**2.9 Degradation assay of β-***O***-4 lignin model compound**

### **2.9.1 Culture experiment**

To assay the abilities of MnP and Lcc to degrade the β-*O*-4 lignin model compound, 4 ethoxy-3-methoxyphenylglycerol- β-guaiacyl ether (Fig. 2; β-*O*-4 compound, hereafter) was synthesized according to the method previously described (Umezawa & Higuchi 1985).

To prepare media containing the β-*O*-4 compound to media, 300 µg of the β-*O*-4 compound was diluted in 50 µl acetone and added to 30 ml MYPG-S. Whole liquid culture media was collected 14, 21 and 42 days after inoculation and assayed for phenol-oxidizing enzyme activity. The β-*O*-4 compound was then recovered from the liquid culture media by the addition of two volumes of ethyl acetate to separate the aqueous phase, before evaporating the ethyl acetate off and then precipitating the compound. To improve subsequent chromatographic analysis, the recovered β-*O*-4 compound was silylated using N-*O*-bis- (trimethylsilyl) trifluoroacetamide (BSTFA) to form a trimethylsilyl (TMS) derivative. This TMS derivative was then subjected to gas chromatography-mass spectrometry (GC-MS) analysis (6890N, Agilent Technologies, CA), which was fitted with an capillary column (HP-5 MS, 30 m × 0.25 mm i.d., 0.25-µm; J&W Scientific, CA) coupled to an MS (JMS-K9, JEOL, Japan) according to the manufacturer's instructions. Helium was used as the carrier gas at 1.5 ml/min. GC oven conditions consisted of 150°C for 1 min, initially ramped at 10°C/min to 200°C and then at 5°C/min to 250°C. The electron impact mass spectra were obtained at an acceleration energy of 70 eV. The degradation rate (%) of the β-*O*-4 compound was calculated using the rate of quantities of the TMS derivative before and after culture, compensating for the recovery of the β-*O*-4 compound with 4,4'-dimethoxybenzoin (anisoin), which was used as an internal standard.

Fig. 2. β-*O*-4 lignin model compound used in this study; 4-ethoxy-3-methoxyphenylglycerolβ-guaiacyl ether (from Umezawa & Higuchi, 1985).
