High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotula

Abstract

An extracellular peroxygenase from Marasmius rotula was produced in liquid culture, chromatographically purified and partially characterized. This is the third aromatic peroxygenase (APO) that has been characterized in detail and the first one that can be produced in high yields. The highest enzyme levels of about 41,000 U l-1 (corresponding to appr. 445 mg l-1 APO protein) exceeded the hitherto reported levels more than 40-fold and were detected in carbon- and nitrogen-rich complex media. The enzyme was purified by FPLC to apparent homogeneity (SDS-PAGE) with a molecular mass of 32 kDa (27 kDa after deglycosylation) and isoelectric points between 4.97 and 5.27. The UV-visible spectrum of the native enzyme showed a characteristic maximum (Soret band) at 418 nm that shifted after reduction with sodium dithionite and flushing with carbon monoxide to 443 nm. The pH optimum of the M. rotula enzyme was found to vary between pH 5 and 6 for most reactions studied. The apparent Km-values for 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, naphthalene and H2O2 were 0.133, 0.118, 0.279, 0.791 and 3.14 mM, respectively. M. rotula APO was found to be highly stable in a pH range from 5 to 10 as well as in the presence of organic solvents (50% vol/vol) such as methanol, acetonitrile and N,N-dimethylformamide. Unlike other APOs, the peroxygenase of M. rotula showed neither brominating nor chlorinating activities.

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone

“…The final purification step in an anion exchanger (MonoQ column) resulted in two fractions with the same molecular mass, thus indicating the presence of two isoforms. Isoforms with almost identical molecular masses were reported for other UPOs (e.g., UPOs of A. aegerita and C. radians ) and fungal heme peroxidases (e.g., manganese peroxidases of Ceriporiopsis subvermispora ) 14, 15, 16, 37…”

“…Another feature of Cgl UPO is the lack of halogenating activity. In contrast to Aae UPO and Cra UPO, which efficiently oxidize bromide, Cgl UPO did not catalyze halide oxidation; in this context it resembles Mro UPO 14, 15, 16. Interestingly, Cgl UPO and Mro UPO belong to group I UPOs (“short” peroxygenases); however, the strongly halogenating chloroperoxidase (CPO) is also in the same subfamily 12…”

“…Peroxygenases of A. aegerita ( Aae UPO) and M. rotula ( Mro UPO) were produced and purified as described previously;14, 16 recombinant UPOs from C. cinerea (r Cci UPO) and H. insolens (r Hin UPO=r novo ) were gifts from Novozymes A/S (Copenhagen, Denmark) 25, 26, 27, 28…”

“…UPOs are relatively stable and versatile biocatalysts, and combine the catalytic cycle of heme peroxidases with the “peroxide shunt” of P450s 12, 13. The first UPO was discovered in the wood‐dwelling agaric fungus Agrocybe aegerita ( Aae UPO); two similar enzymes were described in the mushrooms Coprinellus radians ( Cra UPO) and Marasmius rotula ( Mro UPO) 14, 15, 16. The reactions catalyzed by these UPOs comprise aromatic and aliphatic hydroxylations (including subsequent bond cleavages resulting in N‐ and O‐dealkylations), epoxidation, N‐oxygenation, sulfoxidation, dechlorination, and halide oxidation 11.…”

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

“…UPO-type genes and proteins have been isolated and characterized in Coprinellus radians, Marasmius rotula, and Coprinopsis cinerea (3)(4)(5). Indeed, to date, over 1,000 UPO-like genes have been identified in genetic databases and in basidiomycete genome sequencing, indicating an ancient origin and a widespread distribution of UPO in nature (6,7).…”