臺灣博碩士論文加值系統

本實驗自新竹南寮漁港之土壤中分離具幾丁質分解能力之菌株，經鑑定後並命名為Aeromonas sp. DYU-Too 7。此菌株於30℃下以CB (chitin broth)培養120 h之發酵液，經離心後取其上層液，經飽和硫酸銨沉澱、DEAE Sepharose CL-6B、Sephadex G-100及SDS-PAGE蛋白質純化程序後，可獲得分子量為35 kDa與60 kDa之幾丁質分解酶(chitinase)。35 kDa幾丁質分解酶回收率與純化倍率分別為5.07%與3.66；60 kDa幾丁質分解酶回收率與純化倍率分別為6.76%與2.25。經酵素特性分析，發現35 kDa與60 kDa幾丁質分解酶最適反應pH值分別為pH 5與pH 4，最適反應溫度分別為70℃與60℃；酵素之pH與溫度穩定性實驗結果：35 kDa幾丁質分解酶在pH 5-8與10-60℃時皆可維持原本活性之60%以上；60 kDa幾丁質分解酶在pH 4-8下可維持原本活性之80%以上，於10-50℃時則可維持原本活性之60%以上；金屬離子對酵素活性之影響：35 kDa與60 kDa幾丁質分解酶與10 mM Ba2+, Hg2+, Mg2+及Ag+作用1 h後，皆可維持原本活性之50%以上；酵素水解產物：35 kDa與60 kDa幾丁質分解酶與膠態幾丁質作用24 h，分別得到N-乙醯幾丁二糖2.21 g/L與2.3 g/L；酵素動力學結果：35 kDa幾丁質分解酶Vmax與Km分別為2,000 U/L與8.0 g/L；60 kDa幾丁質分解酶Vmax與Km分別為1,428.6 U/L與4.3 g/L。
本實驗自臺北後山埤土壤取樣，以含0.5%膠態幾丁質之培養基篩菌，分離出一具幾丁質分解能力菌株JR1。此菌株以LB (Lu-
-ria-Bertani)培養基培養2 h達對數增殖期(log phase or exponential
phase)，培養12 h達定常期(stationary phase)。於30℃下，以含2%幾丁質粉末之CB培養120 h，其幾丁質分解酶活性可達366 U/L，發酵液離心後取其上層液，經飽和硫酸銨沉澱、DEAE Sepharose CL-6B、Sephadex G-100及活性電泳染色法蛋白質純化程序後，可獲得分子量為75 kDa之幾丁質分解酶。經酵素特性分析，發現粗酵素液之最適基質濃度為15 g/L，粗酵素液與75 kDa幾丁質分解酶最適反應pH值分別為pH 6與pH 7；最適反應溫度皆為40℃。以CB培養24 h之酵素水解產物為N-乙醯葡萄糖胺(GlcNAc)，4.79 g/L。

Abstract
A chitin-degrading bacterium, DYU-Too 7, was isolated from the soil in Hsin-Chu. The bacterium was identified as Aeromonas sp. The supernatant of 120 h-old culture of Aeromonas sp. DYU-Too 7 cultivated in medium CB (chitin broth) at 30℃ was collected by centrifugation. Two chitinases were purified from the culture super-
natant by ammonium sulfate precipitation, DEAE Sepharose CL-6B and Sephadex G-100. The two chitinases have molecular masses of 35 and 60 kDa, respectively, determined by SDS-PAGE. The 35 and 60 kDa chitinases were purified 3.66-fold with 5.07% yield and 2.25-fold with 6.76% yield, respectively. The optimum pH and temperature were 5 and 70℃ for the 35 kDa chitinase and 4 and 60℃ for the 60 kDa chitinase. Effects of pH and temperature on stability of chitinase have been explored. Experimental results showed that the 35 kDa chitinase was stable for pH 5-8 and 10-60℃, and the 60 kDa chitinase was stable for pH 4-8 and 10-50℃. Effects of metal ions on chitinase activity were also explored. Results showed that the activities of both chitinases of 35 and 60 KDa remained over 50% in a solution containing 10 mM of Ba2+, Hg2+, Mg2+ or Ag+. The main hydrolysate of colloidal chitin was (GlcNAc)2 that had 2.21 g/L for the 35 kDa and 2.3 g/L for the 60 kDa chitinase, respectively. The Km and Vmax were 8 g/L and 2,000 U/L, respectively, for the 35 kDa chitinase, and they were 4.3 g/L and 1,428.6 U/L for the 60 kDa chitinase.
