Characterization of Multi-Enzymes Produced by the Fungus, Dec 1, Responsible for Dye Decolorization

A peroxidase, DyP, produced by the fungus Dec 1 having the ability to decolorize dyes was purified. DyP contains 17% sugar comprising GlcNAc and Man. The molecular mass of DyP was estimated to be 60 kDa and the isoelectric point (pl) was determined to be 3.8. DyP degraded various dyes, and also phenolic compounds of 2,6-dimethoxyphenol and guaiacol. However, veratryl alcohol, abbreviated VA, a substrate of lignin peroxidase, was not degraded by DyP.

Molasses was utilized by Dec 1 to produce DyP as carbon and energy source. Within 10 g/l of molasses concentration, decolorization activity of Dec 1 toward RB5 gradually increased. However, at more than 20 g/l of molasses concentration, the inhibitory effect on the decolorizin g activity of Dec 1 was observed. As the activity of purified DyP was inhibited by 10 g/l of molasses concentration, this indicates that molasses has a stimulative effect on the decolorization activity of Dec 1, but has an inhibitory effect on the DyP activity.

Dec 1 also produced Aryl Alcohol Oxidase (AAO). The role of AAO in the Dec 1 decolorization process of dyes has been observed to be as follows: the first role was that H2O2 was produced by AAO oxidation of Veratryl Alcohol (VA) to veratraldehyde and then utilized by the peroxidase DyP. In the cultivation of Dec l, the presence of H2O2 and veratraldehyde has been detected. The second role was that the polymerization of products produced by DyP oxidation of a simplified RB5, AQ-2’, was prevented by AAO. This was confirmed by the result that the molecular weight of the products was reduced in the mixed decolorization of DyP and AAO.

A new versatile peroxidase named TcVP1 was purified from the Dec 1 culture. Purified TcVP1 behaved as Manganese Peroxidase (MnP) at pH 5, and the enzyme functioned as a Lignin Peroxidase (LiP) at pH 3. As TcVP1 decolorized preferentially azo dyes, co-application of TcVP1 and DyP conducted complete decolorization of anthraquinone dye, RB5, in vitro to colorless products.

Keywords: Peroxidase, DyP, Anthraquinone dye, Aryl alcohol oxidase, Thanatephorus cucumeris Dec 1, Versatile oxidase, TcVP1, Simplified RB5, AQ2’, Veratryl alcohol, VA, Veratraldehyde, Hydrogen peroxide, Molasses, Manganese peroxidase, MnP, Lignin peroxidase, LiP.