Enzymes in "Green" Synthetic Chemistry: Laccase and Lipase

Abstract

Enzymes play an important role in numerous natural processes and are increasingly being utilized as environmentally friendly substitutes and alternatives to many common catalysts. Their essential advantages are high catalytic efficiency, substrate specificity, minimal formation of byproducts, and low energy demand. All of these benefits make enzymes highly desirable targets of academic research and industrial development. This review has the modest aim of briefly overviewing the classification, mechanism of action, basic kinetics and reaction condition effects that are common across all six enzyme classes. Special attention is devoted to immobilization strategies as the main tools to improve the resistance to environmental stress factors (temperature, pH and solvents) and prolong the catalytic lifecycle of these biocatalysts. The advantages and drawbacks of methods such as macromolecular crosslinking, solid scaffold carriers, entrapment, and surface modification (covalent and physical) are discussed and illustrated using numerous examples. Among the hundreds and possibly thousands of known and recently discovered enzymes, hydrolases and oxidoreductases are distinguished by their relative availability, stability, and wide use in synthetic applications, which include pharmaceutics, food and beverage treatments, environmental clean-up, and polymerizations. Two representatives of those groups-laccase (an oxidoreductase) and lipase (a hydrolase)-are discussed at length, including their structure, catalytic mechanism, and diverse usage. Objective representation of the current status and emerging trends are provided in the main conclusions.

Keywords: enzymes; immobilization; laccase; linear-dendritic copolymers; lipase; polymerization; polyphenols; wine making; “green” chemistry.

Scheme 1

Synthesis of product P through formation of enzyme–substrate complex ES from enzyme E and substrate S.

Figure 1

Common methods of enzyme immobilization.

Figure 2

Atomic force microscopy image (left) and schematic structure (right) of fluorescently labeled polycationic, dendronized polymer (denpol) carrying horseradish peroxidase (HRP), and superoxide dismutase (SOD) attached to the backbone by bis-aryl hydrazone (BAH) bonds. Reproduced with permission from [42]. Copyright (2013) American Chemical Society.

Figure 3

Scanning electron microscopy images of poly(amide 6) microparticles with Fe core before (a,b) and after (c,d) laccase adsorption. Modified from the original [68]. This work is licensed under CC BY-NC-ND 4.0.

Figure 4

Schematic diagram of the complex formation between the enzyme and linear–dendritic block copolymer (a); linear–hyperbranched block copolymer (b) and linear–linear block copolymer (c). Modified from the original [77]. This work is licensed under CC BY-NC-ND 4.0.

Figure 5

Conjugation of carboxy-terminated Pluronic 127 to organophosphorus hydrolase (OPH) through sequential modification with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Reproduced with permission from [91]. Copyright (2014) American Chemical Society.

Figure 6

Three-dimensional structure of Trametes versicolor laccase. The protein domains (D1–3) are shown in dark blue, red, and green; the four Cu atoms are depicted in light blue and the glycoside anchoring sites (GL1–3) are marked in yellow. Modified from [120]. This work is licensed under CC BY.

Scheme 2

Laccase catalytic cycle depicting the native intermediate (fully reduced oxygen), the oxidized resting state, the fully reduced form (after substrate oxidation), and the peroxy intermediate (partial oxygen reduction). Phenol is used as a model substrate. Cu^II—blue, Cu^I—green. Modified from the original [125]. Copyright (2010) American Chemical Society.

Figure 7

Selected mediators. 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS; 1-Hydroxybenzotriazole, HOBT; N-hydroxyphthalimide, HOPI; N-hydroxynaphthalimide, HONI and 2,2,6,6-tetramethylpiperidine-N-oxyl, TEMPO.

Scheme 3

Oxidation of HOBT through laccase and electron transfer to substrate with concomitant regeneration of the nitroxide radical mediator.

Scheme 4

Oxidation of ABTS dianion (top) to the radical cation (middle), and finally to the dication (bottom).

Figure 8

Substrate harvesting and migration to laccase active site, as revealed through spectrophotometric investigation of pyrene fluorescence [157].

Scheme 5

Oxidation of fullerene C₆₀. (A). Process mediated by laccase/linear-dendritic complex [157], green arrows indicate oxygen entry to and products exit from the enzyme active site. (B) Process catalyzed through sequential treatment with concentrated H₂SO₄, concentrated HNO₃, and 2N NaOH [158].

Scheme 6

Initial radical formation during the polymerization of tyrosine mediated by laccase/linear-dendritic copolymer complex [171]. Red dots—productive radicals leading to C-C or C-O couplings; blue dot—nonproductive radical.

Scheme 7

Suggested propagation mechanism through addition of tyrosine monomer radicals to the chain ends of unnatural poly(tyrosine). (a) Addition to all C-C coupling chain; (b) addition to all C-O coupling chain.

Figure 9

Polymerization of tyrosine-dendron macromonomers mediated by Trametes versicolor laccase in aqueous medium. The resulting amphiphilic dendronized polymers spontaneously self-assemble in water, as revealed through transmission electron microscopy (bottom right image). Modified with permission from [172]. Copyright (2021) American Chemical Society.

Scheme 8

One-pot synthesis of a penta-block copolymer formed through “quasi-living” copolymerization mediated by native or copolymer-complexed laccase from Trametes versicolor. Modified with permission from [173]. Copyright (2020) American Chemical Society.

Scheme 9

Common reactions mediated by lipases: hydrolysis (top), esterification (middle), and transesterification (bottom).

Figure 10

3D structures of human pancreatic lipase. E*—open conformation at the lipid/water interface; E—closed conformation in solution. Reproduced with permission from [189]. Copyright (1998) Elsevier.

Scheme 10

Lipase-mediated polymerization of lactones (A), oxyacids (B), and dicarboxylic acids and diols (C).

Scheme 11

Alternating copolymerization of catechol and m-xylylenediamine mediated by a laccase–lipase pair co-compartmentalized in supramolecular polymer complexes. Reproduced with permission from [207]. Copyright (2019) American Chemical Society.