A chitin-degrading bacterium, JR1, was isolated from the soil in Taipei with a medium containing 0.5% colloidal chitin. The strain reached its log phase and stationary phase at 2 h and 12 h, respectively, in medium LB. The activity of chitinases produced by strain JR1 was 366 U/L when it was cultured in medium CB containing 2% chitin powder at 30℃ for 120 h. The 75 kDa chitinase was obtained from the purification of the culture supernatant by ammonium sulfate precipitation, DEAE Sepharose CL-6B, Sephadex G-100 and SDS-PAGE. The optimum substrate concentration of crude enzyme was 15 g/L. The optimum pH and temperature were 6 and 40℃ for crude enzyme and 7 and 40℃ for the 75 kDa chitinase. The main hydrolysate of chitin powder was N-acetylglucosamine (GlcNAc), 4.79 g/L, when strain JR1 was cultured in medium CB after 24 h.

目 錄
封面內頁 頁次
簽名頁
授權書……………………….………………………….…………iii
中文摘要…………………………………………….……………..iv
英文摘要…………………………………………….……………..vi
誌謝………………………………………….…….……………..viii
目錄………………………………………………….……………...ix
圖目錄…………………………………………….….…………...xiii
表目錄………………………………………………....……….….xvi
第一章 緒論………………………………………………………..1
第二章 文獻回顧…………………………………………………..2
2-1幾丁質…………………………………………………………2
2-2幾丁質分解酶…………………………………………………7
2.2.1幾丁質分解酶分類……………………………………….7
2.2.2幾丁質分解酶的天然分佈…………………………….10
2.2.3幾丁質分解酶的命名與特性…………………………...10
2.2.4幾丁質分解酶對幾丁質的水解……..…………………11
2.2.5幾丁質分解酶的應用…………………………………...11
2.2.6 N-乙醯幾丁寡醣的特性與應用…………..……………12
2.3 酵素純化……………………………………………………..12
2.3.1硫酸銨沉澱………………………………………………13
2.3.2離子交換層析……………………………….………….13
2.3.3膠體過濾層析…………….…………………………….15
2.4幾丁質分解酶純化與特性分析……………………………16
第三章 材料與方法……………………………………………….25
3.1實驗菌株……………………………………………………25
3.2實驗材料……………………………………………………25
3.3實驗器材……………………………………………………28
3.4實驗方法……………………………………………………29
3.4.1培養基配置…………………………………………….29
3.4.2製備膠態幾丁質……………………………………….29
3.4.3幾丁質分解酶之活性測定…………………………….29
3.4.4幾丁質分解酶之純化分離…………………………….31
第四章 結果與討論………………………………...……………..39
4.1菌株型態……………………………………………………39
4.2幾丁質粉末粒徑大小之測定………………………………39
4.3 Aeromonas sp. DYU-Too 7幾丁質分解酶純化…………..39
4.3.1硫酸銨沈澱……………………………………………39
4.3.2離子交換層析…………………………………………43
4.3.3膠體過濾層析…………………………………………45
4.3.4幾丁質分解酶分子量之測定…………………………45
4.3.5純化總表………………………………………………45
4.4幾丁質分解酶之特性分析…………………………….....49
4.4.1最適反應pH值之測定……………………………….49
4.4.2最適反應溫度之測定…………………………………52
4.4.3 pH穩定性之測定…………………….……..………..52
4.4.4溫度穩定性之測定……………………………………55
4.4.5金屬離子對酵素活性之影響…………………………55
4.4.6酵素水解產物分析……………………………………58
4.4.7酵素動力學……………………………………………61
4.4.8活性電泳染色法………………………………………65
4.5篩選幾丁質分解酶生產菌株………………………………65
4.6篩選菌株之特性分析……………………………………….67
4.7 JR1菌株粗酵素液之特性分析…………………………….71
4.7.1粗酵素液之最適基質濃度……………………………..74
4.7.2粗酵素液之最適反應pH值…………………………...74
4.7.3粗酵素液之最適反應溫度…………………………….74
4.8 JR1菌株幾丁質分解酶純化……………………………….77
4.8.1硫酸銨沈澱……………………………………………..77
4.8.2離子交換層析…………………………………………..77
4.8.3膠體過濾層析………………………………………….79
4.8.4幾丁質分解酶分子量之測定(活染法)………………….79
