Protease enzymes are widely utilizing in industry in laundry detergents to break down proteinaceous stains as well in pharmaceutical and food industries. A particular serine protease called Savinase shows promise for usage in high pH detergent solutions due to its broad substrate specificity. In this study we aim to produce and characterize recombinant Savinase made by Bacillus subtilis. Savinase is naturally secreted by Bacillus sp. As a pro-enzyme. Our goal is to make Savinase extracellularly in the supernatant of B. subtilis since this organism is more genetically tractable for future engineering studies than Bacillus sp. We hypothesize that since the species are related, recombinant Savinase secretion will be at a similar level for both organisms. The gene for Savinase wths its native signal sequence has been incorporated into the genome of B. subtilis 168 in the lacA locus. Additionally, a poly histidine tag has been appended to the C-terminus (Savinase-his) to permit purification using immobilized metal affinity chromatography. To produce the enzyme, a culture of B. subtilis containing the genetic sequence for Savinase-his is grown and induced with xylose, resulting in protein secretion into the extracellular fluid. Once the extracellular and intracellular components are segregated by centrifugation, the extracellular fluid is passed through a nickel resin column using fast protein liquid chromatography (FPLC). The nickel resin interacts with the poly-histidine tag to purify the Savinase from other extracellular components. The purified protein product was then desalted by buffer exchange and characterized by enzyme assays and SDS-PAGE. While purification has proven successful, secretion amounts are lower than expected in B. subtilis. Future steps are to increase the production of Savinase by exploring alternative native secretion tags.
Authors: Nicholas Nocevski, Dr. Jason Boock
Faculty Advisor: Jason T. Boock, Chemical, Paper and Biomedical Engineering
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