Protection strategies for biocatalytic proteins under plasma treatment

Abstract

In plasma-driven biocatalysis, enzymes are employed to carry out reactions using species generated by non-thermal plasmas as the precursors. We have previously demonstrated that this is feasible in principle, but that the approach suffers from the short lifetime of the biocatalyst under operating conditions. In this work, protection strategies were investigated to prevent the dielectric barrier discharge plasma-induced inactivation of biocatalysts, using recombinant unspecific peroxygenase from Agrocybe aegerita (rAaeUPO), one of the most promising enzymes for plasma-driven biocatalysis. Treatment in oxygen-free atmospheres did not provide any advantage over treatment in synthetic air, indicating that the detrimental reactive species did not originate from oxygen in the plasma phase. Chemical scavengers were employed to eliminate undesired reactive species, without any long-term effect on enzyme lifetime. Similarly, chaperones, including the known stress response proteins Hsp33, CnoX, and RidA did not increase the lifetime of rAaeUPO. Immobilization of the biocatalyst proved effective in preserving enzyme activity. The residual activity of rAaeUPO after plasma treatment strongly depended on the specific immobilization support. Essentially complete protection for at least 15 min of plasma exposure was achieved with an epoxy-butyl-functionalized carrier. This study presents new insights into plasma-protein interactions and plots a path forward for protecting biocatalytic proteins from plasma-mediated inactivation.

Plasma‐driven biocatalysis: In situ hydrogen peroxide production with an atmospheric pressure plasma jet increases the performance of OleTJE when compared to adding the same molar amount of hydrogen peroxide in bolus

“…Atmospheric pressure plasmas have been explored for biocatalysis with the enzymes horseradish peroxidase (HRP) and the modified fungal peroxygenase from Agrocybe aegerita (rAaeUPO) in experiments performed by Yayci et al [24][25][26] One of the first methods used was a dielectric barrier discharge (DBD) plasma to generate H 2 O 2 , the reaction mediator for HRP, as a proof of concept. However, HRP lost activity after about 5 min of treatment, which was found to be likely due to heme degradation.…”

“…[25] rAaeUPO was tested as a more industrially relevant enzyme and was able to achieve a TON of 13 787, but this required the buffer solution to be treated separately from the enzyme and then added, obviating the advantage of generation of the required reaction mediator in the solution without dilution. [26] In addition, this method was only able to treat very small volumes of solution at a time (µl), and the solution was exposed to DBD-generated UV photons that can be damaging to enzymes. [26][27][28] To address these DBD limitations, further experiments were conducted by Yayci et al [24] with a microscale atmospheric pressure plasma jet (µ-APPJ), which was used in conjunction with a rotating bed reactor.…”

“…[26] In addition, this method was only able to treat very small volumes of solution at a time (µl), and the solution was exposed to DBD-generated UV photons that can be damaging to enzymes. [26][27][28] To address these DBD limitations, further experiments were conducted by Yayci et al [24] with a microscale atmospheric pressure plasma jet (µ-APPJ), which was used in conjunction with a rotating bed reactor. This was found to be very efficient for rAaeUPO catalysis.…”

Immobilization of the Peroxygenase from Agrocybe aegerita. The Effect of the Immobilization pH on the Features of an Ionically Exchanged Dimeric Peroxygenase

“…rAaeUPO is a highly glycosylated, dimeric enzyme (see Figure 1 and Figure S1) [93,94]. To date, few studies have been undertaken to immobilize the enzyme [95][96][97]. Covalent immobilization on epoxide supports (IB-COV-1 from ChiralVision) was successful but yielded enzyme preparations with dramatically decreased catalytic activity [85].…”

An alginate-confined peroxygenase-CLEA for styrene epoxidation

“…While a limited number of studies report immobilisation of peroxygenases, this technique is not fully explored yet for this enzyme class. 7–12 In previous works, we could demonstrate that immobilised peroxygenases in principle can even be applied in neat ( i.e. almost water-free) reaction media.…”