Identification and Protein Engineering of Galactosidases for the Conversion of Blood Type B to Blood Type O

The supply of blood products such as red blood cells poses a challenge due to rising demand and declining donor numbers. Careful matching of blood products of different types is required. Only type O of the blood types A, B, AB and O can be received by any patient without transfusion incompatibilities. Therefore, O-type blood can be considered "universal blood" and is especially needed in emergency situations. In this study, we focused on the conversion of the B antigen by enzymatic deglycosylation to generate the H antigen determining O-type blood. For this, we characterized several previously unstudied α-1,3-galactosidases belonging to the GH110 family. Our findings revealed that the α-1,3-galactosidase from Pedobacter panaciterrae (PpaGal) exhibits superior efficiency compared to previously described galactosidases. We further increased the activity of PpaGal by 2.5-fold using site-directed mutagenesis. Moreover, we solved two crystal structures of PpaGal, one in the apo-state and another in complex with d-galactose. The combination of our mutagenesis study with the solved crystal structures provides valuable information to guide further optimization of PpaGal or other B antigen converting enzymes paving the way for the easier production of universal blood from B-type blood.