Improving the Performance of Horseradish Peroxidase by Site-Directed Mutagenesis

International Journal of Molecular Sciences
Diana Humer, Oliver Spadiut

Abstract

Horseradish peroxidase (HRP) is an intensely studied enzyme with a wide range of commercial applications. Traditionally, HRP is extracted from plant; however, recombinant HRP (rHRP) production is a promising alternative. Here, non-glycosylated rHRP was produced in Escherichia coli as a DsbA fusion protein including a Dsb signal sequence for translocation to the periplasm and a His tag for purification. The missing N-glycosylation results in reduced catalytic activity and thermal stability, therefore enzyme engineering was used to improve these characteristics. The amino acids at four N-glycosylation sites, namely N13, N57, N255 and N268, were mutated by site-directed mutagenesis and combined to double, triple and quadruple enzyme variants. Subsequently, the rHRP fusion proteins were purified by immobilized metal affinity chromatography (IMAC) and biochemically characterized. We found that the quadruple mutant rHRP N13D/N57S/N255D/N268D showed 2-fold higher thermostability and 8-fold increased catalytic activity with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as reducing substrate when compared to the non-mutated rHRP benchmark enzyme.

References

Dec 9, 1998·International Journal of Radiation Oncology, Biology, Physics·L K FolkesP Wardman
Jan 31, 2004·Phytochemistry·Nigel C Veitch
May 20, 2004·British Journal of Cancer·J TupperG U Dachs
Jul 4, 2006·Trends in Biotechnology·Barry J RyanCiarán O'Fágáin
Jun 17, 2008·Biochimie·Barry J Ryan, Ciarán O'Fágáin
Jan 10, 2009·International Journal of Biological Macromolecules·Nastia VasilevaKatya Gabrovska
Nov 19, 2010·Bioanalysis·Christophe A Marquette, Loïc J Blum
Feb 5, 2011·International Journal of Biological Macromolecules·Sedigheh AsadKhosro Khajeh
Jan 1, 2010·Advances in Experimental Medicine and Biology·Simona Carmen LitescuGabriel Lucian Radu
Apr 17, 2012·Current Opinion in Chemical Biology·Haesik Yang
Jun 30, 2012·Nature Methods·Johannes SchindelinAlbert Cardona
May 27, 2014·Glycobiology·Simona CaponeOliver Spadiut
Jan 13, 2015·Applied Microbiology and Biotechnology·Florian W Krainer, Anton Glieder
Sep 4, 2015·Biotechnology and Applied Biochemistry·Sedigheh AsadKhosro Khajeh
Mar 16, 2017·Journal of Biotechnology·Thomas Gundinger, Oliver Spadiut
Jan 6, 2018·International Journal of Molecular Sciences·Suzanne M HuddyEdward P Rybicki

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Citations

Sep 10, 2019·Expert Opinion on Drug Delivery·Sadia SikderDhananjay Pal
Mar 5, 2020·Molecular & Cellular Proteomics : MCP·Payman Samavarchi-TehraniAnne-Claude Gingras
Jul 3, 2020·International Journal of Molecular Sciences·Diana HumerOliver Spadiut
Jul 24, 2020·International Journal of Molecular Sciences·Vladimír Křen, Pavla Bojarová
Jan 17, 2021·Molecular & Cellular Proteomics : MCP·Payman Samavarchi-TehraniAnne-Claude Gingras
Feb 13, 2021·Molecular Microbiology·Sabrina L Slater, Despoina A I Mavridou
Mar 16, 2021·Journal of Molecular Modeling·Anderson Aparecido do Espírito Santo, Gustavo Troiano Feliciano
Apr 10, 2021·APL Bioengineering·Wonmoon SongNathaniel S Hwang
Apr 13, 2021·Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy·Fatemeh BehrouzifarAzade Ghorbani-HasanSaraei
Jun 3, 2021·Talanta·Marimuthu Citartan
Aug 16, 2021·Biomedicine & Pharmacotherapy = Biomédecine & Pharmacothérapie·Diana HumerOliver Spadiut
Nov 5, 2019·New Biotechnology·Giulio BernardinelliArio de Marco
Sep 30, 2021·Molecular Microbiology·Jessica KimmelTobias Spielmann

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Methods Mentioned

BETA
glycosylation
ABTS
PCR

Software Mentioned

OriginPro
Image [UNK]
Fiji Image Analysis
OriginLab
[UNK] MP System

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