A holistic carrier-bound immobilization approach for unspecific peroxygenase

Abstract

Unspecific peroxygenases (UPOs) are among the most studied enzymes in the last decade and their well-deserved fame owes to the enzyme’s ability of catalyzing the regio- and stereospecific hydroxylation of non-activated C–H bonds at the only expense of H2O2. This leads to more direct routes for the synthesis of different chiral compounds as well as to easier oxyfunctionalization of complex molecules. Unfortunately, due to the high sensitivity towards the process conditions, UPOs’ application at industrial level has been hampered until now. However, this challenge can be overcome by enzyme immobilization, a valid strategy that has been proven to give several benefits. Within this article, we present three different immobilization procedures suitable for UPOs and two of them led to very promising results. The immobilized enzyme, indeed, shows longer stability and increased robustness to reaction conditions. The immobilized enzyme half-life time is 15-fold higher than for the free AaeUPO PaDa-I and no enzyme deactivation occurred when incubated in organic media for 120 h. Moreover, AaeUPO PaDa-I is proved to be recycled and reused up to 7 times when immobilized.

Continuous oxyfunctionalizations catalyzed by unspecific peroxygenase

“…In general, a lower flow rate resulted in a higher standard deviation for the experimental residence time τ exp ; but, overall, the relative deviation from the theoretical residence time τ theo is about 1.5 greater (Table 1, column "τ exp •τ theo −1 "). We were able to show that some degree of stirring was required, as indicated by the significantly high standard deviation for an experiment without stirring (Tables 1, entry 13 and S1, † entries 36,37,38). However, the tank-in-tank number was constant for all investigated flow…”

“…The detailed information on PaDa-I immobilization on the ECR8315F carrier from Purolite Ltd. can be found elsewhere. 38…”

“…Very recently, we reported systematic characterization of the carrier-bound immobilization approaches for UPO and we would like to refer to this study for further information with respect to the immobilization and its investigations (e.g., enzyme loading, recovery of the catalytic activity, etc.). 38 We used a standard and well-accepted ABTS enzymatic assay reaction in continuous operation (Fig. 1).…”

Immobilization of Enzymes on Cyclodextrin-Anchored Dehiscent Mesoporous TiO2 for Efficient Photoenzymatic Hydroxylation

“…As can be seen from Figure S14a,b, the maximum protein loading and specific activity of UPO@ d TiO 2 -CD were 8 mg/g support and 140 U/g support , respectively. Although the enzyme loading was not high, the immobilized enzyme still exhibited high specific activity due to the inherent ultra-high enzyme activity of r Aae UPO . Notably, the protein loading and specific activity of d TiO 2 were significantly increased after the graft of CDs (Figure S15) because CDs can increase the adsorptive properties of the support material and stabilize the immobilized enzyme molecules.…”

“…Although the enzyme loading was not high, the immobilized enzyme still exhibited high specific activity due to the inherent ultra-high enzyme activity of rAaeUPO. 67 Notably, the protein loading and specific activity of dTiO 2 were significantly increased after the graft of CDs (Figure S15) because CDs can increase the adsorptive properties of the support material and stabilize the immobilized enzyme molecules. The immobilization has a positive effect on enzyme stability in extreme environments.…”