Specific immobilization of d-amino acid oxidase on hematin-functionalized support mimicking multi-enzyme catalysis

Abstract

D-Amino acid oxidase (DAAO) catalyzes oxidative deamination of D-amino acids to yield corresponding α-keto acids, producing hydrogen peroxide (H₂O₂). D-Amino acid oxidase was genetically modified by fusion to an elastin-like polypeptide (ELP). For enzyme immobilization, multi-walled carbon nanotubes (MWCNTs) were adopted as the model support. MWCNTs were functionalized with hematin. ELP-DAAO was immobilized on the functionalized CNTs by coupling to the hematin. The specific immobilization enabled ELP-DAAO in proximity to the hematin at a molecular distance. The molecular-distance proximity facilitated the immediate decomposition of H₂O₂ catalyzed by the hematin. The evolved oxygen was efficiently utilized to oxidize the reduced cofactor FDA of DAAO, and H₂O₂ was produced. The forming of a H₂O₂ → O₂ → H₂O₂ circle between the DAAO and hematin has been demonstrated to be the driving force to accelerate the deamination reaction. The enzyme kinetics has shown that the ELP-DAAO/hematin-CNT conjugate exhibited a catalytic efficiency more than three times that of free ELP-DAAO, demonstrating its ability in mimicking multi-enzyme catalysis. The methodology for highly specific enzyme immobilization is not restricted to carbon nanotubes, and can be extended easily to other micro and nanomaterials as supports for specific immobilization of oxidases.