Recombinant Peroxidase Has Similar Substrate Specificity Profiles as, but a Catalytic Efficiency up to 11-Fold Higher than, Horseradish Peroxidase.

Fungal peroxidases are valuable enzymes. peroxidase (ARP) and horseradish peroxidase (HRP) share a conserved catalytic site. Both native ARP and recombinant ARP (rARP) were not commercially available. The substrate specificity and kinetic parameters of rARP and HRP were not well compared, particularly relevent to structure-activity relationship. In this work, rARP expressed by had a production yield of 6.2 mg/L, up to 155-fold higher than ARP and other recombinant peroxidases, and a specific activity of 3240 units/mg toward 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), up to 29-fold higher than HRP and other peroxidases. The Michaelis constant () and first-order rate constant () of rARP showed 10-fold substrate affinity and consequently 6-fold catalytic efficiency of HRP toward ABTS. Under optimal conditions, rARP shared similar substrate specificity profiles as commercial HRP; the second-order rate constants () of rARP showed 2-11-fold catalytic efficiency of HRP toward well-known peroxidase substrates. rARP's higher catalytic efficiency was also in agreement with the shorter binding distance of H/N-His in rARP/substrate in comparison to that of HRP/substrate, as illustrated by docking simulation. The rARP had similar substrate specificity profiles as, but higher specific activity and catalytic efficiency than, HRP, which merits its further structure-functional characterization and applications.