小麥草主要過氧化酶之純化及性質分析

Purification and Characterization of Predominant Peroxidase from Wheat Grass

王黛蓉(Dai-Jung Wang) ； 張珍田(Chen-Tien Chang) ； 王正新(Cheng-Hsin Wang) ； 賴麗旭(Lih-Shiuh Lai)

《臺灣農業化學與食品科學》 43卷5期 (2005/10) Pp. 358-367

https://doi.org/10.6578/TJACFS.2005.045

摘要

本研究針對小麥草汁中所含過氧化酶進行性質分析，並比較純化前後之差異。結果發現，經由熱處理、硫酸銨割分以及等電焦集層析等連續步驟，可提高過氧化酶之純度約87倍，活性收率約為4.8%。將純化之主要過氧化酶通入Superose 12 HR管柱後發現其分子量為23kDa。等電焦集電泳及活性染色分析結果顯示，小麥草主要過氧化酶含有二異構酶，其等電點分別為8.88及8.43，屬鹼性過氧化酶。小麥草粗酵素液之過氧化酶於30-50℃保溫60分鐘或60℃保溫40℃分鐘皆很穩定，活性幾乎無喪失，於60℃下靜置60分鐘活性始明顯降低，殘留活性約為67%。而純化之主要過氧化酶於30-50℃保溫60分鐘，活性亦幾乎無喪失，然當溫度提高至60℃時，活性開始明顯降低，於60℃下靜置10分鐘之殘留活性約為51%。由殘留酵素活性百分率之對數值對保溫靜置時間作圖發現，小麥草過氧化酶之熱失活反應在反應初期為一級反應，且溫度對熱失活速率之影響符合阿瑞尼亞方程式(Arrhenius equation)，粗酵素液之熱失活活化能為2.2×10^5J/mol；純化酵素液之熱失活活化能則為4.2×10^4J/mol。

關鍵字

小麥草 ； 過氧化酶 ； 純化 ； 性質分析

並列摘要

This study aimed at characterizing the peroxidase present from wheat grass before and after purification. It was found that after heat treatment, ammonium sulfate fractionation and chromatofocusing, about 87 purification fold for peroxidase was achieved and the enzyme recovery was about 4.8%. The molecular mass of the predominant peroxidase from wheat grass was about 23kDa as estimated by Superose 12 HR gel filtration. As determined by isoelectric focusing electrophoresis and peroxidase activity staining, two activity bands were obtained, indicating the purified predominant peroxidase consisted of two isoforms. The isoeleetric points of the two peroxidase isforms were 8.88 and 8.43, respectively. Both isoforms belonged to alkali peroxidase. The crude extract of peroxidase from wheat grass was thermally stable after holding at 30-50℃ for 60min or at 60℃ for 40min, nevertheless the peroxidase activity started to drop significantly after holding at 60℃ for 60min. Similarly, the purified predominant peroxidase from wheat grass was thermally stable after holding at 30-50℃ for 60min. However, at temperatures higher than 60℃, the enzyme activity decreased significantly. The residual peroxidase activity was only about 51% after holding at 60°C for 10min. The thermal inactivation reaction of peroxidase during initial thermostat stage followed first-order reaction kinetics, and the temperature dependence of rate constants was in agreement with the Arrhenius equation. The activation energy of thermoinactivation of the crude extract and the purified predominant peroxidase from wheat grass was found to be 2.2×10^5J/mol and 4.2×10^4J/mol, respectively.

並列關鍵字

Wheat grass ； Peroxidase ； Purification ； Characterization