Appl Microbiol Biotechnol (2005) 66: 401–407 DOI 10.1007/s00253-004-1731-2 BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Pauliina Lankinen . Kristiina Hildén . Nina Aro . Mirja Salkinoja-Salonen . Annele Hatakka Manganese peroxidase of Agaricus bisporus: grain bran-promoted production and gene characterization Received: 6 May 2004 / Revised: 22 June 2004 / Accepted: 23 July 2004 / Published online: 6 November 2004 # Springer-Verlag 2004 Abstract The main manganese peroxidase (MnP) isoen- zyme of Agaricus bisporus ATCC 62459 produced in lignocellulose-containing cultures was isolated, cloned and sequenced. In liquid medium, where MnP was previously detected only in trace amounts, the production of MnP was enhanced by rye and wheat bran supplements. The pI (3.25) and N-terminal amino acid sequence (25 aa) of the enzyme from bran-containing cultures were identical to those reported from compost-isolated MnP1. MnP1 is a 328-aa long polypeptide preceded by a 26-aa leader peptide. The nucleotide sequence and putative amino acid sequence of MnP1 reveal its similarity to Pleurotus ostreatus MnP3 (62.5%), Lepista irina versatile peroxi- dase (VP) (61.8%) and Pleurotus eryngii VPs VPL2 and VPL1 (61.9% and 61.2%, respectively). The intron-exon structure resembles that of P. ostreatus MnP1 and P. eryngii VPL1. Despite the sequence similarity to VPs, in the A. bisporus MnP1 sequence, alanine (A163) is present instead of tryptophane (W164), distinguishing it from the veratryl alcohol oxidising P. eryngii VPLs. The MnP sequence can be used as a tool to examine the pattern of ligninolytic gene expression during the growth and fruiting of A. bisporus to optimise compost composition, fungal growth and mushroom production. Introduction Agaricus bisporus, the button mushroom, is economically the most important fungus in the world, with over a one- third share of the 3.2 million tons of mushrooms produced annually (FAOSTAT 2004). The straw- and manure-based compost used for its cultivation is the most complex among media used for edible mushroom production. A. bisporus is a litter decomposer that takes part in lignocellulose degradation in soil together with other fungi, bacteria, and soil animals. Straw is therefore an artificial medium for A. bisporus and requires a pre- composting stage whereby the action of other fungi and bacteria render the substrate more digestible. In compost environment A. bisporus produces at least two ligninolytic enzymes, laccase (EC 1.10.3.2) and manganese peroxidase (MnP; EC 1.11.1.13). The production of MnP and laccase by A. bisporus (Bonnen et al. 1994; Wood 1980) and the degradation of lignin in compost take place mainly during the growth stage of the mycelium, before the production of pin-initials (Durrant et al. 1991). Knowledge of fungal peroxidase-encoding genes and their regulation has increased rapidly in the last 10 years (reviewed by Conesa et al. 2002; Cullen 1997; Martínez 2002). The concentrations of Mn, carbon and nitrogen, as well as the quality of the substrate (wood, sawdust, straw) have been reported to influence the production of MnP and its isoenzyme profile in white-rot fungi (Martínez 2002) and in litter-decomposing fungi (Steffen et al. 2002a,b). Lignocellulose degradation and substrate utilisation by A. bisporus are important for adopting new culture techniques or substrates to improve mushroom production (Stoop and Mooibroek 1999). Factors affecting production of MnP in A. bisporus are unknown. The presence of one, possibly two, MnPs in this fungus was proposed by Bonnen et al. (1994) and Lankinen et al. (2001) but so far genetic data for MnP are lacking. We previously purified A. bisporus MnP1 from compost (Lankinen et al. 2001). Here, we report culture conditions in which A. bisporus ATCC 62459 produces MnP in a defined medium. We also present the nucleotide sequence of the MnP isoenzyme shown to be important during growth of A. bisporus in compost and describe the similarity of this sequence to other fungal MnPs. P. Lankinen (*) . K. Hildén . M. Salkinoja-Salonen . A. Hatakka Department of Applied Chemistry and Microbiology, Viikki, Biocenter 1, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland e-mail: pauliina.lankinen@helsinki.fi Tel.: +358-9-19159561 Fax: +358-9-19159322 N. Aro VTT Biotechnology, P.O. Box 1500, 02044 VTT Espoo, Finland