Department (e.g. History, Chemistry, Finance, etc.)

Plant Sciences

College (e.g. College of Engineering, College of Arts & Sciences, Haslam College of Business, etc.)

Agricultural Sciences and Natural Resources

Abstract

Cellulose is the most abundant carbohydrate in the world and is degraded by the synergistic action of multiple enzymes. One large family of enzymes capable of hydrolyzing cellulose is glycoside hydrolase family 9 (GH9), which includes several endoglucanases. Recent research into the molecular biology of plants has revealed certain genes coding for endo-β-1,4-glucanases (EGases). The EGases in plants are primarily functional during cell elongation through wall stress relaxation. GH9 enzymes have been found in insects, bacteria, oomycetes, and fungi. In insects, EGases enable the organism to digest cellulose; in fungi, EGases are suspected to play an important role in obtaining nutrition for the fungi and may be associated with defense mechanisms. In these systems, EGases play an important role in breaking the internal bonds of cellulose resulting in a disruption of the crystalline structure. EGases are able to cleave cellulose at the β-1,4 linkages in the cellulose chain with a net inversion of anomeric configuration. A putative EGase from switchgrass (Panicum virgatum) was isolated from leaf cDNA. This gene of interest was cloned into Escherichia coli via the pET160 expression system. Selection by antibiotic resistance confirmed transformation of E. coli. Protein expression was detected via SDS-PAGE and tested to confirm cellulose lysing. The optimal pH and temperature were determined using 3,5-Dinitrosalycilic acid (DNSA) assay at different pH and temperature settings measuring reduced sugars released. Functionality in plants was determined through a gene rescue experiment using Arabidopsis thaliana mutants known to be deficient in putative EGase homologs. Switchgrass containing an overexpression of the EGase was compared to wildtype switchgrass via histology and microscopy. The confirmation of a functional EGase from switchgrass may aid in the development of switchgrass transformants with an amorphous cellulose structure, thereby reducing the amount of resources required during biofuel refinery.

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Functional analysis of a putative membrane-bound endo-β-1,4-glucanase from Panicum virgatum

Cellulose is the most abundant carbohydrate in the world and is degraded by the synergistic action of multiple enzymes. One large family of enzymes capable of hydrolyzing cellulose is glycoside hydrolase family 9 (GH9), which includes several endoglucanases. Recent research into the molecular biology of plants has revealed certain genes coding for endo-β-1,4-glucanases (EGases). The EGases in plants are primarily functional during cell elongation through wall stress relaxation. GH9 enzymes have been found in insects, bacteria, oomycetes, and fungi. In insects, EGases enable the organism to digest cellulose; in fungi, EGases are suspected to play an important role in obtaining nutrition for the fungi and may be associated with defense mechanisms. In these systems, EGases play an important role in breaking the internal bonds of cellulose resulting in a disruption of the crystalline structure. EGases are able to cleave cellulose at the β-1,4 linkages in the cellulose chain with a net inversion of anomeric configuration. A putative EGase from switchgrass (Panicum virgatum) was isolated from leaf cDNA. This gene of interest was cloned into Escherichia coli via the pET160 expression system. Selection by antibiotic resistance confirmed transformation of E. coli. Protein expression was detected via SDS-PAGE and tested to confirm cellulose lysing. The optimal pH and temperature were determined using 3,5-Dinitrosalycilic acid (DNSA) assay at different pH and temperature settings measuring reduced sugars released. Functionality in plants was determined through a gene rescue experiment using Arabidopsis thaliana mutants known to be deficient in putative EGase homologs. Switchgrass containing an overexpression of the EGase was compared to wildtype switchgrass via histology and microscopy. The confirmation of a functional EGase from switchgrass may aid in the development of switchgrass transformants with an amorphous cellulose structure, thereby reducing the amount of resources required during biofuel refinery.

 

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