台?9號米糠酯?/脂肪?之純化及生化特性研究

Purification and Characterization of Esterase/Lipase from Tai Keng 9 Rice Bran

吳森源(Sen-Yuan Wu)

指導教授 ： 陳駿季 ； 李雅琳

朝陽科技大學/理工學院/應用化學系生化科技碩博士班/碩士(2011年)

摘要

米糠 (rice bran) 是稻米加工後的副產物，富含油脂、機能性及抗氧化成分，並且存在多種脂質分解酵素 (EC 3.1.1.X.)。酯? (carboxylesterase, EC 3.1.1.1) 與脂肪? (lipase, EC 3.1.1.3) 為其中重要的水解酵素，由於可能引起米糠氧化酸敗 (rancidification)，故本研究以台?九號米糠為材料，純化分離酵素後，探討其生化特性及基質特異性，期望未來可應用於水稻育種之參考。

米糠粗酵素液經SDS-PAGE及酯?活性染色分析，顯示分子量~21.5 kDa處，有一個酵素活性條帶，若以native-PAGE分析，則呈現4個活性條帶；以短碳鏈脂肪酸酯 (p-nitrophenyl butyrate) 及長碳鏈脂肪酸酯 (p-nitrophenyl palmitate) 之酯?及脂肪?基質分析，分別得到比活性157 U/mg protein及1.66 U/mg protein，推測酵素液同時具有酯?及脂肪?，惟前者活性遠高於後者。酵素粗萃液再以20-70%硫酸銨飽和度沉澱劃分，並接續三次管柱層析步驟純化 (Superdex75、Hi-Trap DEAE FF、Superdex75)，可分離得到兩個酵素蛋白質，分別命名為lipase I (21.5 kDa) 及lipase II (22 kDa)，lipase I純化倍率為86.3倍，回收率0.24%，Lipase II純化倍率為7.74倍，回收率2.96%，經二維電泳分析確定其等電點 (pI) 分別為4.5及8.4；lipase I及lipase II皆可水解α-naphthyl butyrate (C4) 及β-naphthyl oleate (C18:1)基質，其中lipase I對β-naphthyl oleate的活性較lipase II高。生化特性分析顯示lipase I及lipase II之最適溫度和pH值相似，分別是40 ℃與pH 9.0，且lipase II 的耐熱性微幅高於lipase I。在基質特異性方面，lipase I偏好短鏈脂肪酸酯，lipase II則偏好短鏈及中鏈者。使用化學試劑處理二酵素，結果顯示：1 mM PMSF 及1 mM EDTA 對二者均有抑制效果；1 mM DTT及1 mM IAA對於二酵素的抑制效果相近，1 mM SDS 可降低lipase I及lipase II的活性，5 mM SDS 幾乎完全抑制lipase II活性。據此推測二酵素之活性中心可能為絲胺酸或半胱胺酸、蛋白質結構可能含有雙硫鍵、需要二價金屬離子協助催化活性之執行，並且lipase I之結構穩定性較lipase II高。

關鍵字

酯? ； 生化特性 ； 基質專一性 ； 脂肪? ； 米糠

並列摘要

Rice bran is a by-product during rice milling, containing abundant oils, functional components, antioxidants, and many lipolytic enzymes (EC 3.1.1.X.) as well. Among the enzymes carboxylesterase (EC 3.1.1.1) and lipase (EC 3.1.1.3) are very important, because they may cause rancidification during rice grain storage. In this study, we have used rice cultivar Tai-Keng 9 (TK9) to isolate and purify esterase/lipase. Through studying the biochemical characteristics and substrate specificities of the enzyme, we expect to employ this information on rice breeding in the future.
The rice bran crude enzymes were analyzed by SDS-PAGE and esterase activity staining, showing a single activity band about 21.5 kDa. When analyzed with native-PAGE four active bands were observed. Short-chain fatty acid ester (p-nitrophenyl butyrate) and long-chain fatty acid ester (p-nitrophenyl palmitate) were used for esterase and lipase activities analyses, and their specific activities were 157 and 1.66 U/mg protein, respectively. It indicated the existence of both activities of esterase and lipase in the extract, but the former was more significant than the latter. The crude extract was subjected to 20-70% ammonium sulfate saturation precipitation and three columns of chromatography were conducted, including resins superdex75, Hi-Trap DEAE FF and then superdex75. Two lipolytic enzymes were separated and dubbed as lipase I (21.5 kDa) and lipase II (22 kDa) with pI 4.5 and 8.4, respectively, resolved by two-dimensional gel electrophoresis. The purification fold and recovery were 86.3 and 0.24% for lipase I, and 7.74 and 2.96% for lipase II. Activity stain of electrophoretic gels showed that both enzymes were able to hydrolyze α-naphthyl butyrate (C4) and β-naphthyl oleate (C18:1), while lipase I showed a higher activity toward β-naphthyl oleate (C18:1) than did lipase II. The study of biochemical characteristics showed that both enzymes had similar optimal temperature and pH at 40 ℃ and pH 9.0, respectively. The thermal stability of lipase II was slightly higher than that of lipase I. The study of substrate specificity showed that lipase I preferred short-chain fatty acid esters, and lipase II short- and medium-chain fatty acid esters. The chemical reagent retreatment experiments showed that both enzymes were effectively inhibited by 1 mM PMSF or 1 mM EDTA; 1 mM of DTT and IAA bore similar inhibition effects; 1 mM SDS significantly reduced the activities of lipase I and lipase II, and 5 mM almost completely inhibited the activity of lipase II. These results may suggest that their active centers are composed of serine or cysteine, their proteins structures contain disulfide bond(s), their activity performance needs divalent metal ion(s), and the structure of lipase I is more tolerable than lipase II.

並列關鍵字

esterase ； biochemical characterization ； substrate specificity ； lipase ； rice bran

參考文獻

許志聖、楊嘉凌 (2009) 水稻篇。台中區農業改良場特刊94號，第40-41頁。

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