A Scavenger Hunt for a DyP-Peroxidase from a Metagenome: Curated Peroxidase Database-Assisted Primer Design and...

Dye-decolorizing peroxidase (DyP)-type peroxidases are heme-containing enzymes that play a role in lignin synthesis and degradation and dye decolorization. Despite numerous studies about this class of enzymes, the enzyme remains under-explored. We used 1000 DyP sequences retrieved from the NCBI database to forge a phylogenetic tree. Nodes in the tree, where sequences displayed a degree of conservation, were used to design degenerate primers to locate DyP-peroxidase sequences from the DNA extract of a tannery wastewater sample. After PCR amplification and visualization using agarose electrophoresis, a band at the expected size of a DyP peroxidase (500-700 bp) was seen. TA cloning followed by blue-white colony selection validated our finding after amplicon sequencing of the PCR product to confirm the presence of an Acinetobacter species DyP-peroxidase. Our metagenomic DyP displayed 99% similarity to the DyP-peroxidase sequence found in the Acinetobacter baumannii ATCC 19606 strain. As a result, and due to the minute differences between our found DyP and the ATCC 19606 strain DyP, we expressed the latter cloned in a pET28b(+) vector and purified it from culture medium using Escherichia coli SoluBl21 as a host strain. A crude oxidation assay using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) deemed the enzyme active as shown by the formation of a green color. The crystal structure of the enzyme was solved at 2.6 Å resolution (PDB ID 9OBR) using X-ray crystallography and presented as a hexamer in solution.