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系統識別號 U0026-2107201516384900
論文名稱(中文) 四種新型重組葉綠素酶的生化特性分析以及酵素固定化之應用
論文名稱(英文) Biochemical characterization of four novel recombinant chlorophyllases and the enzyme affinity immobilization
校院名稱 成功大學
系所名稱(中) 生物科技研究所
系所名稱(英) Institute of Biotechnology
學年度 103
學期 2
出版年 104
研究生(中文) 周宜黎
研究生(英文) Yi-Li Chou
學號 L68951029
學位類別 博士
語文別 英文
論文頁數 111頁
口試委員 指導教授-蕭介夫
口試委員-陳榮芳
口試委員-陳錦樹
口試委員-曲芳華
口試委員-李冠群
口試委員-洪哲穎
口試委員-楊智惠
中文關鍵字 葉綠素酶  葉綠酸衍生物  酵素固定化  固定化金屬離子親和層析法 
英文關鍵字 Chlorophyllase  Chlorophyllide derivatives  Enzyme immobilization  Immobilized metal ion affinity chromatography 
學科別分類
中文摘要 葉綠素酶,主要功能為催化水解葉綠素,產生葉綠酸和植醇,是葉綠素降解途徑中最重要參與水解的酵素。在本研究中,使用固定化金屬離子親和層析法將重組青花菜葉綠素酶1號固定化並且可從粗細胞液中純化出來。與非固定化酵素相比,生化特性分析顯示出固定化酵素在弱鹼環境和高溫環境中具有較高的葉綠素酶活性。此外,固定化酵素在60 ℃下可有效的提高酶的2倍半衰期時間,並且可以提高酵素重複使用17次的操作穩定性。因此,固定化酵素可以重複使用,以降低成本,並且適合用於工業上來生產葉綠酸和植醇的衍生物。
我們分別從馬拉巴栗、莱茵衣藻及固氮藍綠菌ATCC 51142中分離出四種新型重組葉綠素酶基因,並分別在大腸桿菌中表達以及純化。重組馬拉巴栗葉綠素同功酶 (PmCLH1和PmCLH2)以及莱茵衣藻葉綠素酶1號 (CrCLH1)皆包含一個保守脂肪酶區域 (GHSRG) 和催化三元體 (Ser-Asp-His),然而重組固氮藍綠菌ATCC 51142葉綠素酶 (CyanoCLH) 包含一個保守脂肪酶區域 (GHSLG) 和催化二元體 (Ser-Asp)。在生化特性分析下發現PmCLH1,PmCLH2和CrCLH1在pH 6和 40 ℃下具有較高的葉綠素酶活性,而 CyanoCLH在pH 7和60 ℃下具有較高的葉綠素酶活性。酵素動力學分析表明,CrCLH1針對葉綠素a和葉綠素b的催化水解能力高於 PmCLH1和 PmCLH2,而CyanoCLH 則針對菌葉綠素a的催化水解能力遠高於葉綠素a和葉綠素b的水解。CyanoCLH能水解菌葉綠素獲得菌葉綠酸,而此菌葉綠酸可當作光動力學療法的前驅物。因此,由上述結果可知CrCLH1和 CyanoCLH可以當作生物催化劑來生產葉綠酸衍生物和菌葉綠酸前驅物用於醫學應用和製藥工業。
英文摘要 Chlorophyllase (Chlase) is the key catabolic enzyme to catalyze chlorophyll breakdown to produce chlorophyllide and phytol. In this study, immobilized metal ion affinity chromatography technique were used for simultaneous the recombinant Brassica oleracea chlorophyllase 1 (BoCLH1) with poly(His)-tagged at the C-terminal of an enzyme purification and immobilization. Biochemical analysis of the immobilized enzyme showed higher chlorophyllase activity for chlorophyll a hydrolysis in a weak base environment (pH 8.0) and high-temperature environment compared with the free enzyme. In addition, the enzyme half-life (t1/2) of the immobilized BoCLH1 increased from 25.42 to 54.35 min (approximately two-fold) at 60 °C compared with the free enzyme. The immobilized enzyme retained a residual activity of approximately 60% after 17 cycles in a repeated-batch operation. Therefore, the immobilized enzyme can be repeatedly reused to lower costs and is potentially useful in the industrial production of chlorophyllide and phytol.
The four novel recombinant Chlases were isolated from higher plant (Pachira macrocarpa, PmCLH1 and PmCLH2), algae (Chlamydomonas reinhardtii, CrCLH1), and photosynthetic bacterium (Cyanothece sp. ATCC 51142, CyanoCLH), and were expressed in Escherichia coli (DE3) and purified for the biochemical characterization. The recombinant PmCLH1, PmCLH2 and CrCLH1 contained a conserved GHSRG lipase motif and catalytic triad Ser-Asp-His, and the recombinant CyanoCLH comprised a conserved GHSLG lipase motif and a catalytic dyad Ser-Asp. The biochemical characterization of the recombinant PmCLH1, PmCLH2 and CrCLH1 revealed higher activity at pH 6 and 40 °C, whereas the recombinant CyanoCLH exhibited higher activity at pH 7 and 60 °C. Kinetic analysis revealed that the recombinant CrCLH1 exhibited higher activity for chlorophylls hydrolysis than did the recombinant PmCLH1 and PmCLH2, whereas the recombinant CyanoCLH exhibited higher activity for bacteriochlorophyll a hydrolysis to produce bacteriochlorophyllide a, which is a bacteriochlorin a precursor. Therefore, the recombinant CrCLH1 and CyanoCLH can be used as biocatalysts to produce chlorophyllide and bacteriochlorin a precursor for medical and pharmaceutical applications.
論文目次 Contents
Chinese Abstract (中文摘要)............................. I
Abstract.............................................. II
Acknowledgements..................................... III
Contents.............................................. IV
List of Tables........................................ VI
List of Figures...................................... VII
Abbreviation.......................................... IX
Chapter 1 Research background.......................... 1
1-1 Chlorophyllase..................................... 1
1-2 Applications of Chlide and phytol derivatives...... 3
1-3 Enzyme immobilization.............................. 4
1-4 Types of immobilization............................ 5
1-5 Research motivation................................ 9
Chapter 2 Materials and methods....................... 11
2-1 Materials......................................... 11
2-2 Buffer preparation................................ 14
2-3 Experimental section.............................. 20
Chapter 3 Purification and immobilization of the recombinant BoCLH1 using immobilized metal ion affinity chromatography for producing chlorophyllide and phytol................................................ 28
3-1 Introduction...................................... 28
3-2 Results and discussion............................ 31
3-3 Summary........................................... 38
Chapter 4 Gene cloning and biochemical characterization of four novel recombinant chlorophyllases as potential biocatalysts for producing chlorophyllide and phytol derivatives........................................... 39
4-1 Introduction...................................... 39
4-2 Results and discussion............................ 41
4-3 Summary........................................... 51
Chapter 5 Conclusions................................. 52
References............................................ 54
Appendix.............................................. 99
Formulations......................................... 109
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