Enhanced Productivity of a New Peroxidase DyP by Genetic Manipulation and by Cultivation Methods

A novel peroxidase, DyP gene, dyp, was cloned from a cDNA library of a newly isolated fungus, Dec 1. The open reading frame consisting of 1494 nucleotides indicated a primary translation product of 498 amino acids, and the Molecular mass (Mr) was estimated as 53,306. X-ray diffraction data using crystallized DyP revealed DyP to have a unique tertiary structure differing from that of most other well-known peroxidases. The analyzed amino acid sequence of DyP did not share homology with any other peroxidases except that of a peroxidase derived from Polyporaceae sp.

Mature cDNA encoding dyp was fused with the Aspergillus oryzae α-amylase promoter, amyB, and recombinant DyP was produced. The total activity of the purified recombinant DyP, rDyP, was produced. The total activity of rDyP, was about 400-fold higher than that of the native DyP derived from Dec 1.

The further productivity enhancement of rDyP was carried out using the following different cultivation methods and different media. Recombinant A. oryzae holding a DyP gene, dyp, was grown using the repeated batch method. When a synthetic liquid medium containing maltose as a carbon source was used in repeated batch culture, production of high-level rDyP activity continued for 26 repeated cycles of every 1-day batch.

When the production of rDyP by A. oryzae was carried out using complex media containing rice bran powder both in liquid repeated-batch and in fed-batch cultures, average rDyP productivities were similar in the two batch cultures.

The Solid-state Culture (SSC) was also attempted for the production of rDyP by recombinant A. oryzae using wheat bran as a solid medium and the productivity of rDyP was compared to that in the liquid cultures. The maximum productivity of rDyP in SSC reached 5.3g per kg wheat bran, and this productivity value was equivalent to the productivity using a 56 kg liquid culture. When the unit of the productivity per gram carbon of the medium was introduced, the productivity in SSC was 4.1-fold higher than that in the liquid cultures.

In order to overcome the disadvantages of SSC and liquid culture, Dec 1 was grown in an Air Membrane Surface (AMS) reactor. Although the growth of Dec 1 in AMS culture was almost the same as that in liquid culture at optimum temperatures, DyP productivity, DyP activity, and Aryl Alcohol Oxidase (AAO) activity in AMS culture were 18-, 232-, and 108-fold higher than those in liquid culture, respectively. The advantage of AMS culture of Dec 1 1over SSC and liquid culture has been proven, particularly in the production of DyP.

A biofilm was formed in the AMS culture of Dec 1 and the relationship between biofilm formation and DyP and MnP was analyzed. Two new DyP isozymes, DyP2 and DyP3, were purified from the Dec 1 culture in the AMS reactor, and they were characterized and compared with the characteristics of rDyP.

Keywords: Aspergillus oryzae, Air-membrane surface reactor, Biofilm formation, Crystallization, Decolorizing peroxidase gene (dyp), DyP isozymes , Fed-batch culture, Recombinant DyP (rDyP), Repeated-batch culture, Rice bran powder, Solid-state culture, Wheat bran powder, X-ray diffraction, α-amylase promoter (amyB).