Protein Engineering and Biochemical Characterization of Amylopullulanase from Thermophilic Bacteria Thermoanaerobacter ethanolicus 39E

• Main Authors: Yi-Hsuan Ho, 何宜璇
• Other Authors: Fu-Pang Lin
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/42749022147677549141

碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 97 === The functional and structural significance of the C-terminal fibronectin type III (FnIII) motif of Thermoanaerobacter ethanolicus 39E amylopullulanase (TetApu) was explored using C-terminal truncation mutagenesis. Comparative studies among the engineered molecules of TetApuM955 with one FnIII, TetApuR855 with part of the second FnIII and TetApuQ818 without the second FnIII included initial rate kinetics, fluorescence and CD spectrometric properties, thermostability, substrate binding and hydrolysis abilities. Kinetic analysis revealed that the overall catalytic efficiency, kcat/Km, was 30 percent decreased compared with that of TetApuM955 for the TetApuQ818 truncated enzyme toward the soluble starch. Changes of soluble starch and pullulan subatrate affinity (Km) and catalytic rate constant (kcat) of TetApuQ818 were in different directions. TetApuQ818 retained a similar substrate-binding ability against the raw starch substrate as TetApuM955 and TetApuR855 had. Fluorescence and CD spectroscopy analyses indicated that TetApuQ818 retained the active folding conformation similar to TetApuM955 and TetApuR855. A CD-melting unfolding study indicated that the apparent transition temperature among TetApu955, TetApuR855 and TetApuQ818 was similar. These results indicate that the complete removal of the second C-terminal fibronectin type III (FnIII) motif of TetApuM955 was accepted for the amylopullulanase enzyme activity.