過氧化酵素(Coprinus Cinereus peroxidase, CIP)之製備及酵素固定化

Abstract

Coprinus Cinereus 過氧化酵素(CIP)與山葵過氧化酵素（HRP）相似，擁有高度地催化活性及受質的特異性。先前的研究顯示CIP對於氧化胺基苯二醯井（luminol）產生化學冷光的反應上有更高的催化活性 (如常使用的受質ABTS)。這些特質使得過氧化酵素得以廣泛應用於檢測生物分子的檢測器、酵素免疫檢測、工業界防止染料洗脫的洗衣劑及各類生化與臨床檢驗等用途上。 本研究最主要的目標為建構出適合表現真菌屬的Coprinus cinereus 過氧化酵素的原核細胞表現系統。首先我們利用基因選殖與蛋白質工程技術的方式將全長的CIP基因選殖於pTrc99a的原核細胞表現載體中（此質體命名為pTrc-CIP），並以大腸桿菌JM105為宿主進行大量表現。我們發現所建構出的pTrc-CIP表現載體其最適化條件為在30℃下，菌液濃度為OD 0.8、添加0.8 mM IPTG並予以8小時的表現時間。至於CIP純化部份則是使用50%的硫銨進行沉澱，之後再通過陰離子管柱層析。此一以原核表現系統來大量表現具活性的真菌屬的過氧化酵素，目前已知為唯一成功的例子。另利用受質ABTS來計算酵素動力學參數，得知Km 及Vmax 值分別為0.082 mM 及2.1 μmole/ug/min。 在酵素固定化部份，我們已成功運用溶膠凝膠（sol-gel）方法建立一個酵素固定化之平臺，用此技術製成的基質具有優異的光學特性和熱力學、機械穩定性，且它形成的化學條件溫和，尤其適用於包埋生物大分子（酵素、蛋白質）且亦可提昇酵素使用效益。經由實驗數據可知，使用溶膠凝膠法來固定酵素，的確可以使酵素乾燥保存長達二個月以上活性不損而且亦可重覆使用十次。

The Coprinus cinereus peroxidase (CIP)shows a broad specificity for hydrogen donors and a high catalytic efficiency as does the well-known peroixdase from horseradish roots (HRP). Previous studies have shown that CIP exhibited a much higher catalytic activity to the common substrate, luminol as well as ABTS. This property permits a wide range of application for CIP, including high-sensitivity chemiluminescent determination of biological materials, construction of biosensor, enzyme immunoassay, and dye-transfer inhibition during laundering. The major objective of the present study is to establish a bacterial expression system for CIP. We first constructed CIP expression vector by inserting full length CIP cDNA into the bacterial expression vector, pTrc99a, termed pTrc-CIP. The over expression of CIP was performed in E.coli strain JM105. We found that the pTrc-CIP could be expressed as a soluble form under the 30℃,in the presence of 0.8 mM IPTG for 8 h. The purification of recombinant CIP was carried out by 50% ammonium sulfate precipitation, followed by an anion-exchange chromatography. The study of CIP kinetic parameters was also conducted. The Km and Vmax values are 0.082 mM and 2.1 μmole/μg/min, respectively, by using ABTS as substrate. It is the first known so far example that the soluble, active fungal peroxidase can be successfully expressed in the bacterium. In enzyme immobilization study, we successful established a platform technique for enzyme immobilization by using Sol-gel method. Sol-gel glass could be formed under low temperature and mild chemical condition. The result shows that Sol-gel encapsulated HRP is reusable at least 10 times; its 80% activity can be maintained for around 55 days.