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研究生:李政格
研究生(外文):Cheng Ke Li
論文名稱:NUK-15698菌株生產新穎乳糖氧化酵素之純化及性質的研究
論文名稱(外文):Purification and Characterization of a Novel Lactose Oxidase from NUK-15698 strain
指導教授:林順富林順富引用關係
指導教授(外文):Shuen Fuh Lin
口試委員:林順富陳克紹張瑞章楊文仁
口試委員(外文):Shuen Fuh LinKo Shao ChenRay Chang ChangWen Jen Yang
口試日期:2017-07-27
學位類別:碩士
校院名稱:國立高雄大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:98
中文關鍵詞:黴菌乳糖氧化酵素NUK-15698乳糖乳糖酸
外文關鍵詞:fungilactose oxidaseZincNUK-15698lactoselactobionic acid
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為了開發臨床檢驗以及能轉化特殊產物用途之酵素,本實驗室自1990年來不斷從各地土壤中尋找具有此種價值,並且能工業化之相關酵素。近日於高雄楠梓區土壤中搜尋出一株能生產乳糖氧化酵素(Lactose oxidase)之菌種,NUK-15698。
編號NUK-15698菌株利用麩皮培養基進行固態發酵培養四天,經過硫酸銨沉澱劃分、疏水性管柱phenyl-650M、分子篩HW-50、以及Ultrogel-hydroxyapatite管柱層析等步驟,得到相對純之酵素。比活性由原來的0.7 unit/mg 上升至5.3 unit/mg,純化倍率為7.6倍,回收率為5.4%。利用SDS-PAGE鑑定純度,乳糖氧化酵素分子量為47.2 kDa,純化之酵素含有鋅。E_(280 nm)^(1%)為7.58。動力學研究中,此酵素對β-1, 4糖苷鍵之雙糖擁有較好反應性,而對乳糖的km值為0.076 mM。以乳糖為基質下之kcat為 97.08 s-1。最適pH值為7.5,最適溫度為40度,50℃處理一小時仍維持接近100%之活性,酵素在二價鐵離子以及2-bromo-4'-nitroacetophenol處理下會失去部分活性。酵素反應之產物以矽膠薄層色層分析與乳糖酸標準品進行比對,結果與標準品吻合。

To develop novel clinical examination and special chemicals transforming enzymes which are capable of industrialization, our lab has continually screen valuable strains from soil since 1990.Recently, we find a lactose oxidase(LOD) producing fungus NUK-15698 from Kaohsiung, Taiwan.
The production of LOD adopts solid phase fermentation for four days using the wheat brain as the medium. After amino sulfate precipitation, series different column material applied including phenyl-650M, HW-50, Ultrogel-hydroxyapatite column. LOD had purified 7.6-fold and had 5.4% recovery. The specific activity is 5.3 unit/mg. The molecular weight of enzyme determined approximately 47.2 kDa using 10% SDS-PAGE. Purified enzyme contain zinc. E_(280 nm)^(1%) is 7.58. Kinetic studies reveal that the enzyme prefers β-1, 4-glycosidic disaccharide as substrate. The km value of lactose is 0.076 mM. LOD has highest kcat 97.08s-1 among lactose oxidizing oxidases. LOD is stable in the pH range of 6.0 to 9.0 and up to 50 ℃. The optimal reaction is at pH 7.5 and 40 ℃. LOD is inactive by ferrous ion and 2-bromo-4'-nitroacetophenol. The product is lactobionic acid which compares to the standard.

目錄
表目錄
圖目錄
附錄目
中文摘要
英文摘要
第一章 前言
第一節 活性相關酵素 活性相關酵素
第二節 生物科技產乳糖酸 生物科技產乳糖酸 .
第三節 論文研究目的 論文研究目的
第二章 材料與方法
第一節 菌種篩選
第二節 菌學研究
第三節 酵素活性測定法 酵素活性測定法
第四節 菌種培養
第五節 培養探討
第六節 酵素生產以及純化 酵素生產以及純化
第七節 酵素性質
第八節 材料
第九節 儀器以及軟體
第三章 結果與討論
第一節 菌種鑑定
第二節 不同條件培養探討
第三節 酵素純化
第四節 酵素性質
第四章 結論與未來展望 結論與未來展望
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