Alkylation of a cysteine residue in papain with a pyridoxamine (PX) cofactor was carried out. The resulting semisynthetic enzyme (papain-PX) has no detectable protease activity but has the ability to catalyze enantioselective reductive amination of alpha-keto acids. The conjugate was characterized by ion-exchange chromatography, and the optimal reaction conditions were found. We report that papain-PX reductively aminates the alkyl side chain of functionalized alpha-keto acids to give the respective alpha-amino acids with high enantioselectivities, greater than 70%. Based on these studies, we propose a new model for the catalytic activity of the semisynthetic enzyme with Interchem software. The results of the study demonstrate the effectiveness of the modified enzyme and its potential for engineering new catalytic specificity.