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研究生:蔡嘉煌
研究生(外文):Tsai, Chia-Huang
論文名稱:芽孢桿菌屬甲殼素水解酶的立體障礙對其水解產物組成之影響
論文名稱(外文):Influence Of Steric Hindrance On Hdrolysate Distribution Of Bacillus Chitosanase
指導教授:鄭至玉鄭至玉引用關係
指導教授(外文):Cheng, Chih-Yu
口試委員:張瑞璋陳榮輝
口試委員(外文):Chang, Rey-ChangChen, Ronf-Huei
口試日期:2012-07-23
學位類別:碩士
校院名稱:國立高雄海洋科技大學
系所名稱:海洋生物技術研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:76
中文關鍵詞:甲殼素甲殼素水解酶
外文關鍵詞:chitosanchitosanase
相關次數:
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Bacillus circulans MH-K1甲殼素水解酶被分類在醣類水解第46家族,為一內切型水解酶,其水解產物是以甲殼二醣與三醣為主之甲殼寡醣混合物。為得到產物只有甲殼三醣的外切型甲殼素水解酶,本研究重新檢視其蛋白質與受質的結構關係,除模擬設計可限制受質長度的外切擋板(Exo-loop)並加以突變外,也以飽和定點突變第174、175個胺基酸消減此酵素對受質+2醣之吸引力。
外切擋板的設計為在第180至182個胺基酸位置插入2至8個胺基酸,經蛋白質結構模擬後,選定5個設計進行突變。各突變株於E. coli BL21(DE3)誘導表達蛋白質、粗抽蛋白以及進行對甲殼素水解後,以TLC比較各突變株與野生型之水解產物組成。結果顯示,各突變株仍具有內切型活性,其中一株TMH8之水解產物甲殼二醣產量明顯提高,其外切擋板胺基酸序列為RPKL。
為大量篩選飽和定點突變之基因庫,開發了原位篩選法(In-situ screening)。此方法直接挖取甲殼素水解酶檢測盤(Chitosanase-detection agar plate)上的透明環,進行TLC分析水解產物組成。藉由此方法,從第174、175個胺基酸飽和定點突變基因庫中,篩選了218株突變株。其中,只有58株具有活性,能在甲殼素酶檢測盤中產生透明環,此區的突變易造成酵素失活的現象,說明此區胺基酸在此酵素具一定的影響力。進行核苷酸定序後,分別得到2株較高活性(CSN-II及CSN-TI)、2株較低活性(CSN-LN及CSN-NL)及3株未測得活性(CSN-KT、CSN-HG及CSN-IN)的突變株序列。測試結果發現,活性較低的突變株,大多形成包涵體,利用20℃低溫誘導可以使其恢復正確折疊,改善不溶狀態。TLC結果顯示,此區域突變後,大多會使甲殼二醣產量明顯下降。本研究選擇水解產物有明顯改變的兩個突變株CSN-TI及CSN-LN進行產物分析。在不同反應時間(18至144分鐘),野生型主要水解產物為甲殼二醣,而CSN-TI與CSN-LN為甲殼三醣。將反應終點(30小時)的水解產物TLC圖量化,並計算其反應終產物甲殼三醣與甲殼二醣之比值,顯示野生型、CSN-TI及CSN-LN分別為0.61±0.04、0.94±0.06及1.4±0.1。並且,野生型之反應終產物中,甲殼四醣已幾乎完全被水解,而CSN-TI及CSN-LN尚有約10%未被水解。這證實突變後酵素對受質+2醣的吸引力已然下降。
此研究成功消減酵素對受質+2醣的吸引力並改變水解產物組成,並且發展出可快速判讀水解產物組成之原位篩選法,以及用於快速純化甲殼素水解酶的選擇性沉澱法。以此為基礎,將有機會得到產生單一水解產物為甲殼三醣之外切型水解酶,或是水解產物更靠近甲殼六醣或七醣之甲殼素水解酶。

The chitosanase from Bacillus circulans MH-K1 is classified in glycoside hydrolase family 46. The degree of polymerization of hydrolysates are higher than 2 causing by its endo-splicing pattern (-2)(-1)(1)(2)(3)(4). In order to generate an exo-chitosanase with major product of trimer, the structure relationship between enzyme and substrate were investigated. According to the structures, two strategies were designed—an exo-loop mutagenesis and a saturation mutagenesis at A174/L175.
Firstly, 32 exo-loops with 2-8 amino acids were inserted computationally between Thr180 and Gly182. Five of them were selected and further mutated by site-directed mutagenesis. After over-expression in E. coli BL21(DE3), crude extraction of exo-loop mutants was obtained for chitosan hydrolysis, and the hydrolysates were assayed by TLC. All of the mutants have the ability of endo-type chitosanase activity. However, the hydrolysis products were trended to (GlcN)2 by mutant TMH8 in which the sequence of exo-loop was RPKL.
