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Directed Evolution of Hemicellulosic Hydrolases for Conversion of Biomass for Production on Biofuels and Bioproducts
L. Yuan
Department of Plant and Soil Sciences
Project Description
In the 12-month project period, we have fulfilled most of the proposed tasks and designed and performed additional experiments that further advanced the project. We have isolated additional genes encoding the xylosidase and arabinofuranosidase and expressed these genes in E. coli for recombinant protein productions. We have developed a number of high throughput assays that enable the screening of large numbers of enzyme variants. We have constructed and screened mutant libraries for the arabinofuranosidase; enzyme variants have been identified to potentially have xylosdiase activity. We have developed a capillary electrophoresis method to assay the hydrolases using natural substrate. We have also generated chimeric and trimeric fusion enzymes and characterized the kinetic properties of these enzymes. Furthermore, we have transformed these fusion enzyme genes into tobacco plants to test the feasibility of producing these multifunctional proteins in transgenic plants. We have presented our results in a national conference, and a manuscript describing the construction and characterization of the chimeric enzymes has been submitted to a peer-reviewed journal.
Impact
Biofuels derived from cellulose/hemicellulose have the advantages of high-level sustainability, obtaining from non-food portions of renewable feedstocks and potential to have large-scale impact to our state's and national agriculture. Although not the only enzymes needed in the process, xylosidase and arabinofuranosidase play important and synergetic roles in hemicellulose degradation. The ability to optimize the enzyme activities at a milder temperature can lead to reduction of energy for the bioconversion. Development of an enzyme with dual-specificity will not only provide insight into protein structure/function relationships, but will also result in significant cost and energy savings for the production of the hydrolases. More importantly the approaches, if successful, will be applicable to other enzymes involved in the process such as xylanase and glucanronidase.
Publications
Fan, Z., Wagschal K., Lee, CC., Kong, Q., Maiti, IB. and Yuan, L. (2008)The Construction and Characterization of Two Xylan-degrading Chimeric Enzymes. (submitted for publication)