Construction of a horseradish peroxidase resistant toward hydrogen peroxide by saturation mutagenesis

Biotechnology and Applied Biochemistry
Sedigheh AsadKhosro Khajeh

Abstract

Horseradish peroxidase (HRP) with a variety of potential biotechnological applications is still isolated from the horseradish root as a mixture of different isoenzymes with different biochemical properties. There is an increasing demand for preparations of high amounts of pure enzyme but its recombinant production is limited because of the lack of glycosylation in Escherichia coli and different glycosylation patterns in yeasts which affects its stability parameters. The goal of this study was to increase the stability of non-glycosylated enzyme, which is produced in E. coli, toward hydrogen peroxide via mutagenesis. Asparagine 268, one of the N-glycosylation sites of the enzyme, has been mutated via saturation mutagenesis using the megaprimer method. Modification and miniaturization of previously described protocols enabled screening of a library propagated in E. coli XJb (DE3). The library of mutants was screened for stability toward hydrogen peroxide with azinobis (ethylbenzthiazoline sulfonate) as a reducing substrate. Asn268Gly mutant, the top variant from the screening, exhibited 18-fold increased stability toward hydrogen peroxide and twice improved thermal stability compared with the recombinant HRP. Moreover, the substi...Continue Reading

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Citations

Nov 16, 2017·MBio·Gur PinesRyan T Gill
Aug 23, 2018·Biotechnology and Applied Biochemistry·Narjes NakhaeeMohammad Ali Amoozegar
Feb 23, 2019·International Journal of Molecular Sciences·Diana Humer, Oliver Spadiut
Aug 13, 2017·Applied Microbiology and Biotechnology·Ali GhasemiBahareh Dabirmanesh
Aug 16, 2021·Biomedicine & Pharmacotherapy = Biomédecine & Pharmacothérapie·Diana HumerOliver Spadiut

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