Blood transfusions are an integral component of healthcare; however, availability of viable blood is limited by patient-donor blood type specificity, which contributes to seasonal shortages as well as shortages worldwide, especially in developing countries, and during pandemics or natural disasters. Attempts to increase blood supply with commercial incentives have raised ethical concerns, and current proposed artificial blood substitutes are unable to fully replicate the function of native red blood cells (RBCs). In this study, we explore the potential strategy of alleviating blood shortages through enzymatic conversion of A, B, and AB blood types to blood type O. In theory, this process eliminates ABO patient-donor incompatibility, which increases the supply of universal donor blood. Three glycoside hydrolases, α-N-acetylgalactosaminidase, α-galactosidase, and endo-β-galactosidase, were selected to act as molecular scissors to cleave terminal residues on A and B RBC surface antigens and catalyze the conversion process. These enzymes were recombinantly expressed in BL21(DE3) Escherichia coli and purified through nickel ion affinity chromatography. A combination of colorimetric substrate assays, thin-layer chromatography, and mass spectroscopy were utilized to evaluate enzyme functionality. Enzyme efficiency was modeled using Michaelis-Menten kinetics. Partial enzymatic A-to-O blood type conversion on porcine red blood cells was observed with slide agglutination tests. Results confirm recombinant enzyme-mediated blood type conversion as a potential strategy for alleviating blood shortages.