Coprinus cinereus過氧化氫酵素於大腸桿菌中之表現及特性研究

Abstract

Coprinus cinereus peroxidase (E.C.1.11.1.7)為一種含heme的過氧化氫酵素，可將過氧化氫催化成水及氧化酚類化合物及其衍生物。Coprinus cinereus peroxidase (CIP) 與山葵過氧化氫酵素(horseradish peroxidase)相似，擁有高催化活性及受質特異性。根據之前的研究顯示，CIP對某些呈色劑(chromgens)有極高的催化活性，藉由這些觀察而推測出CIP有極高的應用性於工業上，例如生物分子檢測器，酵素免疫檢測，及除去酚類污染物的工具。 本次研究中，主要的目標是讓CIP大量表現於大腸桿菌表現系統中，藉由之前實驗室建構所pTrc99a-CIP的質體下表現，我們研究數種會對表現有影響的參數，如誘導溫度，IPTG的濃度，誘導時間。不幸地，基因重組蛋白CIP的產量過少導致於在純化過程中易失去活性。此外我們利用基因選殖及蛋白質工程技術將CIP選殖到pET 30 b(+)質體上，發現將pET 30 b(+)-CIP的質體表現在誘導溫度25℃，菌液濃度O.D.600為0.9時，添加0.6 mM IPTG表現19小時，有較高的催化活性。並將表現出來的CIP以西方墨點法加以確認。將表現出來的CIP以陰離子交換管柱層析，及His-tag親和力管柱層析加以純化。我們發現另一種未知過的氧化氫酵素大量存在於大腸桿菌且會在純化的過程中產生干擾。 在酵素熱穩定性方面，則發現CIP含兩個His-tag及一個S-tag在30℃時，活性喪失80%。未來，我們將研究能強化CIP表現於大腸桿菌中的表現條件。

Coprinus cinereus peroxidase (CIP) is the heme containing enzyme that catalyzes the oxidation of a phenolic compounds and derivatives in the presence of hydrogen peroxide. It shows a broad specificity for hydrogen donors and high catalytic efficiency as does the Horseradish peroxidase (HRP). Previous study has shown that CIP has higher catalytic activity for several chromagens than HRP. This observation suggests that CIP has high potential in industrial application, such as enzymatic immunoassay, biosensor and polluent remove. In this study, our aim is to overexpress fungal peroxidase (CIP) in E.coli. Previously constructed pTrc99a-CIP to investigate the effect of various parameters, such as temperature, IPTG concentration, and induction time in the expression of CIP. Unfortunately, the yield of recombinant CIP was too low to sustain the lost during the purification. We further constructed a new CIP expression vector, pET30 b(+)-CIP and used to express recombinant CIP. We found that CIP could be optimally induced with bacterial population of O.D. 600 = 0.9 in the present of 1.2 mM IPTG for 19 h. The CIP expression was confirmed by westerm blotting. The purification of expressed CIP was first performed on an anion exchange column, Q-Sepharose and His-tag affinity column. Interestingly, we found an unknown peroxidase might exist in E.coli that made in interference during the purification of CIP. In thermal stability aspect, the CIP carried two His-tags and one S-tag lost 80% activity at 30℃. In the future, we will further investigate the conditions that may enhance the expression of CIP in bacteria