Applied Biochemistry and Biotechnology

, Volume 164, Issue 4, pp 454–463

Investigating the Structural and Functional Effects of Mutating Asn Glycosylation Sites of Horseradish Peroxidase to Asp


DOI: 10.1007/s12010-010-9147-1

Cite this article as:
Asad, S., Khajeh, K. & Ghaemi, N. Appl Biochem Biotechnol (2011) 164: 454. doi:10.1007/s12010-010-9147-1


Horseradish peroxidase (HRP) has long attracted intense research interest and is used in many biotechnological fields, including diagnostics, biosensors, and biocatalysis. Enhancement of HRP catalytic activity and/or stability would further increase its applications. One of the problems with heterologus expression of HRP especially in prokaryotic host is lack of glycosylation that affects it's stability toward H2O2 and thermal inactivation. In this study, two asparagine residues which constitute two of the eight glycosylation sites in native HRP (Asn 13 and 268) with respectively 83% and 65% surface accessibility were substituted with aspartic acid in recombinant HRP. Both mutant proteins expressed in Escherichia coli showed increased stabilities against heat (increase in t1/2 from 20 min in native rHRP to 32 and 67 min in N13D and N268D) and H2O2 (up to threefold). Unexpectedly, despite the distance of the mutated positions from the active site, notable alterations in steady-state kcat and Km values occurred with phenol/4-aminoantipyrine as reducing substrate which might be due to conformational changes. No significant alteration in flexibility was detected by acrylamide quenching analyses, but ANS binding experiments purposed lesser binding of ANS to hydrophobic patches in mutated HRPs. Double mutation was non-additive and non-synergistic.


Recombinant horseradish peroxidase Site-directed mutagenesis Glycosylation site Protein stability H2O2 inactivation 

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of Biotechnology, University College of ScienceUniversity of TehranTehranIran
  2. 2.Department of Biochemistry, Faculty of Biological ScienceTarbiat Modares UniversityTehranIran

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