- 學位論文
雙功能酵素1,3-1,4-β-D-葡聚醣水解酶與β-1,4-聚木醣酶 純化與特性分析
Purification and characterization of a chimeric 1,3-1,4-β-D-glucanase and β-1,4-xylanase enzyme
摘要
Fibrobacter succinogenes 1,3-1,4-β-D-葡聚醣水解酶(Fsβ-glucanase , E.C. 3.2.1.73)及uncultured Neocallimastigales β-1,4-聚木醣酶(XynR8, E.C. 3.2.1.8)皆是從牛胃中選殖出來的酵素。它們能有效且專一性極高地分別水解穀類植物中的β-D-葡聚醣(β-D-glucan)、地衣聚醣(lichenan)中緊鄰β-1,3鍵結之後β-1,4鍵結位置及水解聚木醣之主鏈沒有取代基或支鏈之β-1,4鍵結位置。我們想要製造出一雙功能酵素，於是利用基因重組的方式將兩種不同來源、不同性質的截短型酵素接在一起後，藉由大腸桿菌去大量表現，並利用親合層析管柱(Ni-NTA)純化，將所純化出來之重組融合酵素進行生化特性之分析。分析結果顯示此重組融合酵素同時具有葡聚醣水解酶與聚木醣酶之活性，與個別酵素相比，此重組融合酵素之熱穩定範圍為30~55℃且耐酸鹼範圍一樣頗寬(pH 3~10)。就葡聚糖水解酶部份而言，此重組融合酵素之催化效率增加約1.8倍；而以聚木醣酶的部份來說降低約0.9倍。此重組融合酵素於X-ray結晶學研究正在進行當中。
並列摘要
Truncated Fibrobacter succinogenes 1,3-1,4-β-D-glucanase (TG) specifically hydrolyzes 1,4-β-D-glucosidic bonds adjacent to 1,3-β-linkages in mix-linked β-glucans or lichenan. Truncated uncultured Neocallimastigales xylanase (TX) is a hydrolytic enzyme which cleaves the β-1,4 glycosidic bonds of the complex plant cell wall polysaccharide xylan. A chimeric TG and TX was expressed successfully as a 6xHis tagged recombinant protein in Escherichia coli BL21 (DE3). The purified recombinant chimeric protein exhibited both TG and TX catalytic activities. Optimal temperature 45 ℃ and pH 7.5 were found for the TG activity, whereas 55 ℃ and pH 7.0 were determined for the TX activity. Compared with the wild type single enzyme activity, the TG increased 1.8-fold and TX activity decreased 0.9-fold in catalytic efficiency value (kcat/Km), respectively. Recombinant chimeric enzyme was stable when incubated in 30-55 ℃ and pH 3-10. This is the first successful attempt to fuse glucanase and xylanase as one chimera while retaining individual enzymatic activities. X-ray crystallographic study for the recombinant protein is in progress.