Characterization and site-directed mutagenesis from rumen fungi of the recombinant XynR8 xylanase

• Main Authors: CHIANG YU-CHUAN, 江育全
• Other Authors: CHENG HSUEH-LING
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/74861804486305486897

碩士 === 國立屏東科技大學 === 生物科技研究所 === 94 === Previously, a xylanase (EC 3.2.1.8) gene was subcloned from rumen fungi of Bubalus bubalis, named xynR8. The catalytic activity of XynR8 was found to be relatively estimated tobe at least 120-fold higher than other previously published xylanases. Thus, in this study, the gene of xynR8 was subcloned into pET21c vector and expressed in E.coli BL21(DE3). The resulting protein was purified by methods including affinity chromatography and characterized for its biochemical and kinetic properties. Km and kcat of the enzyme were determined to be 11.1 mg/ml and 389432.4 sec-1 respectively. The specific activity of the enzyme was estimated tobe at least 200-fold higher than those of commercially available xylanases. In addition, XynR8 appeared to have a broader range of pH tolerance (pH3~11). To increase its value in industrial application and to study its structure-function relationship, site-directed mutagenesis was conducted. Mutant XynR8(N41D) and XynR8(N58D) showed a better pH stability than the wild-type enzyme, whereas mutants XynR8(N85D), XynR8(I165K) and XynR8(I165R) showed no catalytic activities. Structure modeling by Deep view showed that the three-dimensional structure of XynR8 contained one additional α-helix than the general structure of family 11 xylanases. Whether this structural variation confers the high catalytic activity of XynR8 needs further investigation. In addition, N41 and N58 both were predicted to locate within two different loops the influence of these loop on the pH stability of XynR8 may be further explored.