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Open Access Research article

Peroxidase gene discovery from the horseradish transcriptome

Laura Näätsaari1, Florian W Krainer1, Michael Schubert13, Anton Glieder2 and Gerhard G Thallinger23*

Author Affiliations

1 Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria

2 Austrian Centre of Industrial Biotechnology (ACIB GmbH), Petersgasse 14, 8010 Graz, Austria

3 Institute for Genomics and Bioinformatics, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria

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BMC Genomics 2014, 15:227  doi:10.1186/1471-2164-15-227

Published: 24 March 2014

Abstract

Background

Horseradish peroxidases (HRPs) from Armoracia rusticana have long been utilized as reporters in various diagnostic assays and histochemical stainings. Regardless of their increasing importance in the field of life sciences and suggested uses in medical applications, chemical synthesis and other industrial applications, the HRP isoenzymes, their substrate specificities and enzymatic properties are poorly characterized. Due to lacking sequence information of natural isoenzymes and the low levels of HRP expression in heterologous hosts, commercially available HRP is still extracted as a mixture of isoenzymes from the roots of A. rusticana.

Results

In this study, a normalized, size-selected A. rusticana transcriptome library was sequenced using 454 Titanium technology. The resulting reads were assembled into 14871 isotigs with an average length of 1133 bp. Sequence databases, ORF finding and ORF characterization were utilized to identify peroxidase genes from the 14871 isotigs generated by de novo assembly. The sequences were manually reviewed and verified with Sanger sequencing of PCR amplified genomic fragments, resulting in the discovery of 28 secretory peroxidases, 23 of them previously unknown. A total of 22 isoenzymes including allelic variants were successfully expressed in Pichia pastoris and showed peroxidase activity with at least one of the substrates tested, thus enabling their development into commercial pure isoenzymes.

Conclusions

This study demonstrates that transcriptome sequencing combined with sequence motif search is a powerful concept for the discovery and quick supply of new enzymes and isoenzymes from any plant or other eukaryotic organisms. Identification and manual verification of the sequences of 28 HRP isoenzymes do not only contribute a set of peroxidases for industrial, biological and biomedical applications, but also provide valuable information on the reliability of the approach in identifying and characterizing a large group of isoenzymes.

Keywords:
Horseradish peroxidase; Armoracia rusticana; Transcriptome sequencing; Pichia pastoris; 454 sequencing