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  1. NTU Theses and Dissertations Repository
  2. 生物資源暨農學院
  3. 生物科技研究所
請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8039
標題: 海棲熱袍菌纖維素酶 Cel12A 之結晶結構研究及其基因改造
Crystal structural studies and genetic engineering of Thermotoga maritima cellulase Cel12A
作者: Ya-Shan Cheng
鄭雅珊
指導教授: 劉?睿
關鍵字: 耐熱纖維素&#37238,蛋白結構,基因改造,定點突變,
thermostable cellulase,endoglucanase,protein structure,X-ray crystallography,protein engineering,enzymatic activity,site-directed mutagenesis,
出版年 : 2012
學位: 博士
摘要: Cellulose is the major component of plant cell wall and the most abundant renewable biomass on earth. Cellulases which can hydrolyze cellulose into simple sugar have been used in many industrial applications such as biofuel production and food industry for a long time. Despite cellulases have been widely utilized, most of current industrial cellulases are from mesophile and do not have good thermostability. Cellulase 12A, which was isolated from an anaerobic hyperthermophilic bacterium Thermotoga maritima (TmCel12A), belongs to glycoside hydrolase family 12. It is a hyperthermostable β-1,4-endoglucanase which can randomly degrade cellulose molecules into smaller fragments, facilitating further utilization of carbohydrate. In this study, the crystal structures of TmCel12A and its complex form with oligosaccharides were both determined by X-ray crystallography. The TmCel12A structure shows a typical β-jelly roll protein folding of the GH12 enzymes, with two antiparallel β-sheets and a central active-site cleft for substrate binding. A unique surface loop A3-B3 that contains Arg60 and Tyr61 interacting with the substrate by hydrogen bonds and aromatic residue stacking is observed in the complex structures of TmCel12A with cellobiose and cellotetraose. The role of Arg60 and Tyr61 in the substrate binding and catalytic reaction of TmCel12A was further investigated by site-directed mutagenesis. The results showed that the Y61G mutant had highest enzymatic activity when compared with the wild-type enzyme and other mutants. It also exhibited wider range of working temperature than the wild type, while its hyperthermostability resembled to the wild type. Besides, the kcat and Km values of Y61G were both higher than those of the wild type. In combination with the crystal structure of Y61G-substrate complex, the kinetic data suggested that the higher endoglucanase activity of Y61G is probably due to easier dissociation of cleaved sugar moiety at the reducing end. Further structural analyses of Y61GG and Y61del mutants indicated that the insertion and deletion mutations of the Tyr61 site did not affect the overall protein structure. However, their local perturbations might reduce the substrate binding affinity. It seems that the catalytic reaction of TmCel12A is a subtle modulation of substrate binding and product release. To summarize, the enhancement of enzymatic activity by single mutation of Y61G is a good example of enzyme engineering for industrial application. It also provides information regarding developing a better industrial enzyme by subtle changes of protein structure.
URI: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8039
全文授權: 同意授權(全球公開)
顯示於系所單位:生物科技研究所

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