Construction and characterization of bifunctional fibrolytic enzymes

• Main Authors: Meng-Shan Wu, 吳孟珊
• Other Authors: 劉嚞睿
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/40179850858592933442

碩士 === 國立臺灣大學 === 動物科學技術學研究所 === 100 === 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.