Purification and Characterization of Chitinase and Protease from a Bacteria Strain TKU008 張旻璇、顏裕鴻 ; 王三郎
E-mail: 9511323@mail.dyu.edu.tw
ABSTRACT
The purpose of this study is to isolate an indigenous microorganism to degrade Shrimp and Crab shell Powder (SCSP) chitin and to secrete extracellular protease and chitinase. To found the optimal condition for protease and chitinase production. Extracellular proteins were purified by ammonium sulfate precipitation, dialysis to remove salts , ionic exchange of DEAE-sepharose CL-6B and sephadex S-100 gel filtration . The molecular mass of TKU008 protease and chitinase was determined by SDS-PAGE and gel filtration was 40 kDa and 57 kDa , approximately . Bacterium TKU 008 was isolated from soil of southern of Taiwan. Shrimp and Crab shell Powder was used to be the carbon souse of TKU008. The optimized culture condition for protease and chitinase production was found when culture was shaken in 100 mL of medium（pH6）at 30°? containing : 1 % SCSP, 0.1 % K2HPO4 , 0.05 % MgSO4．7H2O . The highest yield of protease and chitinase was produced under the optimum culture condition. The optimum pH, optimum temperature, pH stability and thermal stability of protease were pH 7, 50°?, pH 6 and 30°? . The optimum pH, optimum temperature, pH stability and thermal stability of chitinase were pH 6, 50°?, pH 7 and 30~40°? . After the purification, the yield, fold and specific activity of protease are 1%, 2-fold and 0.36 U/mL and the yield, fold and specific activity of chitinase are 50%,134-fold and 7.56 U/mL by purification procedures. The protease was characterized as a metalloprotease , due to it was inactivated by EDTA. Both of protease and chitinase activities were inhibited by Cu2+、Mn2+ . When enzymes were treated of various surfsctants , either protease or chitinase activity were stable. The effect of organic solvents on protease and
chitinase activity were also explored. The remaining activity of TKU008 protease was 90 % after keeping it at 25°? and 4°? for 10 days. Enzyme had different specific proteolytic activity with casein 、elastin、human albumin、hemoglobin as the substrates.
Keywords : chitin ; protease ; chitinase ; shrimp and crab shall power Table of Contents
