Downstream processing to obtain enantiopure compounds from a racemic mixture relies mainly on crystallization. Natural transporters can specifically translocate enantiomers through membranes. Here a β-barrel transmembrane protein FhuA is re-engineered into a chiral channel protein (FhuAF4) to resolve racemic mixtures of d-/l-arginine. The engineered FhuAF4 variant exhibits an enantioselectivity (E-value) of 1.92 and an enantiomeric excess percentage (ee%) of 23.91 at 52.39% conversion. OmniChange mutant libraries at the computationally identified "filter-regions" likely help to identify FhuA variants for enantiomeric separation of other compounds.