Abstract
A dye-decolorizing peroxidase (DyP) from Irpex lacteus was cloned and heterologously expressed as inclusion bodies in Escherichia coli. The protein was purified in one chromatographic step after its in vitro activation. It was active on ABTS, 2,6-dimethoxyphenol (DMP), and anthraquinoid and azo dyes as reported for other fungal DyPs, but it was also able to oxidize Mn2+ (as manganese peroxidases and versatile peroxidases) and veratryl alcohol (VA) (as lignin peroxidases and versatile peroxidases). This corroborated that I. lacteus DyPs are the only enzymes able to oxidize high redox potential dyes, VA and Mn+2. Phylogenetic analysis grouped this enzyme with other type D-DyPs from basidiomycetes. In addition to its interest for dye decolorization, the results of the transformation of softwood and hardwood lignosulfonates suggest a putative biological role of this enzyme in the degradation of phenolic lignin.
Highlights
The arsenal of oxidoreductases in these organisms is the main driver of lignin degradation, leaving a whitish material enriched in cellulose and hemicellulose [4]
After sequencing of the gene coding for I. lacteus DyP from the fungal cDNA, the sequence was translated in the correct reading frame to obtain a 447 amino acids protein
IrlacDyP molecular mass was estimated to be around 50 kDa by SDS–PAGE (Figure 4B). This is in agreement with previous findings describing the native enzyme Mw after deglycosylating (51.11 kDa, [21]), and it is similar to those reported for other fungal DyPs, such as those from A. auricula-judae [38], P. ostreatus [39], and other I. lacteus strains [40]
Summary
N-terminal sequencing and peptide mass fingerprinting analyses of IrlacDyP were previously performed [21]. Protein sequences of the DyPs displaying higher identity to IrlacDyP (and their coding nucleotide sequences (mRNA)) were retrieved from the database, and the nucleotides coding for the predicted signal peptide were removed after analysis using the SignalP 4.1 server [25]. After transformation of the recombinant vectors into the E. coli DH5α strain, 3 clones containing the inserted fragments were sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit and the automated ABI Prism 3730 DNA sequencer (Applied Biosystems, MA, USA). Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model and selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site This analysis involved 68 amino acid sequences. The models were analyzed using PyMol 1.1 (http://pymol.org/)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
