Unspecific peroxygenases (UPOs) are glycosylated fungal enzymes that can selectively oxidize C–H bonds. UPOs employ hydrogen peroxide as the oxygen donor and reductant. With such an easy-to-handle cosubstrate and without the need for a reducing agent, UPOs are emerging as convenient oxidative biocatalysts. Here, an unspecific peroxygenase from Hypoxylon sp. EC38 (HspUPO) was identified in an activity-based screen of six putative peroxygenase enzymes that were heterologously expressed in Pichia pastoris. The enzyme was found to tolerate selected organic solvents such as acetonitrile and acetone. HspUPO is a versatile catalyst performing various reactions, such as the oxidation of prim- and sec-alcohols, epoxidations, and hydroxylations. Semipreparative biotransformations were demonstrated for the nonenantioselective oxidation of racemic 1-phenylethanol rac-1b (TON = 13 000), giving the product with 88% isolated yield, and the oxidation of indole 6a to give indigo 6b (TON = 2800) with 98% isolated yield. HspUPO features a compact and rigid three-dimensional conformation that wraps around the heme and defines a funnel-shaped tunnel that leads to the heme iron from the protein surface. The tunnel extends along a distance of about 12 Å with a fairly constant diameter in its innermost segment. Its surface comprises both hydrophobic and hydrophilic groups for dealing with substrates of variable polarities. The structural investigation of several protein–ligand complexes revealed that the active site of HspUPO is accessible to molecules of varying bulkiness with minimal or no conformational changes, explaining the relatively broad substrate scope of the enzyme. With its convenient expression system, robust operational properties, relatively small size, well-defined structural features, and diverse reaction scope, HspUPO is an exploitable candidate for peroxygenase-based biocatalysis.

Green leaf volatiles (GLVs) are mainly C 6 -and in rare cases also C 9 -aldehydes, -alcohols, and -esters, which are released by plants in response to biotic or abiotic stresses. These compounds are named for their characteristic smell reminiscent of freshly mowed grass. This review focuses on GLVs and the two major pathway enzymes responsible for their formation: lipoxygenases (LOXs) and fatty acid hydroperoxide lyases (HPLs). LOXs catalyze the peroxidation of unsaturated fatty acids, such as linoleic and α-linolenic acids. Hydroperoxy fatty acids are further converted by HPLs into aldehydes and oxo-acids. In many industrial applications, plant extracts have been used as LOX and HPL sources. However, these processes are limited by low enzyme concentration, stability, and specificity. Alternatively, recombinant enzymes can be used as biocatalysts for GLV synthesis. The increasing number of well-characterized enzymes efficiently expressed by microbial hosts will foster the development of innovative biocatalytic processes for GLV production.

Since 2004, unspecific peroxygenases, in short UPOs (EC. 1.11.2.1), have been explored. UPOs are closing a gap between P450 monooxygenases and chloroperoxidases. These enzymes are highly active biocatalysts for the selective oxyfunctionalisation of C–H, C=C and C-C bonds. UPOs are secreted fungal proteins and Komagataella phaffii (Pichia pastoris) is an ideal host for high throughput screening approaches and UPO production. Heterologous overexpression of 26 new UPOs by K. phaffii was performed in deep well plate cultivation and shake flask cultivation up to 50 mL volume. Enzymes were screened using colorimetric assays with 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (DMP), naphthalene and 5-nitro-1,3-benzodioxole (NBD) as reporter substrates. The PaDa-I (AaeUPO mutant) and HspUPO were used as benchmarks to find interesting new enzymes with complementary activity profiles as well as good producing strains. Herein we show that six UPOs from Psathyrella aberdarensis, Coprinopsis marcescibilis, Aspergillus novoparasiticus, Dendrothele bispora and Aspergillus brasiliensis are particularly active.

Among nature's arsenal of oxidative enzymes, cytochrome P450s (CYPs) catalyze the most challenging reactions, the hydroxylations of non-activated CÀ H bonds. Human CYPs are studied in drug development due to their physiological role at the forefront of metabolic detoxification, but their challenging handling makes them unsuitable for application. CYPs have a great potential for biocatalysis, but often lack appropriate features such as high and soluble expression, self-sufficient internal electron transport, high stability, and an engineerable substrate scope. We have probed these characteristics for a recently described CYP that originates from the thermophilic fungus Thermothelomyces thermophila (CYP505A30), a homolog of the well-known P450-BM3 from Bacillus megaterium. CYP505A30 is a natural monooxygenase-reductase fusion, is well expressed, and moderately tolerant towards temperature and solvent exposure. Although overall comparable, we found the stability of the enzyme's domains to be inverse to P450-BM3, with a more stable reductase compared to the heme domain. After analysis of a homology model, we created mutants of the enzyme based on literature data for P450-BM3. We then probed the enzyme variants in bioconversions using a panel of active pharmaceutical ingredients, and activities were detected for a number of structurally diverse compounds. Ibuprofen was biooxidized in a preparative scale whole cell bioconversion to 1-, 2-and 3hydroxyibuprofen.