Production and characterisation of a novel actinobacterial DyP-type peroxidase and its application in coupling of phenolic monomers

Amos Musengi; Kim Durrell; Alaric Prins; Nuraan Khan; Mayowa Agunbiade; Tukayi Kudanga; Bronwyn Kirby-McCullough; Brett I Pletschke; Stephanie G Burton; Marilize Le Roes-Hill

doi:10.1016/j.enzmictec.2020.109654

Production and characterisation of a novel actinobacterial DyP-type peroxidase and its application in coupling of phenolic monomers

Enzyme Microb Technol. 2020 Nov:141:109654. doi: 10.1016/j.enzmictec.2020.109654. Epub 2020 Sep 3.

Authors

Affiliations

¹ Applied Microbiology and Health Biotechnology Institute, Cape Peninsula University of Technology, PO Box 1906, Bellville, 7535, South Africa; Biotechnology Department, Harare Institute of Technology, P. O. Box BE 277, Belvedere, Harare, Zimbabwe.
² Applied Microbiology and Health Biotechnology Institute, Cape Peninsula University of Technology, PO Box 1906, Bellville, 7535, South Africa.
³ Applied Microbiology and Health Biotechnology Institute, Cape Peninsula University of Technology, PO Box 1906, Bellville, 7535, South Africa; Institute for Microbial Biotechnology and Metagenomics, Department of Biotechnology, University of the Western Cape, Bellville, 7535, South Africa.
⁴ Applied Microbiology and Health Biotechnology Institute, Cape Peninsula University of Technology, PO Box 1906, Bellville, 7535, South Africa; Department of Biotechnology and Food Technology, Durban University of Technology, PO Box 1334, Durban, 4000, South Africa.
⁵ Institute for Microbial Biotechnology and Metagenomics, Department of Biotechnology, University of the Western Cape, Bellville, 7535, South Africa.
⁶ Department of Biochemistry and Microbiology, Rhodes University, PO Box 94, Makhanda (Grahamstown), 6140, South Africa.
⁷ Vice-Principal: Research and Postgraduate Education and Department of Biochemistry, University of Pretoria, Private Bag X20, Hatfield, Pretoria, 0028, South Africa.
⁸ Applied Microbiology and Health Biotechnology Institute, Cape Peninsula University of Technology, PO Box 1906, Bellville, 7535, South Africa. Electronic address: leroesm@cput.ac.za.

PMID: 33051013
DOI: 10.1016/j.enzmictec.2020.109654

Abstract

The extracellular peroxidase from Streptomyces albidoflavus BSII#1 was purified to near homogeneity using sequential steps of acid and acetone precipitation, followed by ultrafiltration. The purified peroxidase was characterised and tested for the ability to catalyse coupling reactions between selected phenolic monomer pairs. A 46-fold purification of the peroxidase was achieved, and it was shown to be a 46 kDa haem peroxidase. Unlike other actinobacteria-derived peroxidases, it was only inhibited (27 % inhibition) by relatively high concentrations of sodium azide (5 mM) and was capable of oxidising eleven (2,4-dichlorophenol, 2,6-dimethoxyphenol, 4-tert-butylcatechol, ABTS, caffeic acid, catechol, guaiacol, l-DOPA, o-aminophenol, phenol, pyrogallol) of the seventeen substrates tested. The peroxidase remained stable at temperatures of up to 80 °C for 60 min and retained >50 % activity after 24 h between pH 5.0-9.0, but was most sensitive to incubation with hydrogen peroxide (H₂O₂; 0.01 mM), l-cysteine (0.02 mM) and ascorbate (0.05 mM) for one hour. It was significantly inhibited by all organic solvents tested (p ≤ 0.05). The K_m and V_max values of the partially purified peroxidase with the substrate 2,4-DCP were 0.95 mM and 0.12 mmol min^-1, respectively. The dyes reactive blue 4, reactive black 5, and Azure B, were all decolourised to a certain extent: approximately 30 % decolourisation was observed after 24 h (1 μM dye). The peroxidase successfully catalysed coupling reactions between several phenolic monomer pairs including catechin-caffeic acid, catechin-catechol, catechin-guaiacol and guaiacol-syringaldazine under the non-optimised conditions used in this study. Genome sequencing confirmed the identity of strain BSII#1 as a S. albidoflavus strain. In addition, the genome sequence revealed the presence of one peroxidase gene that includes the twin arginine translocation signal sequence of extracellular proteins. Functional studies confirmed that the peroxidase produced by S. albidoflavus BSII#1 is part of the dye-decolourising peroxidase (DyP-type) family.

Keywords: Actinobacteria; Biocatalysis; DyP-type peroxidase; Genome; Purification; Streptomyces albidoflavus.

MeSH terms

Amino Acid Sequence
Bacterial Proteins / chemistry
Bacterial Proteins / isolation & purification
Bacterial Proteins / metabolism*
Biocatalysis
Coloring Agents / metabolism*
Enzyme Inhibitors / pharmacology
Genome, Bacterial / genetics
Hydrogen-Ion Concentration
Kinetics
Oxidative Coupling
Peroxidase / chemistry
Peroxidase / genetics
Peroxidase / isolation & purification
Peroxidase / metabolism*
Phenols / chemistry
Phenols / metabolism*
Protein Sorting Signals
Streptomyces / enzymology
Streptomyces / genetics
Streptomyces / metabolism
Substrate Specificity
Temperature

Substances

Bacterial Proteins
Coloring Agents
Enzyme Inhibitors
Phenols
Protein Sorting Signals
Peroxidase

Supplementary concepts

Streptomyces albidoflavus