Characterization of Arabidopsis Glycoside Hydrolases Family 9 Genes

• Main Authors: Ya-ru Li, 李雅茹
• Other Authors: Zin-Huang, Liu
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/15421696963854225892

碩士 === 國立中山大學 === 生物科學系研究所 === 98 === Generation of alcohol for biofuels from fermentation of sugar or starch has several economic disadvantages such as high cost of sugar processing and land usage competing with staple food. The solution may reside in hydrolysis of cellulose from crop waste such as stalks of rice and corn or non-crop plants such as weeds or wood. Our goal is to identify cellulases that can degrade cellulosic biomass more efficiently. Studies of microbial Family 9 glycoside hydrolase (GH9) proteins, including both endo-glucanases (EC 3.2.1.4) and cellobiohydrolases (EC 3.2.1.91), have shown that they function through an inverting mechanism to cleave the 1, 4-β-glucosidic bond between two unsubstituted Glc units. The main function of plant glycoside hydrolases are involved in polysaccharide metabolism of cell wall during cell growth. Twelve Arabidopsis thaliana (Columbia) endo-1,4-β-glucanases that belong to the GH9, were cloned and expressed in Pichia pastoris in order to produce cellulases to facilitate efficient bio-alcohol production. The recombinant proteins do not show in vitro endo-1, 4-β-glucanase activity, but we can detect the recombinant proteins expression in supernatant or in pellet. The lack of enzymatic activity from recombinant proteins is probably due to improper folding or glycosylation, or fast degradation resulted from the above reasons. Other bioreactor will be tested in the future. Genetic engineering to modify Arabidopsis thaliana (Columbia) endo-β-1, 4-glucanases is another approach to produce functional cellulases with economic efficiency that can be adapted to industrial scale for alcohol generation. On the other hand, we use semi-quantitative PCR method to study the Arabidopsis GH9 genes expression level in different tissue. At4g39000 and At3g43860 were found only in flowers and inflorescence, and At1g65610 expression in roots and shoots of the amount of more. Other genes in different tissues, was no found significant difference.