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

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 substitution led to 2.5-fold improvement in the catalytic efficiency with phenol/4-aminoantipyrine. Constructed mutant represents a stable biocatalyst, which may find use in medical diagnostics, biosensing, and bioprocesses.

Improving the Performance of Horseradish Peroxidase by Site-Directed Mutagenesis

“…For N255D on the other hand, Capone et al [19] observed almost the same catalytic activity compared to the benchmark enzyme (1.1-fold increase). N268D had a 2-fold increased turnover number (k cat ) when compared to the non-mutated rHRP and the same trend was shown by Asad et al [20], where a 2.6-fold enhanced k cat with phenol/4-aminoantipyrine was reported. The slightly enhanced catalytic efficiency of N57S, when compared to the benchmark rHRP (1.2-fold), is in accordance with Capone et al [19] (1.4-fold).…”

“…They described variants N13D and N268D, which showed increased catalytic efficiency with phenol/4-aminoantipyrine and were both more stable in terms of hydrogen peroxide and heat tolerance. A follow-up study identified N268G, which showed 18-fold higher resistance towards hydrogen peroxide and 2.5-fold higher thermal stability [20]. Capone et al [19] performed a profound investigation of N -glycosylation mutants in P. pastoris , where asparagines at eight sites were replaced by aspartic acid, serine or glutamine.…”

Refactoring the Genetic Code for Increased Evolvability

“…Saturation mutagenesis techniques provide full access to all amino acids by using degenerate codons covering a comprehensive collection of amino acids (15)(16)(17). Such approaches frequently identify multiple nucleotide replacements (MNRs) within a codon to be superior to SNRs in a variety of contexts (16,(18)(19)(20)(21)(22)(23)(24)(25)(26). Recent technological advances in DNA synthesis and sequencing have enabled more detailed surveys of a complete systematic saturation mutagenesis of a gene, permitting the interrogation of the full mutational landscape at single amino acid resolution (27)(28)(29).…”

N-linked glycosylation increases horse radish peroxidase rigidity leading to enhanced activity and stability

“…Enhancement of activity upon glycosylation has been observed for various enzymes 5,52 but little is known how glycan addition at sites remote from the active site are influencing enzyme activity. Indeed, it has been observed previously for HRP when plant produced enzyme is compared to the recombinant form 16,21,28,57 but with little attention paid and no rationale provided for such an observation. Thus, the main cause of this activity enhancement is largely unknown.…”