Construction and characterization of bifunctional fibrolytic enzymes

植物的細胞壁主要由纖維素、半纖維素與木質素等構成，而這種複雜的結構較不易被單一纖維分解酵素所分解，進而造成利用上的困難，因此近年來許多研究利用基因工程技術融合多種纖維素分解酵素或半纖維素分解酵素基因，建構出具雙功能或多功能之纖維分解嵌合酵素，與單一功能纖維分解酵素相比，多功能或雙功能纖維分解酵素除了可提升分解植物細胞壁纖維的效率之外，在酵素製程方面亦節省了時間與金錢成本。 本研究係將兩段分別由瘤胃真菌Piromyces rhizinflatus選殖出的β-聚葡萄糖酶 (β-glucanase) cbhYW23-2基因與由Neocallimastix patriciarum選殖出的聚木糖酶 (xylanase) xynCDBFV基因以兩種不同排列順序進行嵌合，表達出分別為β-聚葡萄糖酶在胺端的Cbh-Xyn以及聚木糖在胺端的Xyn-Cbh兩種嵌合纖維分解酵素。β-聚葡萄糖酶與聚木糖酶兩酵素之間由富含甘氨酸的連接胜肽 (GGGGS)2作為連結，以確保酵素間保有各自正確摺疊的空間以及彼此構形的完整性。所得到之嵌合酵素基因序列長度皆為2202個含氮鹼基，表達出的酵素由734個胺基酸組成，分子量約為87 kDa。嵌合酵素以大腸桿菌表達並以親和性管柱純化後，測試酵素活性並與單一酵素進行比較，再以反應曲面法 (response surface methodology) 結合中央合成設計 (central composite design) 及迴歸分析，測定兩嵌合酵素之最適溫度及pH值，結果測得Cbh-Xyn在pH 5.9與52 ℃的條件下有最佳的聚木糖酶活性，比活性為740.6 ± 24 U/mg，而在pH 6.0與44 ℃時有最佳的β-聚葡萄糖酶活性，比活性為1518.3 ± 31 U/mg；而Xyn-Cbh的最適反應條件則是在pH 6.2與50 ℃的條件下有最佳的聚木糖酶活性，比活性為3121 ± 153 U/mg，而在pH 6.1與46 ℃時有最佳的β-聚葡萄糖酶活性，比活性則為2526 ± 206 U/mg。另以天然稻稈作為受質進行酵素水解，亦證實在此最適反應條件下，嵌合纖維分解酵素Xyn-Cbh處理所釋出的還原糖量較單一酵素處理所釋出的還原糖量高。 綜上所述，本研究成功利用反應曲面法得到嵌合纖維分解酵素之最適反應條件，並且證實嵌合酵素Xyn-Cbh具有較佳的酵素活性，有潛力廣泛應用於各種不同條件的生物技術與工業用途。Plant cell walls are comprised of cellulose, hemicellulose and lignin. This complex structure acts as a barrier for degradation by fibrolytic enzymes. Therefore, bifunctional or multifunctional fibrolytic enzymes are more efficient in hydrolysis of plant cell walls and more cost and time saving in enzyme production as compared to the single functional enzymes. In this study, two chimeric fibrolytic enzymes were constructed by fusion of cbhYW23-2, a β-glucanase gene from ruminal fungus Piromyces rhizinflatus, and xynCDBFV, a xylanase gene from Neocallimastix patriciarum. One of the chimeric enzymes was fused the CbhYW23-2 with the N-terminus of XynCDBFV (Cbh-Xyn) and the other was fused the CbhYW23-2 with the C-terminus of XynCDBFV (Xyn-Cbh). A Gly-rich flexible linker (GGGGS)2 was introduced between CbhYW23-2 and XynCDBFV in order to retain the independent folding of domains and the conformational freedom relative to one another. The resultant chimeric enzymes were composed of 734 amino acid residues with a predicted molecular weight of 87 kDa. To examine the enzyme activities, the parental enzymes, CbhYW23-2 and XynCDBFV, and the chimeric enzymes, Cbh-Xyn and Xyn-Cbh, were heterologously expressed by Escherichia coli and purified by immobilized metal ion-affinity chromatography. Response surface modeling (RSM) combined with central composite design (CCD) and regression analysis were then employed for the planned statistical optimization of the enzyme activities of these two chimeric enzymes. As the results, the optimal reaction condition for the highest xylanase activity of Cbh-Xyn was observed at pH 5.9 and 52 ℃ with specific activity of 740.6 ± 24 U/mg, whereas the highest β-glucanase activity was observed at pH 6.0 and 44 ℃ with specific activity of 1518.3 ± 31 U/mg. The optimal reaction conditions for the highest xylanase and β-glucanase activity of Xyn-Cbh were observed at 50 ℃ and pH 6.2 and at 46 ℃ and pH 6.1 with specific activity of 3121 ± 153 and 2526 ± 205.7 U/mg, respectively. Under the optimal conditions, the chimeric enzyme Xyn-Cbh had higher hydrolytic activities toward rice straw than the parental enzymes. In conclusion, the results suggested that the chimeric enzyme Xyn-Cbh showed a high hydrolytic activity and has potential for use in a range of various biotechnological and industrial applications