Abstract
In 2004, the fungal heme-thiolate enzyme subfamily of unspecific peroxygenases (UPOs) was first described in the basidiomycete Agrocybe aegerita. As UPOs naturally catalyze a broad range of oxidative transformations by using hydrogen peroxide as electron acceptor and thus possess a great application potential, they have been extensively studied in recent years. However, despite their versatility to catalyze challenging selective oxyfunctionalizations, the availability of UPOs for potential biotechnological applications is restricted. Particularly limiting are the identification of novel natural biocatalysts, their production, and the description of their properties. It is hence of great interest to further characterize the enzyme subfamily as well as to identify promising new candidates. Therefore, this review provides an overview of the state of the art in identification, expression, and screening approaches of fungal UPOs, challenges associated with current protein production and screening strategies, as well as potential solutions and opportunities.
Highlights
The selective oxyfunctionalization of organic molecules is one of the most challenging tasks in synthetic chemistry
Droplet microfluidics combined with electrospray ionization (ESI)mass spectrometry (MS) can provide label-free, high-throughput screening with the opportunity to test simultaneously substrate libraries (Diefenbach et al, 2018)
Fungal unspecific peroxygenases (UPOs) catalyze both, one-electron oxidations and two-electron oxidations with peroxidederived oxygen transfer, the latter being more interesting with respect to synthesis of pharmaceuticals
Summary
The selective oxyfunctionalization of organic molecules is one of the most challenging tasks in synthetic chemistry. For a general overview of the fungal peroxygenase superfamily, Hofrichter et al (2020) summarized the state of knowledge of basic and applied UPO research including phylogeny, protein structure, and catalytic activity The aim of this contribution was to review the current state of the art in UPO identification, recombinant expression, and screening approaches along with a brief overview of the status of the sustainable biotechnological applicability of UPOs. In particular, the following tools for selection of putative UPOs as well as UPO synthesis and characterization are discussed: (1) genome mining approaches to identify new candidates, (2) efficient expression systems for protein production, and (3) high-throughput methods for time-saving evaluation of enzyme activity. AC, affinity chromatography; BR, bioreactor; f.p., for purification; GPC, gel permeation chromatography; HIC, hydrophobic interaction chromatography; IEX, ion exchange chromatography; n.a., not available; n.d., not detectable; n.p., not purified; SEC, size exclusion chromatography; SF, shake flask
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