頁次 封面內頁 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xii 表目錄xiv 第一章 緒言 1 第二章 文獻回顧 3 2.1幾丁質與幾丁聚醣 3 2.1.1 幾丁質與幾丁聚醣的發現 3 2.1.2 幾丁質與幾丁聚醣之化學構造與特性 3 2.1.3 幾丁質與幾丁 聚醣之製備 5 2.1.4 幾丁質與幾丁聚醣之應用 6 2.2蛋白? 13 2.2.1 蛋白?﹞岑略 13 2.2.2 蛋白?﹞坐擭?抩睇P其命名 13 2.2.3 蛋 白?﹞妖S性 15 2.2.4 蛋白質之應用 18 2.3 幾丁質? 23 2.3.1 幾丁質? 略 23 2.3.2 幾丁質?﹞擭 23 2.3.3 幾丁質?〞漱捄M分布 25 2.3.4 幾丁質?★鼢X丁質之水解 27 2.3.5 幾丁質?﹞尾野 27 第三章 材料與方法 32 3.1儀器與藥品 32 3.1.1 儀器 32 3.1.2 藥 品 32 3.2菌株 34 3.2.1 生產菌株之篩選與分離 34 3.2.2 菌株鑑定 35 3.3實驗方法 35 3.3.1懸浮態幾丁質(Colloidal chitin)之製備 35 3.3.2 蛋白質?′〝吨妥?w 35 3.3.3 幾丁質?′〝妠?w 36 3.3.4 蛋白質定量 37 3.4 蛋白?◆P幾丁質?☆?A生長條件探討 38 3.4.1 最適培養基組成之研究 38 3.4.2 最適培養條件的探討 39 3.4.3 酵素之生化特性分析 39 3.5 蛋白?◆P幾丁質之純化分離 40 3.5.1 層析膠體之前處理 40 3.5.2 酵素純化分離 41 3.5.3 純化酵素之生化特性分析 44 第四章 結果與討論 47 4.1菌株之篩 選 47 4.2菌種鑑定 47 4.3最適培養基組成之研究 47 4.3.1碳源的選擇 48 4.3.2蝦蟹殼粉含量對酵素產量之影響 48 4.4蛋白?◆P 幾丁質?☆?A生產條件探討 48 4.5粗酵素之生化特性分析 50 4.6蛋白?◆P幾丁質?﹞妖瞻 擢 61 4.6.1 粗酵素液製備 61 4.6.2 離子交換樹脂層析法 61 4.6.3 膠體過濾層析 64 4.6.4 綜合結果 64 4.6.5 SDS-PAGE 64 4.7純化酵素之生化特性分析 65 第五 章 結論 90 參考文獻 92 附錄ㄧ 製備SDS-PAGE相關藥品配法 103 圖 目 錄 頁次 圖2.1 幾丁質之化學構造 7 圖2.2 纖維素、
幾丁質與幾丁聚糖的構造 8 圖2.3 幾丁質製備流程 9 圖2.4 幾丁質進行生物轉化為單細胞蛋白之程序 31 圖4.1 不同來源之碳 源對蛋白?﹞帤X丁質?〃ㄥq的影響 52 圖4.2 蝦蟹殼粉添加對蛋白?﹞帤X丁質?〃ㄥq之影響 53 圖4.3 培養天數對酵素產量之 影響 54 圖4.4 溫度對蛋白?﹞帤X丁質?★鼤ㄥq之影響 55 圖4.5 pH對酵素產量之影響 56 圖4.6 通氣量對酵素產量之影響 57 圖4.7 TKU008所產蛋白?﹞帤X丁質?﹞坏耵囍掃u 58 圖4.8 粗酵素蛋白?◆P幾丁質?﹞妊抩A反應溫度 59 圖4.9 粗酵素蛋白?
◆P幾丁質?﹞妊抩A反應pH值 60 圖4.10 TKU008所生產蛋白?◆P幾丁質?﹞妖瞻 擢鰿y程圖 62 圖4.11 蛋白?◆P幾丁質?﹞
伶EAE-Sepharose CL-6B層析圖譜 63 圖4.12 蛋白質?◆P幾丁質?﹞刨ephacryl S-100層析圖譜 67 圖4.13 SDS-PAGE檢
測TKU008所產蛋白?◆P幾丁質?○J白質帶 70 圖4.14 SDS-PAGE 分子量與相對移動速率之關係 71 圖4.15 TKU008蛋白?◆P 幾丁質?﹞妊抩A反應溫度 72 圖4.16 TKU008蛋白?◆P幾丁質?﹞妊抩A反應pH值 73 圖4.17 TKU008蛋白?◆P幾丁質?﹞尬 鬖w定性 74 圖4.18 TKU008蛋白?◆P幾丁質?﹞吠H安定性 76 圖4.19有機溶劑對酵素活性之影響 82 圖4.20蛋白?’b有機溶 劑中儲存安定性 83 圖4.21幾丁質?’b中有機溶劑儲存安定性 84 圖4.22 TKU008蛋白?﹞妊抩A反應基質濃度 86 圖4.23 TKU008幾丁質?﹞妊抩A反應基質濃度 87 圖4.24蛋白?‥ineweaver-Burk雙倒數作圖之Km與Vmax 88 圖4.25幾丁質?
‥ineweaver-Burk雙倒數作圖之Km與Vmax 89 表 目 錄 頁次 表2.1製備不同去乙醯度之幾丁聚醣所需鹼液濃度及溫度 10 表2.2幾丁質及幾丁聚醣之特性與應用 11 表2.3不同來源的蛋白?﹞巹S性 17 表2.4不同微生物所產金屬型蛋白?﹞坐騆 20 表2.5蛋白?’b工業上的應用 21 表2.6不同微生物所產幾丁質?﹞坐騆 30 表4.1 TKU008蛋白?﹞妖瞻 妣n 68 表4.2 TKU008 幾丁質?﹞妖瞻 妣n 69 表4.3 數種化學物質對蛋白?◆P幾丁質?′〝吨尬v響 77 表4.4 界面活性劑對蛋?◆P幾丁質?′〝吨 尬v響 78 表4.5 TKU008所生產蛋白?﹞妍羷霂S異性 79
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