臺灣博碩士論文加值系統

本研究使用一株具有生產半纖維素酶能力之分離株，Aureobasidium pullulans NCH-218，以大豆豆渣為主要基質進行聚甘露糖酶生產、培養條件、純化及酵素特性分析。首先以搖瓶培養方式（載液量 50 mL，於 250 mL 之平底三角瓶）探討 A. pullulans NCH-218 生產聚甘露糖酶較適液態培養基組成與培養條件。實驗結果顯示，以大豆豆渣濃度 3%（w/v）為基礎，添加 1%（w/v）刺槐豆膠作為生產酵素誘導物，0.4 % (NH4)2SO4 為額外氮源，培養溫度30℃，起始 pH 5.4-5.5，菌液接種量 1%（v/v）（1.0 × 105 cell/mL），振盪速率 130 rpm，收集培養三天後之酵素液，其聚甘露糖酶活性為 3.55 U/mL。A. pullulans NCH-218 聚甘露糖酶經超過濾濃縮、硫酸銨（60-80 %）及DEAE-Sepharose Fast Flow 離子交換層析等步驟純化後，進行純化聚甘露糖酶性質分析。由 SDS-PAGE 結果推論此酵素分子量約 34 kDa，最適反應 pH 為 3.0，在 pH 3.0-6.0 間活性仍高且穩定性良好，最適反應溫度為 60℃，溫度低於 40℃ 熱穩定性較佳。對聚甘露糖類具專一性，以刺槐豆膠作為酵素基質之水解產物為甘露寡糖，以甘露二糖及甘露三糖為主。

A hemicellulase-producing fungal isolate, Aureobasidium pullulans NCH-218, isolated from our lab（Huang, 2012） was used in the study. The purpose of the study was to produce, purify and characterize the mannanase from A. pullulans NCH-218. The optimal medium and cultivation conditions for A. pullulans NCH-218 was determined in shaking flask（capacity 50mL/250 mL）. The results showed that 3% soybean okara was used as basal medium with 1% locust bean gum as additional carbon source, 0.4% (NH4)2SO4 as additional nitrogen source. Incubation temperature at 30℃, initial pH at 5.4-5.5, inoculum size 1%（v/v）（1.0 × 105 cell/mL）, and shaking rate at 130 rpm gave the best result for the enzyme production. The maximum activity approximately 3.55 U/mL in the culture broth was obtained after 3 days under the above conditions. The broth filtrate from A. pullulans NCH-218 was subsequently purified by ultrafiltration, ammonium sulfate precipitation（60-80%） and DEAE-Sepharose Fast Flow ion exchange chromatography. The nearly purified mannanase had molecular weight around 34 kDa. The optimum pH was pH 3.0, while the enzyme remained quite stable in pH ranging pH from 3.0 to 6.0. The optimum temperature was 60℃, and the enzyme was stable at temperature under 40℃. The purified mannanase demonstrate a specificity towards mannans-based substrates. The hydrolysis products from locust
bean gum was manno-oligosaccharides, mainly mannobiose and mannotriose.

摘要 ii
Abstract iii
目次 iv
圖目次 vii
表目次 ix
第一章 前言 1
第二章 文獻回顧 2
一、 大豆 2
（一） 大豆之簡介 2
（二） 大豆之機能性 2
二、 大豆豆渣 5
（一） 大豆豆渣之組成 5
（二） 大豆豆渣之應用與相關研究 5
三、 半纖維素之簡介 8
四、 聚甘露糖之介紹 9
（一） 聚甘露糖簡介 9
（二） 聚甘露糖結構與種類 9
五、 聚甘露糖酶之介紹 14
（一） 聚甘露糖酶來源 14
（二） 聚甘露糖酶種類 14
（三） 聚甘露糖酶之應用 18
六、 Aureobasidium pullulans之介紹 21
（一） Aureobasidium pullulans 之生長與分布 21
（二） Aureobasidium pullulans 之應用 22
第三章 材料與方法 26
一、 實驗材料 26
（一） 實驗原料 26
（二） 實驗菌株 26
（三） 培養基 26
（四） 化學藥劑 27
二、 儀器設備 28
三、 實驗架構 30
第四章 結果與討論 39
一、 大豆豆渣基本成分分析 39
二、 A. pullulans NCH-218 之生長曲線 39
三、 生產聚甘露糖酶較適培養條件之探討 42
（一） 豆渣濃度 42
（二） 碳源種類 44
（三） 碳源濃度 45
（四） 氮源種類 48
（五） 起始 pH 50
（六） 培養溫度 52
（七） 菌液接種量 53
（八） 振盪速率 56
（九） 較適培養條件下時間對聚甘露糖酶活性影響 56
四、 聚甘露糖酶粗酵素液性質分析 59
（一） 粗酵素液製備 59
（二） 最適反應 pH 59
（三） pH 穩定性 59
（四） 最適反應溫度 62
（五） 熱穩定性 62
五、 酵素純化 66
（一） 超過濾濃縮 66
（二） 硫酸銨沉澱劃分 66
（三） DEAE-Sepharose FF 離子交換層析 66
六、 純化後聚甘露糖酶性質分析 69
（一） 酵素純度鑑定 69
（二） 原態電泳（native-PAGE） 69
（三） 最適反應 pH 73
（四） pH 穩定性 73
（五） 最適反應溫度 76
（六） 熱穩定性 76
（七） 基質特異性 76
（八） 水解產物分析 79
第五章 結論 82
第六章 未來展望 83
第七章 參考文獻 84

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