The use of glucoamylase-bioconjugated nanoparticles to facilitate starch fermentation

This project concerns the cloning and synthesis of an enzyme, glucoamylase, for bioconjugation to magnetic particles, which could potentially lead to a more efficient process for biofuel production. There are many methods currently utilized for biomass pre-processing; however, this project particularly focuses on the creation of glucoamylase-conjugated magnetic nanoparticles, which allows for the enzymes to be retrievable enabling novel reuse. Glucoamylase is an enzyme that breaks the bonds of large carbohydrates (starches). Particularly, glucoamylase is an amylase that cleaves the last alpha-1,4-glycosidic linkage at the nonreducing end of amylose and amylopectin to yield glucose. The resulting monomeric glucose molecules can then be fermented by organisms such as yeast into biofuels of interest such as ethanol. The glucoamylase from this project is specifically targeted from Trichoderma reesei, an organism commonly used in industry for the production of cellulytic enzymes. Glucoamylase has not been widely isolated from this organism before, so the development of a method for isolating and cloning this enzyme has been formulated and tested for this project. Various methods have been tested in order to create a cDNA library containing the gene for this enzyme from Trichoderma reesei; however, none have extremely high and successful yields. These low yields from Trichoderma reesei have led to the production of glucoamylase similar to that of Trichoderma reesei from two organisms, Pichia stipitis and Debaryomycese hansenii. In addition, experiments have been performed with fluorescent silica-coated iron oxide particles to test for separation capabilities. Based on preliminary data, it is hopeful that when these enzymes are conjugated to the particles, a successful, high-yielding magnetic recovery will be observed.