Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20218
標題: Phanerochaete chrysosporium ATCC 24725 之木質素過氧化氫酶H8基因的選殖與異源表現
Cloning and heterologous expression of the lignin peroxidase isozyme H8 gene of Phanerochaete chrysosporium ATCC 24725
作者: 劉盈秀
Liu, Yin-Hsiu
關鍵字: 生質能源
lignocellulose
白腐真菌
黃孢亮毛伏革菌
木質素過氧化氫酶
藜蘆醛生成法
ABTS陽離子生成法
bioenergy
Phanerochaete chrysosporium
LiP H8
Clostridium xylanolyticum Ter3
VA assay
ABTS assay
出版社: 生命科學系所
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摘要: 自二十世紀中石化燃料迅速地被開發,直到今日,人類過度使用石化燃料,不僅造成溫室效應等嚴重的環境破壞,也使全球蘊藏量日漸枯竭,因此積極尋找乾淨且可再生的替代能源成了當務之急,生質能源之開發即為其中之一。生質能源指的是將生物質加工製造或轉換而得到之能源,其中,以木質纖維素為原料來製造產生的生質能源將會是可見未來的主力,因為木質纖維素是地球上含量最多的碳水化合物,大量存在於農林業作物及其廢棄物中。木質纖維素包含纖維素、半纖維素以及木質素,其中纖維素以及半纖維素外面包覆著一層難以被分解的木質素,使得纖維素與半纖維素很難被利用。目前僅知白腐真菌是地球上最能分解木質素的微生物,尤其是Phanerochaete chrysosporium所產生的木質素分解酶能完整且有效率地分解木質素。實驗室有一株具有纖維素酶和半纖維素酶且又能產生氫氣和乙醇的梭狀芽孢菌屬菌株Clostridium xylanolyticum Ter3,為了讓Ter3有效率地利用木質纖維素當原料來生產氫氣與乙醇,本研究嘗試選殖P. chrysosporium的木質素分解酶LiP H8基因,使其能在C. xylanolyticum Ter3表現分解活性。另外一方面,也選殖lip H8基因到E. coli與 Pichia pastoris,目的是希望利用它們生長快速、容易培養以及可大量表現蛋白等優點產生大量LiP H8蛋白。利用E. coli與Clostridium屬細菌的穿梭載體pIMPI構築成一個帶有LiP H8基因的重組質體pIMPI-lac+lip,以電穿孔轉形方式送入C. xylanolyticum Ter3,發現沒有得到任何的轉形株,推測C. xylanolyticum Ter3可能含有會辨認並切割外來DNA上特別序列的未知限制酶系統,以致送入的重組質體pIMPI-lac+lip無法存在於C. xylanolyticum Ter3。本研究利用藜蘆醛生成法與ABTS陽離子生成法檢測LiP H8蛋白之活性,用藜蘆醛生成法測得原始菌株P. chrysosporium 的LiP H8 蛋白最高活性為50 U/L,用ABTS陽離子生成法測得的活性是101.83 U/L。將lip H8基因構築於表現載體pET43.1a(+)中,使重組質體pET43.1a-lip表現於E. coli BL21 (DE3),能做出於N端加上Nus-tag後約108 kD大小的正確重組蛋白LiP H8,用藜蘆醛生成法測得到的最高LiP H8蛋白活性為0.56 U/L。另外將LiP H8基因構築於質體pPICZαA中,將重組質體pPICZαA-mlip電穿孔轉形方式表現於P. pastoris中,用藜蘆醛生成法有測得LiP H8蛋白最高活性為1.04 U/L,但在SDS-PAGE圖上則是沒有看到重組LiP H8蛋白的表現。
The exploitation of fossil fuels have rapidly grown since the middle of twentieth century. To today, excessive use of fossil fuels by human beings not only caused greenhouse effect and serious environmental pollution but also gradually exhaust its deposits in the world. So, it has become a top priority to search for clean and renewable alternative energy sources, the development bioenergy is one of them. Bioenergy is renewable energy made available from materials derived from biological source, lignocellulose is particularly well-suited for production because of it is the most abundant carbohydrate on the earth and rich in agricultural and forestry plants and their waste. Lignocellulose is composed of cellulose, hemicellulose and lignin, the aggregates of cellulose and hemicellulose are surrounded by rigid and hard- decomposed lignin that hinder cellulose and hemicellulose to be utilized. No better than the white rot fungi especially Phanerochaete chrysosporium can produce ligninase to decompose lignin completely and efficiently in the microbial world. Clostridium xylanolyticum Ter3 is an isolate of our lab that not only can synthesize cellulase and hemicellulase to decompose cellulose and hemicellulose, but also produce H2 and ethanol very well. In order to let C. xylanolyticum Ter3 utilize lignocellulose to produce H2 and ethanol efficiently, an aim of this study is to clone and express the ligninase gene (lip H8 gene) of P. chrysosporium in C. xylanolyticum Ter3. The lip H8 gene was also tried to express in E. coli and Pichia pastoris, owing to as a host they have many advantages such as ease of culture, rapid growth, and high-level expression of a cloned gene. By using the shuttle vector pIMPI of E. coli and Clostridium spp. to clone the lip H8 gene, recombinant plasmid pIMPI-lac+lip was constructed, but the following effort to transform this recombinant plasmid to C. xylanolyticum Ter3 by electroporation was unsuccessful, it seems that C. xylanolyticum Ter3 contained unknown restriction enzyme system to digest pIMPI-lac+lip. Two methods was used to measure the LiP H8 activity in this study:VA and ABTS assay. The highest LiP H8 activity expressed by P. chrysosporium was 50 U/L by VA assay and 101.83 U/L by ABTS assay. When the lip H8 gene was cloned into expression vector pET43.1a(+) to form recombinant plasmid pET43.1a-lip and transformed to E. coli BL21 (DE3), a 108-kDa fusion protein with Nus-tag linking to the N terminal of LiP H8 enzyme protein was produced, the highest enzyme activity shown by this fusion protein was 0.56 U/L by VA assay. The lip H8 gene was cloned into vector pPICZαA to form recombinant plasmid pPICZαA -mlip, when it was transformed to P. pastoris, the highest LiP H8 activity of 1.04 U/L was measured by VA assay, but no fusion protein with correct size was found by SDS-PAGE.
URI: http://hdl.handle.net/11455/20218
其他識別: U0005-2108201323235500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2108201323235500
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