The Induction, Generation, Purification And Decolorization Of Dyes By Bilirubin Oxidase From Myrothecium Verrucaria

Bilirubin oxidase (EC.1.3.3.5) produced by Myrothecium verrucaria is a multicopper oxidase. It can catalyze the oxidation of biliverdin and several laccase substrates, showing a promising potential in environmental applications. This paper was focused on fermentation process and purification of bilirubin oxidase form M. verrucaria and decolorization of dyes.The result showed that the optimal culture conditions were as follow: cultured for 4days, culture temperature was 26℃, broth initial pH was pH6.0, spores liquid dilution was 10-3,250 mL Erlenmeyer flasks contained 100 mL,20% liquid potato culture medium. The effects of carbon source, nitrogen source, metal ions and inducers on enzyme production of M. verrucaria were investigated using one factor method and orthogonal design. The results showed that glucose and soya peptone were the optimal carbon source and nitrogen source, respectively. A higher enzyme activity was achieved with the addition of 2 mmol/L Cu2+ and 0.1 mmol/L of inducers. The orthogonal design experiment indicated that the optimal medium was:glucose 10 g/L, soya peptone 7.5 g/L, CuSO4 1 mmol/L, gallic acid 0.1 mmol/L. The enzyme activity was enhanced 3 times over the initial values after the fermentation medium was optimized using orthogonal design.Furthermore, the bilirubin oxidase was purified using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The purification fold was 3.12 and recovery of total bilirubin oxidase activity was 27.92%. The molecular mass of the enzyme was 68.1 kDa as determined by SDS-PAGE, and single protein band appeared on the Native PAGE gel identifying the enzyme had been purified to electrophoretic homogeneity. The Native PAGE stained with ABTS and bilirubin showed the purified bilirubin oxidase was able to catalyze the oxidation of ABTS and bilirubin. The Native PAGE gel which was stained with indigo carmine, it further demonstrated purified BOD indeed had ability to degrade indigo carmine. The purified enzyme was identified by N-terminal amino acid sequence and MALDI-TOF-MS, homologies of up 100% were found with bilirubin oxidase from M. verrucaria.The optimum reaction pH of the purified enzyme for bilirubin was 7.5 and maximum enzyme activity was at 40℃. The optimum pH of the enzyme reaction for ABTS was approximate to pH 3.4. The enzyme activity showed slight decrease after incubation at 25℃for 1 h at pH 7.5～8.0; the enzyme activity was stable after incubation for 1 h at temperature up to 40℃. The purified enzyme can catalyze the oxidation of bilirubin and ABTS, it catalytic efficiency for bilirubin was also higher than for ABTS. In addition the enzyme activity was strongly inhibited by 1 mmol/L L-cysteine,10 mmol/L sodium azide and 1 mmol/L DTT. Bilirubin oxidase activity was decreased by the addition of most metal ions. However, only the copper ion made the enzyme activity increased remarkably.Co-culture system showed that M. verrucaria can degrade a variety of dyes. The biosorption and biodegradation of the dye were detected in the process of decolorization, finally more than 90% decolorization efficiency was achieved by incubating for 7 d at 26℃. Additionally, The partial purified bilirubin oxidase can efficiently decolorize indigo carmine at 30℃～50℃, pH5.5～9.5 and dye concentration of 50 mg/L-200 mg/L. The partial purified bilirubin oxidase was able to decolorize more than 90% of indigo carmine (100 mg/L) in only 50 min with an enzymatic activity of 1000 U/L, suggesting a broad application prospect of bilirubin oxidase in dye effluents decolorization.