4.9幾丁質分解酶之特性分析…………………………………84
4.9.1最適反應pH值………………………………………..84
4.9.2最適反應溫度…………………………..……………….84
4.9.3 pH穩定性之測定…………………..……...………….84
4.9.4溫度穩定性之測定…………………………..………….87
4.9.5金屬離子對酵素活性之影響……..…....…….………….87
4.9.6酵素動力學..…………….………………….………….91
4.9.7幾丁質分解酶分子量之測定(SDS-PAGE).…….……….91
第五章 結論………………………………………………………96
參考文獻……………………………………………………………97
圖 目 錄
圖2.1 幾丁質、幾丁聚醣、纖維素之化學構造 3
圖2.2 幾丁質、幾丁聚醣之製備 …5
圖2.3 幾丁質的水解物 8
圖4.1 Aeromonas sp. DYU-Too 7之革蘭氏染色顯微照片…… 40
圖4.2 以位相差顯微鏡測定幾丁質粉末之粒徑大小………….…41
圖4.3 Aeromonas sp. DYU-Too 7以CB培養所得
粗酵素液之幾丁質分解酶活性與還原醣含
量變化…………………………………………………...42
圖4.4 Aeromonas sp. DYU-Too 7培養120 h之酵
素液經DEAE Sepharose CL-6B分離之結果………….….44
圖4.5 活性波峰D1以Sephadex G-100分離之結果………….….46
圖4.6 活性波峰D2以Sephadex G-100分離之結果.…….….…...47
圖4.7 以SDS-PAGE測定Aeromonas sp.DYU-Too 7
純化過程中具活性波峰酵素液之分子量………………….48
圖4.8 pH值對35 kDa與60 kDa幾丁質分解酶
反應活性之影響………………………………………….51
圖4.9 溫度對35 kDa與60 kDa幾丁質分解酶
反應活性之影響………………………………………….53
圖4.10 幾丁質分解酶(35 kDa與60 kDa)之pH穩定性……….....54
圖4.11 幾丁質分解酶(35 kDa與60 kDa)之溫度穩定性…….…..56
圖4.12 幾丁質分解酶(35 kDa)對不同基質作用
所得之N-乙醯幾丁二糖……………...….…..………….59
圖4.13 幾丁質分解酶(60 kDa)對不同基質作用
所得之N-乙醯幾丁二糖…………….…………..………...60
圖4.14 不同基質濃度對35 kDa與60 kDa幾丁質
分解酶活性之影響……………………………….......62
圖4.15 幾丁質分解酶(35 kDa) Lineweaver-Burk
雙倒數作圖之Km與Vmax………………………………..63
圖4.16 幾丁質分解酶(60 kDa) Lineweaver-Burk
雙倒數作圖之Km與Vmax………………………………..64
圖4.17 以活性電泳染色法檢定純化過程中具活性
波峰酵素液之幾丁質分解酶……………………………66
圖4.18 篩選菌株JR1與JR2以CB培養所得粗酵素
液之幾丁質分解酶活性…………………………………68
圖4.19 篩選菌株JR1與JR2以CB培養所得之
N-乙醯葡萄糖胺…………………………………..……….69
圖4.20 篩選菌株JR1與JR2以LB培養之生長曲線……….……70
圖4.21 JR1菌株於膠態幾丁質培養基產生透明環之型態…........72
圖4.22 JR1菌株以CB培養所得粗酵素液之幾丁質
分解酵素活性、還原醣及蛋白質含量關係圖………….73
圖4.23 基質濃度對JR1菌株幾丁質粗酵素液反應
活性之影響……………………………………………….75
圖4.24 pH值對JR1菌株幾丁質粗酵素液反應活性
之影響…………………………………………………….76
圖4.25 溫度對JR1菌株幾丁質粗酵素液反應活性之影響…..….78
圖4.26 JR1菌株培養120 h之酵素液經DEAE
Sepharose CL-6B分離之結果…………………………..80
圖4.27 活性波峰A1以Sephadex G-100分離之結果…………...81
圖4.28 以活性電泳染色法測定JR1菌株粗酵素液
純化過程中具活性波峰酵素液之分子量……………...82
圖4.29 pH值對75 kDa幾丁質分解酶反應活性之影響…….......85
圖4.30 溫度對75 kDa幾丁質分解酶反應活性之影響…………..86
圖4.31 幾丁質分解酶(75 kDa)之pH穩定性……………………..88
圖4.32 幾丁質分解酶(75 kDa)之溫度穩定性………………...…..89
圖4.33 不同基質濃度對75 kDa幾丁質分解酶活性之影響……..92
圖4.34 幾丁質分解酶(75 kDa) Lineweaver-Burk
雙倒數作圖之Km與Vmax…………………………………..93
圖4.35 以SDS-PAGE測定JR1菌株純化過程中
具活性波峰酵素液之分子量………………………….…..95
表 目 錄
表2.1 幾丁質和幾丁聚醣的應用………………………………….6
表2.2 依照不同蛋白質性質所使用之分離方法………………..14
表2.3 微生物幾丁質分解酶特性之比較………………………..22
表2.3 微生物幾丁質分解酶特性之比較(續)………….…………23
表2.3 微生物幾丁質分解酶特性之比較(續)……………..……...24
表3.1 Aeromonas sp. DYU-Too 7之簡單鑑定試驗........…....26
表3.2 培養基組成………………………………………………..30
表3.3 分離膠組成………………………………………………..37
表3.4 堆積膠組成………………………………………………..38
表4.1 Aeromonas sp. DYU-Too 7 chitinases之純化總表……50
表4.2 金屬離子對35 kDa與60 kDa幾丁質
分解酵素活性之影響…………………………………...…57
表4.3 金屬離子對75 kDa幾丁質分解酶活性之影響…………90

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