Development of radical-stable peroxidases based on peroxidase inactivation mechanism

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공과대학 협동과정 바이오엔지니어링전공
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서울대학교 대학원
Peroxidase inactivationRadical stabilityRadical couplingMass spectrometrySite-directed mutagenesisCoprinus cinereus peroxidaseHorseradish peroxidase
학위논문 (박사)-- 서울대학교 대학원 : 협동과정 바이오엔지니어링전공, 2015. 2. 한지숙.
Peroxidases catalyze a variety of oxidative transformations of many aromatic compounds and thus have potential in biosynthesis and other biotechnological applications. However, the usefulness of these versatile enzymes is limited, as the enzyme is quickly inactivated during the oxidation reaction of aromatic compounds. This low stability of peroxidases results in low product yield due to the incomplete reaction and increased production costs. Many researchers have studied this, and three possible pathways for peroxidase inactivation have been proposed: reaction with excess hydrogen peroxide, sorption by polymer product, and reaction with radical intermediates.

The first two pathways have been corroborated with extensive evidence
however, the free radical-mediated mechanism of peroxidase inactivation has not been fully elucidated. Thus, the dominant inactivation mechanism in the oxidation reaction of phenolic compounds must be revealed. An understanding of the molecular mechanism of radical-mediated inactivation is necessary for protein engineering to improve peroxidase stability.
Firstly, the dominant mechanism of peroxidase inactivation during phenol oxidation was determined. Two peroxidases, Coprinus cinereus peroxidase (CiP) and horseradish peroxidase isozyme C (HRPC), showed much higher inactivation rates after the simultaneous addition of phenol and hydrogen peroxide. After the oxidation reaction of phenol, the molecular weights of polypeptides originating from the inactivated peroxidases were slightly increased, and a large fraction of heme from the two inactivated peroxidases remained intact. These findings support the hypothesis that the inactivation of peroxidase during the oxidation of phenol occurs by the coupling of phenoxyl radicals with peroxidase polypeptides.

Secondly, the radical coupling site of CiP was identified, and the radical stability of CiP was improved by site-directed mutagenesis. The residue F230 of CiP modified with the phenoxyl radical was mutated to amino acids (Ala) that resist radical coupling. The F230A mutant showed the highest stability against the radical attack, retaining 80% of its initial activity, while the wild-type protein was almost completely inactivated. In addition, no structural changes were observed in CiP after radical coupling.

Thirdly, HRPC was also engineered to enhance the radical stability. Phenylalanine residues that are vulnerable to modification by phenoxyl radicals were identified and then changed to Ala to prevent radical coupling. The F68A/F142A/F143A/F179A mutant exhibited dramatic enhancement of radical stability, retaining 41% of its initial activity compared to the wild type, which was completely inactivated. Radical coupling did not change the secondary structure or the active site structure of HRPC. Structure and sequence alignment revealed that radical-vulnerable Phe residues were conserved in homologous peroxidases.

Fourthly, the radical-stable CiP mutant, F230A, was applied to the major practical applications, such as the removal of phenol, the decolorization of dye, and the synthesis of polymers. As expected, the removal efficiency of phenol and the decolorization efficiency of Reactive Black 5 were increased four- and five-fold, respectively, compared with that of the wild type. In addition, the phenolic polymer having the highest molecular mass (8850 Da) was synthesized by the F230A mutant in a 50% v/v isopropanol-buffer mixture.

A novel engineering strategy to eliminate the radical coupling site increased the radical stability of two peroxidases, CiP and HRPC. This implies that phenoxyl radicals covalently bind to critical Phe residues and inactivate peroxidase by blocking substrate access to the active site of the enzyme.
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College of Engineering/Engineering Practice School (공과대학/대학원)Program in Bioengineering (협동과정-바이오엔지니어링전공)Theses (Ph.D. / Sc.D._협동과정-바이오엔지니어링전공)
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