Secondly, a saturation mutagenesis was performed on residues 174 and 175 for weakening the binding between enzyme and the +2 position of substrate. For high-throughput screening of the mutation library, an in-situ screening was established. It is an efficient screening method that analyzes the hydrolysate of clear zone from chitosanase-detection agar plate by TLC. After in-situ screening of 216 mutants, only 58 mutants with chitosanase activities were obtained. Low colony-count with chitosanase indicated that these two residues are critical for Bacillus chitosanase. After DNA sequencing, 2 higher activity mutants (CSN-II and CSN-TI), 2 lower activity mutants (CSN-LN and CSN-NL), and 3 non-activity mutants (CSN-KT, CSN-HG and CSN-IN) were identified. When cultured in LB medium, most mutated chitosanase were expressed as inclusion bodies, and most of them were resolved by lower induction temperature. According to TLC results, (GlcN)2 of hydrolysate distribution was almost reduced by mutants. Two mutants, CSN-TI and CSN-LN, were selected for their particularly different products. The hydrolysate distribution of wild type, CSN-TI and CSN-LN were analyzed by TLC from 18 to 144 minutes. During the hydrolysis reaction, the main product of wild type, CSN-TI and CSN-LN were (GlcN)2, (GlcN)3 and (GlcN)3, respectively. The end product (after 30 hours) was analyzed by TLC and quantification. The (GlcN)3 / (GlcN)2 ratio of wild type, CSN-TI and CSN-LN were 0.61 ± 0.04、0.94 ± 0.06 and 1.4 ± 0.1, respectively. Besides, (GlcN)4 was almost hydrolysed by wild type; but remaining about 10% by CSN-TI and CSN-LN. It represents that mutagenesis on residues 174 and 175, especially A174T/L175I and A174L/L175N, do weaken the binding of the +2 position of substrate. Based on the mutagenesis of exo-loop and residues corresponding to the +2 position of substrate, more chitosanase with potential hydrolysate can be designed in the near future.
中文摘要 i
英文摘要 iii
誌謝 v
目錄 vi
表目錄 viii
圖目錄 ix
第一章 前言 1
1.1甲殼素之來源、結構與功能 1
1.2甲殼寡醣之定義、功能與來源 2
1.3甲殼素水解酶(Chitosanase)的分類 2
1.3.1依受質(Substrate)專一性分類 2
1.3.2依一級結構分類 3
1.3.3依內切型(Endo-type)、外切型(Exo-type)分類 4
1.4水解產物與酵素截切方式之關係 4
1.4.1外切型葡萄糖胺水解酶之水解產物 4
1.4.2內切型甲殼素水解酶之水解產物 5
1.5外切擋板(Exo-loop)的計設與限制 5
1.5.1外切擋板限制受質長度的研究 5
1.5.2使用外切擋板限制受質長度的局限 8
1.6醣類水解第46家族之蛋白質三維結構與胺基酸殘基的功能 9
1.6.1 GH 46之蛋白質三維結構 9
1.6.2 GH 46胺基酸活性催化殘基 10
1.6.3 GH 46胺基酸殘基功能之研究 10
1.7 B. circulans MH-K1甲殼素水解酶與受質的作用力 12
1.7.1可能形成氫鍵的胺基酸位置 12
1.7.2酵素與受質+2醣吸引之胺基酸 15
1.8研究目的及策略 16
第二章 實驗材料與方法 17
2.1實驗器材與材料 17
2.2實驗方法 17
2.2.1質體的建構 17
2.2.2質體的轉形作用 18
2.2.3蛋白質的誘導表達 19
2.2.4酵素的純化 19
2.2.5酵素活性與產物組成分析 20
2.2.6酵素基因突變 22
2.2.7原位篩選法(In-situ screening) 23
2.2.8蛋白質結構預測與模擬 24
第三章 實驗結果與討論 26
3.1野生型甲殼素水解酶的製備與純化 26
3.2野生型甲殼素水解酶之水解產物組成 28
3.3外切擋板的設計與其水解產物組成 29
3.3.1外切擋板的設計 29
3.3.2外切擋板突變株及其水解產物組成 31
3.4原位篩選法的建立 33
3.5受質+2醣引力的消減 34
3.5.1受質+2醣引力消減之設計與突變 34
3.5.2飽和定點突變的篩選及其產物組成 35
3.5.3飽和定點突變的核酸定序與蛋白質表現 36
3.5.4 CSN-TI與CSN-LN的酵素純化及水解產物組成 39
3.5.5第174、175個胺基酸對酵素結構與受質黏合的影響 45
第四章 結論 49
第五章 未來發展 51
參考文獻 52
附錄 54
附錄1:二十種胺基酸的分類及性質 54
附錄2:不含訊息胜肽之DNA及胺基酸序列 55
附錄3:選擇性沉澱法 56
附錄4:不同反應時間之還原醣產物量 62

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