5 g extractive-free beech wood meal (60–80 mesh) and 1.25 mL distilled water in 50-ml Erlenmeyer flasks, which were then incubated at 30 °C for 28 days. After incubation, weight loss, Klason lignin content, and acid-soluble lignin content of the fungal-treated wood meal suspensions were determined, as previously described (Hirai et al., 1994). The selection factor (SF), which is an indicator of ligninolytic selectivity, was calculated Selleck Inhibitor Library as follows: SF = lignin

loss/holocellulose loss. Holocellulose loss was calculated as follows: total weight loss − lignin loss. Phanerochaete chrysosporium ME-446, P. sordida YK-624, and BM-65 were cultured in wood meal suspensions, as described above, and were incubated at 30 °C for 7, 14, 21, and 28 days. After incubation, weight loss, Klason lignin content, and acid-soluble lignin content of the fungal-treated wood meals were Selleckchem CT99021 determined, as described above. Phanerochaete sordida YK-624 and BM-65 were cultured in wood meal suspensions, as described above, and were incubated at 30 °C for 4, 8, 12, 16, 20, 24, and 28 days. Following the culture period, the method described by Hirai et al. (1994) was modified for enzyme extraction. Briefly, fungal-treated wood meal was homogenized with

25 mL of 50 mM malonate buffer (pH 4.0) containing 0.05% Tween 20 (Wako) using a Polytron PT1200 homogenizer for a total of 5 min (20-s blending with 10-min intervals) at 4 °C. Modified methods described by Périé & Gold (1992) and Wariishi et al. (1994) were used for the determination of MnP and LiP activities, respectively, and details are described in Appendix S1. Phanerochaete sordida YK-624 and BM-65 were cultured in wood meal suspensions, as described above, and were incubated at 30 °C for 4, 8, 12, 16, 20, 24, and 28 days. Fungal-treated wood

meals were stored at −80 °C. The purification of total RNA from the two fungal cultures was performed as described above. The concentration and purity of total RNA were estimated by measuring the absorbance at 260 and 280 nm. Two hundred nanograms of tuclazepam total RNA was reverse-transcribed using a Takara Prime Script RT-PCR kit (TaKaRa Bio). The synthesized cDNA was amplified by PCR using a LightCycler System (Roche Applied Science) with primer pairs targeting native mnp4 (mnp4F2–mnp4R4) and recombinant mnp4 (mnp4F2–gpdR1), and gpd (gpdF1–gpdR2), which was used as an endogenous reference gene. Details of primers design and the LightCycler reaction are described in Appendix S1. The nucleotide sequences of the gene mnp4, full-length cDNA of bee2, and 5′ flanking region of bee2 derived from P. sordida YK-624 have been deposited in the DDBJ database (http://www.ddbj.nig.ac.jp/) under accession numbers AB585997, AB638492, and AB638493, respectively. When P. sordida YK-624 was cultured under wood-rotting conditions, large amounts of proteins were produced, as determined by 2-DE.