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    請使用永久網址來引用或連結此文件: https://ir.cnu.edu.tw/handle/310902800/9074


    標題: 固定化香蕉葉多酚氧化酶之特性及於酚類化合物轉化之應用
    Characterization of immobilized polyphenol oxidase from banana leaf and its application on the transformation of phenolic compounds.
    作者: 陶申秋
    Shen-chiu Tao
    貢獻者: 朱惠鈴
    嘉南藥理科技大學:生物科技研究所
    關鍵字: 
    酪胺酸酶
    多酚氧化酶
    香蕉葉
    phenol
    tyrosinase
    polyphenol oxidase
    banana leaf
    日期: 2005
    上傳時間: 2008-11-24 17:00:52 (UTC+8)
    摘要: 本研究目的在探討固定化香蕉葉多酚氧化酶之特性及於酚類化合物轉化之應用,首先探討不同品種香蕉葉多酚氧化酶(PPO)並比較不同之固定化方法,結果以北蕉的單酚酶比活性最高315(units/mg),活性約為其他品種之2-20倍。以Amberlite XAD-7、褐藻酸及殼糖胺為固定化酵素之擔體,吸附之PPO相對活性分別為100%、41.4%及20.6%,固定化酵素之操作穩定性分別為18次、5次及9次,以Amberlite XAD-7為擔體之固定化效果最佳。
    固定化擔體Amberlite XAD-7與北蕉香蕉葉PPO酵素之最適作用時間為120分鐘。兩者之最適固定化比例為1:5(w/v),其酵素吸附量為 4200 units/g。比較固定化北蕉香蕉葉PPO經冷凍乾燥、真空乾燥、室溫乾燥、熱風乾燥後,PPO殘留活性分別為98.7、98.6、98.6和94.7%,以經濟效益為考量,故選擇室溫乾燥。固定化北蕉香蕉葉PPO儲存在-20℃及室溫下,經60天後其殘留PPO相對活性分別為97%和35%。可知固定化酵素儲藏於-20℃時,其穩定性較佳。
    可溶性及固定化北蕉香蕉葉PPO以L-DOPA為基質,最適pH分別為6.8及6,最適溫度為50℃和70℃。在pH穩定性方面,可溶性北蕉香蕉葉PPO於pH6-6.8之PPO相對活性皆在70%以上,而固定化北蕉香蕉葉PPO於pH4-8之PPO相對活性均大於20%。在熱安定性方面,可溶性北蕉香蕉葉PPO於65℃以下反應30分鐘其PPO殘留活性皆在50%以上,固定化北蕉香蕉葉PPO於75℃以下反應30分鐘其PPO殘留活性皆在50%以上,兩者在75℃下反應70分鐘,固定化北蕉香蕉葉之PPO殘留活性仍較可溶性北蕉香蕉葉PPO高出三倍。固定化北蕉香蕉葉PPO與基質L-DOPA反覆操作8次仍保有50%以上之殘留活性,而與基質反覆操作18次後仍保有7%殘留活性。
    可溶性北蕉香蕉葉PPO對4-氯酚、3-氯酚、對-甲酚、4-甲氧基酚、酚、2-氯酚之相對反應性分別為100%、94%、83%、53.2%、50.7%及46.9%,固定化北蕉香蕉葉PPO者分別為100%、97%、96.7%、79.4%、82.5%及81.5%,故兩者對4-氯酚反應性最高。以4-氯酚為基質進行反應,可溶性北蕉香蕉葉PPO於pH6-6.8之PPO相對活性均為最高66%,固定化北蕉香蕉葉PPO於pH4-9之PPO相對活性均大於95%。可溶性北蕉香蕉葉PPO之最適溫度為50℃,固定化北蕉香蕉葉PPO於20-80℃之PPO相對活性皆在80%以上。固定化北蕉香蕉葉PPO與基質4-氯酚反覆操作15次後仍有60%殘留活性,反覆操作18次後仍有32%殘留活性。以4-氯酚為基質,可溶性香蕉葉多酚氧化酶之Km值為1.88 mM,固定化香蕉葉多酚氧化酶之Km值為13.1 mM。
    The objective of this work is to study the characterization of immobilized polyphenol oxidase from banana leaves and its application on the transformation of phenolic compounds. Polyphenol oxidases (PPO) of banana leaves from a variety of sources were compared. Among them, PPO from Giant cavedishii leaves showed the highest specific activity (315 units/mg). PPO from Giant cavendishii leaves was further immobilized with different matrix, PPO activity and operating stability were examined. The relative PPO activity immobilized with Amberlite XAD-7, alginate and chitosan were 100, 41.4 and 20.6%, and the reused time was 18, 5 and 9 respectively. PPO immobilized with Amberlite XAD-7 exhibited the highest activity.
    The optimal preincubation time of Amberlite XAD-7 with polyphenol oxidase from the Giant cavendishii was 120 minutes. The optimal absorbed PPO activity was 4200 units/g Amberlite XAD-7. The residual activity after freezing drying, vacuum drying, room temperature drying, and hot-air drying of immobilized PPO were 98.7, 98.6, 98.6 and 94.7% respectively. The dried immobilized PPO was then stored at -20℃ and room temperature for 60 days, the residual PPO activity were 97% and 35% respectively.
    The optimal pH of soluble and immobilized PPO from Giant cavendishii leaves were 6.8 and 6 using L-DOPA as substrate, and the optimal temperature were 50℃ and 70℃ respectively. The PPO was preincubated at different pH for 60 minutes, the soluble PPO retained more than 70% activity at pH ranged 6-6.8 while immobilized PPO showed more than 20% activity at pH ranged 4-8. The soluble PPO revealed more than 50% activity at temperature below 65℃ for 30 mins, while immobilized PPO showed more than 50% activity at temperature below 75℃ for 30 mins. The immobilized PPO at 75℃ for 70 mins displayed tree folds of activity compared to soluble PPO. Moreover, the immobilized PPO retained 50% activity while reacted with L-DOPA for 8 times; 7% activity for 18 times.
    When 4-chlorophenol, 3-chlorophenol, p-cresol, 4-methoxyphenol and 2-chlorophenol were used as substrate, the relative activities of soluble PPO were 100%, 94%, 83%, 53.2%, 50.7% and 46.9%; immobilized PPO, 100%, 97%, 96.7%, 79.4%, 82.5% and 81.5% respectively. 4-Chlorophenol showed the highest reactivity to PPO. The optimal pH for soluble PPO was at pH6, using 4-chlorophenol as substrate, and more than 66% activity was found between pH6-6.8. The optimal pH of immobilized PPO was at pH6, more than 95% activity was found between pH4-8. The optimal temperature of soluble and immobilized PPO were all at 50℃, while immobilized PPO showed more than 80% activity between 20-80℃. The immobilized PPO retained 60% activity while reacted with 4-chlorophenol for 15 times, 32% activity for 18 times. The Km of soluble PPO was 1.88 mM using 4-chlorophenol as substrate, and that of immobilized PPO was 13.1 mM.
    關聯: 校內校外均不公開
    顯示於類別:[生物科技系(所)] 博碩士論文

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