Date Published: October 11, 2011
Author(s): Glenn Gröbe, René Ullrich, Marek J Pecyna, Danuta Kapturska, Stephanie Friedrich, Martin Hofrichter, Katrin Scheibner.
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.
Enzymes catalyzing oxygen-transfer reactions are of great interest for chemical synthesis since they work selectively and under ambient conditions (Joo et al. 1999). Despite this fact, production of respective biocatalysts is still limited to small scale in the laboratory and the few enzymes available are just provided as expensive fine chemicals. One reason for this is the intracellular nature of oxygenases, which is connected with complex cofactor requirements and low enzyme stability (Urlacher and Eiben 2006). Thus cytochrome P450 monooxygenases (P450s), which represent the most versatile group of oxygen-transferring biocatalysts, need NAD(P)H as electron donating co-substrate and – at least – one accessory protein, a flavin reductase, as electron-transferring partner enzyme (Smith et al. 1994). Most P450s tend to lose their activity rapidly outside the protecting cell, which hampers their purification and use in cell-free reaction systems (Urlacher et al. 2004). Extracellular biocatalysts of the heme peroxidase type could help to overcome this problem, since they are secretory proteins and hence relatively stable, they have the same prosthetic group (protoporphyrine IX = heme) as P450s and form similar activated oxo-intermediates (compound I) (Pickard et al. 1991, McCarthy and White 1983).
The agaric basidiomycete M. rotula produced a novel aromatic peroxygenase (MroAPO) during growth in agitated liquid culture. The enzyme was purified to apparent homogeneity and characterized. It is a relatively small (Mw: 32 kDa), glycosylated (16%) heme-thiolate protein that peroxygenates naphthalene, toluene, benzyl and veratryl alcohol as well as oxidizes typical peroxidase substrates such as ABTS and DMP (but neither chloride nor bromide). The fungus produced up to 41.000 U L-1 (445 mg L-1) of MroAPO in a complex medium rich in organic carbon and nitrogen (4.2% glucose, 4.5% soybean peptone and 0.45% yeast extract). To our best knowledge, the calculated amount of 445 mg L-1 peroxygenase protein is one of the highest levels of a secreted heme peroxidase reported for a wild-type basidiomycete so far.
The authors declare that they have no competing interests.
a In earlier publications, the enzyme was abbreviated AaP (Agrocybe aegerita peroxidase/peroxygenase; Ullrich et al. 2004, Hofrichter and Ullrich 2006)