Date of Award
Master of Science
Food Science and Human Nutrition
Food Science and Technology
Joey N. Talbert
Fusion tags are genetically coded protein or peptides that can be expressed as attached moieties to the desired enzyme to improve downstream purification of the target protein. Fusion tags have the potential to provide specific immobilization with repeatable attachment of the enzyme, eliminate the need for purification and post-modification and enable dissociation of spent enzyme and regeneration of the support. Lipase is an industry-relevant enzyme utilized for the production of natural flavors, biodegradable polyesters for food packaging, structured lipids, antioxidant esters, sugar esters and several other important compounds used in food and agriculture industries. In this research work, we are targeting the immobilization of lipase B from Candida antarctica (CALB) using novel Strep-tag II fusion technology as a means to enhance its functional properties.
The gene encoding CALB was codon-optimized and synthesized for expression in E. coli. Multiple plasmid constructs were designed to allow for the direct fusion of a Strep-tag II to lipase. The corresponding bacterial expression systems were optimized to promote lipase expression. SDS-PAGE was employed to monitor protein expression. The expressed recombinant lipase having Strep-tag II was purified using Strep-Tactin (engineered streptavidin) column and further used for immobilization to Strep-Tactin based supports. Enzyme specific activity and kinetics were evaluated using colorimetric assays.
The optimized bacterial expression system yielded a recombinant CALB with a Strep-II fusion tag. The expressed enzyme displayed specific affinity to Strep-Tactin and could be attached to a Strep-Tactin coated support. The resulting enzyme was catalytically active and was purified and characterized for immobilization studies using bio-affinity interaction.
Sumreet Singh Johar
Johar, Sumreet Singh, "Strep-tag II fusion technology for the immobilization of lipase B from Candida antarctica" (2016). Graduate Theses and Dissertations. 16055.