Heme-containing unspecific peroxygenases (UPOs) have attracted significant attention as biocatalysts for oxidation reactions due to their ability to function without expensive nicotinamide cofactors. In the recent study, the UPO from aspergillus brasiliensis (AbrUPO) is found to catalyze the aromatic hydroxylation of substituted benzenes, a feature that distinguishes AbrUPO from other reported wild-type UPOs. To elucidate the underlying factors in the active site and substrate access channel of AbrUPO—which contains fewer phenylalanine residues compared to other UPOs that primarily catalyze benzylic hydroxylation—twenty two AbrUPO variants with single, double, triple, or quadruple amino acid substitutions were constructed to mimic the active sites or substrate access channels of other UPOs. A number of mutated variants exhibited altered activity and selectivity, and several positions were identified that influence enzyme chemoselectivity. Among them, substitution of alanine at position 186 with bulkier residues such as phenylalanine or leucine lead to a shift in chemoselectivity toward alkyl chain hydroxylation of substituted benzenes. Molecular docking studies indicated that the A186F mutation restricts the flexibility and reorientation of ethylbenzene in the active site of AbrUPO, thereby preventing oxidation at the aromatic ring while promoting benzylic hydroxylation.

An enzymatic method for the selective hydroxylation of phenols using a peroxygenase from Aspergillus brasiliensis (AbrUPO) is reported. A broad range of phenolic starting materials can be selectively transformed into the corresponding hydroquinones. Semi-preparative syntheses of several hydroquinones were realised without further optimization pointing out the applicability of this enzyme as biocatalyst.

Selective aromatic hydroxylation of substituted benzenes provides access to versatile phenolic synthons. Unspecific peroxygenases (UPOs) have been recognised as promising biocatalysts for synthetic chemistry. While UPOs accept diverse substrates and enable a broad range of oxygenation reactions, aromatic hydroxylation reactions catalysed by these enzymes have been rarely described. Here, we report on a UPO from Aspergillus brasiliensis (AbrUPO) heterologously expressed in Pichia pastoris at a concentration of 742 mg per litre that is able to catalyse aromatic hydroxylation of substituted benzenes. The preference of AbrUPO for aromatic or benzylic hydroxylation was found to depend on the number, chemical properties and length of existing ring substituents. While oxidation of ethylbenzene gave ring- and side-chain hydroxylation products at a 1 : 1 ratio, increasing the chain-length of the alkyl substituent enhanced the preference for benzylic hydroxylation. With the para-disubstituted p-cymene as a substrate, the chemoselectivity of AbrUPO strongly shifted towards aromatic hydroxylation. All tested substituted phenols resulted in exclusive aromatic hydroxylation. The observed formation of low quantities of quinones was attributed to the inherent peroxidase activity, while further oxidation of benzylic alcohols to ketones was suggested to occur due to both peroxidase and peroxygenase activity of AbrUPO. ‘Overoxidation’ due to peroxidase activity could be completely avoided by adding ascorbic acid and shortening reaction time.

Laccases have gained significant attention due to their emerging applications including bioremediation, biomass degradation and biofuel cells. One of the prerequisites for the industrial application of laccases is their sufficient availability. However, expression levels of recombinantly expressed laccases are often low. In this study Mrl2, a new laccase from the basidiomycete Moniliophthora roreri, was cloned in Pichia pastoris and produced in an optimized fed-batch process at an exceptionally high yield of 1.05 g l⁻¹. With a redox potential of 0.58 V, Mrl2 belongs to mid-redox potential laccases. However, Mrl2 demonstrated high k_cat values of 316, 20, 74, and 36 s⁻¹ towards 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ), 2,6-dimethoxyphenol (2,6-DMP) and guaiacol, respectively. Mrl2 remained stable above pH 6 and in the presence of many metal ions, which is important for application in bioremediation. Mrl2 was investigated for the ability to degrade endocrine disrupting chemicals (EDCs) and non-steroidal anti-inflammatory drugs (NSDAIs) at neutral pH value. The enzyme accepted and converted estrone, 17β-estradiol, estriol, the synthetic contraceptive 17α-ethinyl estradiol and bisphenol A at pH 7 faster than high-potential laccases from Trametes versicolor. For example, within 30 min Mrl2 removed more than 90% bisphenol A, 17ß-estradiol, 17α-ethinyl estradiol and estriol, respectively. The concentration of the recalcitrant drug diclofenac dropped by 56% after 20 h incubation with Mrl2.