Immobilization of unspecific peroxygenase expressed in Pichia pastoris by metal affinity binding

S. Bormann, B. O. Burek, R. Ulber, D. Holtmann

 Mol. Catal., 492 (2020), 110999, doi: 10.1016/j.mcat.2020.110999

Abstract

Unspecific peroxygenases (UPOs) catalyze oxidation and oxygenation reactions using only hydrogen peroxide as a co-substrate. Especially their ability to oxygenate unactivated C–H bonds has attracted interest in their synthetic application. Due to the high production cost of these heme-enzymes, special care has to be taken to fully utilize the catalyst. Immobilization is one strategy to achieve this as it allows recovery and reuse of the costly biocatalyst. Herein, we investigate the immobilization of an UPO-variant (AaeUPO-PaDa-I) originally from Agrocybe aegerita by fusion of a C-terminal hexa-histidine tag and subsequent metal affinity binding on porous glass carriers (EnginZyme EziG). The fusion protein was expressed in Pichia pastoris, purified and characterized. The specific activity did not change considerably with slightly reduced activities ranging from 78% to 53% of the untagged enzyme. Using this immobilization procedure, it was possible to retain the full catalytic potential of the enzyme in terms of product produced, which is shown for a benzylic hydroxylation process. The reusability of the immobilized catalyst was demonstrated by carrying out repeated fed-batch operation for multiple cycles. As a proof of concept, we demonstrate the immobilization directly from crude culture broth, which has the potential to greatly simplify immobilization as no purification steps are necessary.

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