2 6 dmp

Methyl orange (4-[4-(dimethylamino)phenylazo]benzene sulphonic acid sodium salt), crystal violet (hexamethylpararosaniline chloride), malachite green (N,N,N′,N′-tetramethy-l-4,4′-diaminotriphenylcarbenium chloride), ABTS, 2,6-DMP and guaiacol were obtained from Sigma-Aldrich commercially. The P. pastoris strain GS115 (his4) and plasmid pPIC9K were purchased from Invitrogen (San Diego, CA, USA). Unless otherwise stated, all other chemicals were from commercial sources and were of analytical grade.

Daunomycin hydrochlorine (≥90%), doxorubicin hydrochlorine (≥98%), mitoxantrone hydrochlorine (≥90%), 2.6-dimethoxyphenol (99%; 2.6-DMP), nitrotetrazolium blue (99%; NBT), protocatechuic acid (97%), sodium alginate, calcium chloride (93%), 30% hydrogen peroxide, malonic acid (99%), DPPH^• (2.2-diphenyl-1-picrylhydrazyl), Trolox (97%), and the glucose oxidase assay were purchased from Sigma-Aldrich (St. Louis, MO, USA). The catalase assay kit was purchased from Merck Millipore. All other chemicals and reagents were of analytical grade.

ABTS, syringaldazine and 2,6 DMP (2,6-dimethoxyphenol) and other chemicals were from Sigma-Aldrich (St. Louis, MO, USA). Restriction enzymes and molecular biology reagents were from New England Biolabs (Ipswich, MA, United States). All chemicals were of analytical grade. The Myrothecium verrucaria bilirubine oxidase (MvBOD, 3.61 mg/mL) was from Novozymes (Bagsværd, Denmark). The osmium polymer ([Os (4,4′-dichloro-2,2′-bipyridine)2(poly-vinylimidazole)10Cl]·Cl, E°′ = 0.350 V vs. Ag|AgCl_sat) (Additional file 1: Figure S1) was kindly provided by Prof. Dónal Leech and Dr. Peter Ó Conghaile from Biomolecular Electronics Research Laboratory, National University of Ireland (Galway, Ireland) and prepared as previously reported [23 (link)].
The standard yeast selection medium YPDS (Yeast Extract Peptone Dextrose with Sorbitol) comprised 1% yeast extract, 2% peptone, 2% dextrose (glucose), 1 M sorbitol, 2% agar (all w/v, Invitrogen, Carlsbad, CA, USA). The standard yeast expression media BMGY/BMMY (Buffered Glycerol-complex Medium/Buffered Methanol-complex Medium, both from Invitrogen) comprised 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10⁻⁵% biotin (all w/v), 1% glycerol or 0.5% methanol (both v/v).

Purified AfAA9_B activity was analyzed as reported by Breslmayr et al. (2018) [80 (link)]. The assay consisted of a reaction mixture containing 1 mM 2,6-dimethoxyphenol (2,6-DMP) (Sigma–Aldrich, St. Louis, MO, USA), 100 μM H₂O₂, and recombinant purified AfAA9_B in 50 mM sodium phosphate buffer (pH 8.0). For the blank, the enzyme was denatured by incubation at 99 °C for 30 min before the reaction mixture was added. After 5 min at 30 °C, absorbance was read at 469 nm to calculate the LPMO peroxidase activity.

Usual laccase substrates [2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol (2,6-DMP)], amines histamine, tyramine, and putrescine, expression inducers isopropyl-β-D-thiogalactopyranoside (IPTG) and arabinose, antibiotics kanamycin, and chloramphenicol, and also standard proteins used for molecular weight determination were obtained from Sigma (Madrid, Spain). Nickel-chelating nitrilotriacetic acid (Ni-NTA) agarose was from Qiagen (Hilden, Germany). All other chemicals and reagents were of analytical grade.

Filamentous fungus G. lucidum 77002 was preserved at culture collection of the School of Life Sciences, Anhui University, China (China Center for Type Culture Collection No. AF2013025). S. cerevisiae was from China Center of Industrial Culture Collection, Beijing, China (No. CICC31014). Syringaldazine, 2,6-DMP, guaiacol, catechol, L-dopamine, ABTS, and K₄Fe(CN)₆ were purchased from Sigma-Aldrich (St. Louis, MO, USA). Wheat bran was from Zhangyuan (Bozhou, China), and peanut powder was purchased from Huiyou (Hefei, China).
G. lucidum 77002 was maintained on potato dextrose agar (PDA, contained 20.0 g glucose, 1.0 g KH₂PO₄, 1.5 g MgSO₄ · 7H₂O, 50.0 μg vitamin B₁, 15.0 g agar powder, and 200.0 g potato extract liquid/L) slants at 4°C. YMG liquid medium (contained 10.0 g malt extract, 4.0 g yeast extract, and 10.0 g glucose/L) was used to prepare G. lucidum 77002 inoculation. The liquid fermentation medium used for producing laccase contained 30.0 g peanut powder, 30.0 g wheat bran, and 1.5 g KH₂PO₄/L. YNB medium (contained 1.7 g yeast nitrogen base, 5.0 g ammonia sulfate, and 20.0 g glucose/L) was used for culture of S. cerevisiae.

Strain K. huakuii LAM0618^T was provided by the Agricultural Culture Collection of China (ACCC 06121). E. coli strains DH5α and BL21 (DE3) (Tian Gen Co., Ltd, China) were used as host strains for plasmid propagation and protein expression, respectively. The E. coli strains were routinely grown in Luria-Bertani medium. Antibiotics were added at desired concentrations (50 μg/mL kanamycin or 100 μg/mL ampicillin). A genomic DNA kit, a gel extraction kit and a plasmid kit were purchased from Omega Bio-Tek (Norcross, GA, USA). Enzymes for cloning procedures and protein ladders were obtained from Fermentas (St. Leon-Rot, Germany). ABTS, SGZ, 2,6-DMP, guaiacol and l-dopamine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals were of analytical grade or higher and were purchased from commercial sources.