Unprecedented Enzymatic Synthesis of Perfectly Structured Alternating Copolymers via “Green” Reaction Cocatalyzed by Laccase and Lipase Compartmentalized within Supramolecular Complexes

Abstract

This study describes the first use of laccase–lipase enzymatic reaction for the synthesis of novel perfectly structured alternating copolymers. Initially, six types of complexing agents, linear(A)–linear(B), linear(A)–linear(B)–linear(A), linear–dendritic, dendritic–linear–dendritic, linear–hyperbranched, and hyperbranched–linear–hyperbranched amphiphilic block copolymers, are proven to significantly enhance enzyme activity of three different types of lipases - Penicillium camemberti, Candida rugosa, and Burkholderia cepacia (up to 1400%, 1700%, and 870% increase with respect to the native enzymes). The copolymerization is performed in several consecutive steps: (a) lipase and laccase are dissolved in aqueous medium at neutral pH; (b) a complexing agent is added leading to cocompartmentalization of the two enzymes within a micelle or physical network; (c) the two comonomers are introduced simultaneously to the tandem enzyme complex. The reaction proceeds in the following pathway: laccase catalyzes the oxidation of catechol to o-quinone followed by lipase comediated Michael addition of a diamine. While laccase could catalyze the entire process, addition of lipase is able to increase copolymer yield up to 30.7%. Addition of a complexing agent improves the yield further up to 67.9% (23.2% yield obtained for native laccase). Complexing agents significantly increase polymer molecular mass (M w = 130 900 vs 35 500 Da for the native enzymes reaction system). The resulting copolymers are highly fluorescent (quantum yield up to 0.733) and demonstrate pH sensitive behavior, properties that hint toward their potential as imaging agents.

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

“…The atom transfer radical polymerization of N-vinylimidazole catalyzed by T. versicolor laccase showcased its ability to produce a polymer with a well-defined molecular mass and potential applications in drug and gene delivery, fuel cell membranes, and polyionic liquids [150]. Another laccase-mediated polymerization in a highly controlled manner produced alternating copolymers of catechol and xylylenediamines with potential applications as imaging agents [151]. 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].…”

Chemical Design of Hydrogels with Immobilized Laccase for the Reduction of Persistent Trace Compounds in Wastewater

“…In particular, oxidoreductases like peroxidases and laccases (LAC) are capable of converting a wide range of organic compounds. ,, LAC is a multicopper oxidase and catalyzes the oxidation of various substrates in the presence of dissolved oxygen as a cosubstrate. LAC is able to oxidize phenolic compounds via one-electron oxidation as well as non-phenolic compounds in the presence of electron-transfer mediator molecules. − Owing to these unique biocatalytic properties, laccases have become increasingly attractive in research and industry. ,− For application in water treatment, immobilization of the applied enzyme is indispensable to provide the prerequisite for separation of the cleaned water and the biocatalyst. Enzyme immobilization techniques can be basically divided into two main methods, immobilization by physical and chemical interactions, both with advantages and disadvantages. , Physical enzyme immobilization involves entrapment or encapsulation in a three-dimensional matrix, for instance, in a polymer gel .…”

“…Enzyme immobilization techniques can be basically divided into two main methods, immobilization by physical and chemical interactions, both with advantages and disadvantages. , Physical enzyme immobilization involves entrapment or encapsulation in a three-dimensional matrix, for instance, in a polymer gel . Chemical interactions include complexation, cross-linking, adsorption of the enzyme, and covalent bonding to a support . In many cases, immobilization enhances the enzyme activity, durability, and repeatability of conversions. , Entrapment, encapsulation, adsorption, and complexation represent cost-effective, easy to handle immobilization techniques that require neither structural modification of the enzyme, which often leads to a decrease in activity, nor chemical modification or pretreatment of the carrier.…”

“…Hydrogels show excellent adsorption capacities as well as nontoxic, biodegradable, and biocompatible properties, making them environmentally friendly and interesting for many applications, among them water treatment. As a consequence, hydrogels have been studied frequently for application in the removal of environmental pollutants such as dyes , and heavy metal ions. , In addition, hydrogels represent suitable materials for the immobilization of enzymes since their properties are adjustable by the chemical structure. ,,,, Very often, enzyme-immobilized hydrogels are prepared by mixing the enzyme into the preparation formulation of the hydrogel . Simon et al found in the case of this noncovalent immobilization an enzyme leakage of about 50% after the first washing.…”

Magnetically Responsive PA6 Microparticles with Immobilized Laccase Show High Catalytic Efficiency in the Enzymatic Treatment of Catechol

“…Laccases from different sources can be useful in a large number of applications ranging from food additives and beverage processing to biomedical diagnosis, pulp delignification, wood fiber modification, chemical, or medicinal synthesis, for production of biofuels [3,13,14]. Laccases have shown great promise in "green" synthesis and as polymerization mediators [15][16][17][18], in industrial effluent treatment including dye decolorization and degradation [19,20], as well as in wastewater and soil remediation [21].…”