Reactive green 19

Amylose, 2-hydroxyethyl-cellulose (Sigma 434965, molar 2-hydroxyethyl substitution 2.5 mol per mol cellulose), curdlan, laminarin, amylopectin, pectolyase from A. japonicus and α-amylase from bovine pancreas were obtained from Sigma (Brøndby, Denmark). All other polysaccharides were obtained from Megazyme (Bray, Ireland). The enzymes used are listed with the supplier in Table 2. The dyes reactive red 4, reactive blue 4, reactive green 19 and reactive yellow 2, cross-linker 1,4-butanediol diglycidyl ether, NaOH and all salts for buffers were obtained from Sigma (Brøndby, Denmark). Two pathogenic fungi C. acutatum (isolate SA 0-1) and P. expansum (isolate IK2020) and the apple pomace media were kindly provided by Birgit Jensen and Daniel Buchvaldt Amby (Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, 1871 Frederiksberg, Denmark).

This method was established by using Phanerochaete velutina (FBCC 941, previous number 244i) and can be applied to other white rot fungi. We present here the results of four persistent textile dyes. However, the method can be applied to on any dye and any white rot fungus. The investigated dyes were Reactive Orange 16 (Sigma-Aldrich, Germany, CAS, no. 12225-83-1, referred as “RO16”), Reactive Black 5 (Sigma-Aldrich, Germany, CAS, no. 17095-24-8, referred as “RB5”), Reactive Blue 4 (Sigma-Aldrich, Germany, CAS, no. 13324-20-4, referred as “RB4”), and Reactive Green 19 (Sigma-Aldrich, Germany, CAS, no. 61931-49-5, referred as “RG19”).
We present and discuss the method step by step providing methodological steps that are necessary to obtain kinetic data on the overall decolorization, and the decolorization caused by non-enzymatic degradation, as well as the decolorization caused by enzymatic degradation.

Saccharomonospora viridis DSM 43017 was obtained from the China General Microbiological Culture Collection Center, Beijing (reference number CGMCC 4.1324). The strain was cultivated in a shaker flask at 45°C in STS medium (1.0% (w/v) soy peptone, 1.0% (w/v) glucose, 0.2% (w/v) yeast extract, 0.2% (w/v) NaCl, and 0.2% casein enzymatic hydrolysate, all from Biodee, Beijing, China), adjusted pH to 8.0 with NaOH prior to autoclaving.
Escherichia coli DH5α and E. coli BL21 (Tiangen, Beijing, China) (were cultivated in Luria Bertani (LB) medium at 37°C for gene cloning, sequencing, and expression. The pEASY-T3 vector (TransGen, Beijing, China) was used for plasmid gene cloning and sequencing. The plasmid pET-28a (+) (Takara Bio, Otsu, Japan) was used as an expression vector. Manganese peroxidase (MnP), azure B, brilliant green, reactive blue 19, reactive green 19, reactive yellow 2, reactive black 5, reactive red 120, malachite green and crystal violet were purchased from Sigma (St. Louis, MO, USA). All other reagents used were of analytical grade unless otherwise stated.