建立漆酶固定化系統降解銀杏葉萃取物中銀杏酸

Immobilization of Laccase for Ginkgolic Acid Degradation in Ginkgo biloba Leaf Extract

陳宏岳(Hung-Yuen Chen)

指導教授 ： 鄭光成

共同指導教授 ： 丁俞文(Yuwen Ting)

國立臺灣大學/生物資源暨農學院/食品科技研究所/博士(2021年)

https://doi.org/10.6342/NTU202100635

摘要

銀杏為全世界常用且廣泛應用及研究的中藥材，其萃取物中主要成分為類黃酮和萜類，具抗氧化、清除游離自由基等能力，廣泛應用於治療冠狀動脈硬化及阿茲海默症等臨床疾病。然而銀杏萃取過程常伴隨有毒性物質，其中以銀杏酸為主，銀杏酸具有嚴重過敏性與毒性，於德國 Commission E規定，銀杏相關產品中銀杏酸驗出量不得高於5 ppm。目前移除銀杏酸的方式主要有三種方法，相較於有機溶劑分離及奈米粒子吸附，酵素降解法對於環境較為友善，但因酵素提取不易且多為水溶性，造成成本高昂、回收不易通常為單次使用等缺點，因此難以實際應用於產業界。本研究將利用酵素固定化的方式，固定漆酶於利用靜電紡絲技術所製成的尼龍載體上，進行銀杏酸的降解反應。靜電紡絲技術成功製備納米碳管和尼龍6,6混合製成的奈米纖維膜，固定在此載體上的漆酶活性較尼龍6,6顆粒高，且漆酶的pH和溫度穩定性得到顯著改善。並且於儲存40天或重複使用10次後仍保持相對活性50％以上。結果成功證明使用奈米纖維膜作為酶固定平台可以顯著提高酶的活性和穩定性。進一步開發使用響應表面方法及同軸靜電噴灑方法，以製備Fe3O4 /尼龍6,6複合納米顆粒，其平均直徑為376 ± 102 nm，並可藉由磁力簡單地回收。固定在複合載體的漆酶量約為60 mg / g。結果顯示漆酶固定後於反應溫度60–90°C範圍內，其熱穩定性得到了顯著改善。此外，固定在複合載體的漆酶在儲存21天或重複使用5個循環後，相對活性仍具有50%，顯示出良好的儲存穩定性和可重複使用性，並且仍具有降解銀杏酸能力。因此以同軸靜電噴灑製備之高性能固定化納米載體具有應用於酵素固定化的潛力。

關鍵字

銀杏酸 ； 酵素固定化 ； 漆酶 ； 靜電紡絲 ； 靜電噴灑

並列摘要

Ginkgo biloba is a common Chinese medicinal material that is widely used and researched all over the world. The main extracted ingredients are flavonoids and terpenes, which have antioxidant and free radical scavenging activity. G. biloba extract is widely used in the treatment of coronary atherosclerosis, Alzheimer's, and other clinical diseases. However, the extraction process of G. biloba is often accompanied by the production of toxic by-products. Ginkgolic acid is one of the main toxic components among G. biloba extract, and it is severely allergenic and poisonous. According to German Commission E regulations, the content of ginkgolic acid in G. biloba related products must be less than 5 ppm. At present, there are three main ways removing ginkgolic acid. Compared to organic solvent separation and nanoparticle adsorption, enzyme degradation is more environmentally friendly. However, enzymes are difficult to extract and are mostly water-soluble; furthermore, they are expensive and difficult to recycle. Due to the expensive and disposable nature of enzymes, enzyme degradation is rarely used in industrial application. Novel electrospinning technology successfully produced high-quality nanoscopic fiber mats composed of a mixture of multi-walled carbon nanotube and nylon 6,6. Laccase immobilized onto the NFMs exhibited a much higher level of efficiency in the catalysis of ABTS than nylon 6,6 pellet. After NFMs immobilization, the pH and temperature stability of laccase were significantly improved. The nanofiber mat immobilized laccase could maintain more than 50% of its original activity even after 40-day storage or after 10 operational cycles. The results successfully demonstrated the great potential in using novel electrospun nanofiber mat as an enzyme immobilization platform, which could significantly enhance enzyme activity and stability. Core/shell Fe3O4/nylon 6,6 composite nanoparticles (FNCNs) was further developed by the one-step coaxial electrospraying and optimized by response surface methodology (RSM). As results, FNCNs had an average diameter of 376 ± 102 nm and could be recovered easily using magnetic force. The amount of the laccase immobilized on the FNCNs was approximately 60 mg/g FNCNs. As a result, thermal stability of the free laccase was significantly improved in the range of 60–90°C after immobilization. Furthermore, the laccase immobilized on the FNCNs exhibited a relative activity higher than 50% after being stored for 21 days or reused for 5 cycles, showing good storage stability and reusability. Therefore, the high performance immobilization nanocarrier has potential for application in enzyme immobilization.

並列關鍵字

Ginkgolic acid ； Enzyme immobilization ； Laccase ； Electrospinning ； Electrospraying

參考文獻

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