 化學工程學系所
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本研究是以再生纖維薄膜(RC)利用epichlorohydrin (EPI)來改質官能基,再以不同鍵結方式將酵素DCase固定上去。四種固定方式分別為RC-EPI、RC-EPI-IDA (iminodiacetic acid)、RC-EPI-DA (diamine)-GA (glutaraldehyde)和RC-EPI-IDA-Fe3+來固定化酵素,其中以RC-EPI-DA-GA的固定化效果最好。

其最適化固定化條件為,5 ml EPI與20 ml 1.4 M NaOH 於24 ℃、150 rpm下反應14小時,之後將膜置於10 ml的1M 1,4-Diaminobutane (溶於1M Na2CO3, pH 11)於80 ℃、100 rpm下反應12小時,再將膜置於25 ml的 0.1% glutaraldehyde於pH 7.0、15℃、100 rpm下反應2小時,使用SEM分析薄膜表面型態,改質前後的薄膜有相似的結構和孔洞分佈。

以此最適化條件製作之RC-EPI-DA-GA膜探討DCase固定化之最佳條件,其結果為:10ml, 100mM Tris-HCl buffer (pH 7.0)溶液中含DCase和1mM DTT,與一片RC-EPI-DA-GA膜固定2.5小時。其固定化完可得12.80倍的純化倍率和17.815的高比活性。將固定化酵素和自由酵素相比,最適化溫度可從40℃提升到50℃。固定化最適反應pH 值為7-9與自由態相比(pH 9)相比,範圍增加兩個單位。於重複批次反應上,固定化 DCase 可維持16 個循環,依然保有約90%的初始活性,利用酵素固定化方法生產D-p-HPG,除了對環境的耐抗性有所提昇外,重複使用也是一大優點,可減少酵素使用量,更符合經濟效益。

The regenrated cellulose (RC) membane as solid matrix was modified via a series of reactions to conetruct the modified membrand for immobilization of enzyme (DCase). Four kinds of immobiization methods: RC-EPI、RC-EPI-IDA (iminodiacetic acid)、RC-EPI-DA (diamine)-GA (glutaraldehyde) and RC-EPI-IDA-Fe3+ were use to immobilized enzyme (DCase). The membrane with RC-EPI-DA-GA gave the best result for DCase immobilization and was selected in this study.

The optimal preparation conditions were concluded as follows: for one RC membrane immersed in 20 ml, 1.4 M NaOH, 5 ml EPI and operated at 24 °C, 150 rpm for 14 h. After that, the membrane was immersed in 10ml of 1M 1,4-Diaminobutane (dissolved in 1M sodium carbonate) and allowed to react at 80℃ for 12h. Then, the membrane was immersed in 10ml of 0.1% glutaraldehyde (dissolved in 0.1M phosphate buffer) and allowed to react for 2h. From the surace morphologies scanned by SEM, the original membrane and the modified membrand had the similar structure and pore distribution.

In order to evaluate the performance of RC-EPI-DA-GA for immobilization of DCase, the crude enzymes wewe immobilied to the RC-EPI-DA-GA in the presence of 10ml, 100mM Tris-HCl buffer (pH 7.0) containing 1mM DTT for 2.5h. A 12.8-fold DCase purification was obtained. Compared to the free enzyme, the optimal reaction temperature of immobilized DCase was increased from 40℃ to 50℃. While the optimal pH of DCase was increased from pH 7.0 to pH 9.0. While the immobilized DCase retained 90% of the initial activity after 16-time repeated use. It is very feasible to use the immobilized DCase in industrial application.
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