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Polymerization of Horseradish Peroxidase by a Laccase‐Catalyzed Tyrosine Coupling Reaction
Author(s) -
Permana Dani,
Minamihata Kosuke,
Tatsuke Tsuneyuki,
Lee Jae M.,
Kusakabe Takahiro,
Goto Masahiro,
Kamiya Noriho
Publication year - 2019
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201800531
Subject(s) - horseradish peroxidase , polymerization , chemistry , covalent bond , tyrosine , tyrosinase , peroxidase , laccase , combinatorial chemistry , polymer , polymer chemistry , biochemistry , enzyme , organic chemistry
The polymerization of proteins can create newly active and large bio‐macromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine‐tag (Y‐tag) through a flexible linker at the N‐ and/or C ‐ termini are expressed in silkworm, Bombyx mori . Trametes sp. laccase (TL) is used to activate the tyrosine of Y‐tagged HRPs with molecular O 2 to form a tyrosyl‐free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP‐catalyzed self‐crosslinking reaction in the presence of H 2 O 2 . The addition of H 2 O 2 in the self‐polymerization of Y‐tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site‐selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y‐tagged HRPs and HRP‐protein G (Y‐HRP‐pG) units catalyzed by TL shows a higher signal in enzyme‐linked immunosorbent assay (ELISA) than the genetically pG‐fused HRP, Y‐HRP‐pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes.