Polymerization of Horseradish Peroxidase by a Laccase‐Catalyzed Tyrosine Coupling Reaction

Abstract

The polymerization of proteins can create newly active and large biomacromolecular 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.

Tree bark scrape fungus: A potential source of laccase for application in bioremediation of non-textile dyes

“…Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2), belonging to a group of enzymes called multicopper blue oxidasehas been noted to exhibit a wide substrate specificity [1]. It has been applied in various sectors, such as biomedical [2], dye degradation [3], paper industries for delignification [4][5], bioremediation [6], in biosensors [7], as melanin degraders in the cosmetic industry [8], as an enzymatic biofuel [9] and used in juice clarification [10]. Furthermore, laccase is a key biological mediator and the best alternative for chemical mediators; thus, it is regarded as a green enzyme in dye degradation, which is a new era for dye degradation [11].…”

Applications of Microbial Laccases: Patent Review of the Past Decade (2009–2019)

“…A recent report showcased the ability of laccase to catalyze the polymerization of dopamine, yielding a more uniform and stable polydopamine homopolymer compared to the one obtained by conventional methods [152]. Furthermore, laccases have been employed in the synthesis of biomacromolecular assemblies such as tyrosine-tagged crosslinked proteins with promising biomedical applications [153,154]. Recent laccase-related patents within this application field are covering inventions such as the production of protein/polysaccharide composite film using thermostable recombinant bacteria laccase or methods for preparing seals for automobile instrument panels (CN109111584A, CN109096736A) ( Table S1).…”

Tree bark scrape fungus: A potential source of laccase for application in bioremediation of non-textile dyes

“…The physical parameters viz. pH (2)(3)(4)(5)(6)(7)(8)(9)(10), temperature (20- were optimized for optimum production of laccase [30].…”

“…Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) belonging to a group of an enzyme called a multicopper blue oxidase (MCBO) exhibits wide substrate specificity ranging from phenolic to non-phenolic compounds [1]. It finds applications in various sectors such as biomedical [2], dye degradation [3] (paper industries for delignification [4][5], bioremediation [6], in biosensors [7] in cosmetic industry as melanin degraders [8] and as a enzymatic biofuel [9]. It is the major ligninolytic enzyme used in juice clarification [10].…”