Peroxidase gene discovery from the horseradish transcriptome

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Horseradish peroxidases (HRPs) are vital reporters in diagnostics and histochemistry.
• Current commercial HRP is a mixture of isoenzymes due to poor characterization and low expression levels.
• Limited sequence data hinders the development of specific HRP isoenzymes for various applications.

Purpose of the Study:

• To identify and characterize novel horseradish peroxidase (HRP) isoenzymes from Armoracia rusticana.
• To enable the production of pure HRP isoenzymes for industrial, biological, and biomedical uses.
• To validate transcriptome sequencing as a method for discovering new enzymes.

Main Methods:

• Sequencing of a normalized, size-selected A. rusticana transcriptome library using 454 Titanium technology.
• De novo assembly of reads into isotigs, followed by ORF finding and characterization.
• Manual verification of sequences via Sanger sequencing of PCR-amplified genomic fragments.
• Expression of identified isoenzymes in Pichia pastoris for activity testing.

Main Results:

• Assembly yielded 14871 isotigs, leading to the identification of 28 secretory peroxidase sequences.
• 23 of the identified peroxidase sequences were previously unknown.
• 22 distinct isoenzymes, including allelic variants, were successfully expressed and demonstrated peroxidase activity.

Conclusions:

• Transcriptome sequencing combined with sequence motif search is an effective strategy for discovering new enzymes.
• The study provides a valuable set of 28 HRP isoenzymes for diverse applications.
• This approach offers a reliable method for identifying and characterizing large groups of isoenzymes.