Enzymes in "Green" Synthetic Chemistry: Laccase and Lipase.

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.

Enzymatic Synthesis and Antimicrobial Activity of Oligomer Analogues of Medicinal Biopolymers from Comfrey and Other Species of the Boraginaceae Family.

This study reports the first enzymatic synthesis leading to several oligomer analogues of poly[3-(3,4-dihydroxyphenyl)glyceric acid]. This biopolymer, extracted from plants of the Boraginaceae family has shown a wide spectrum of pharmacological properties, including antimicrobial activity. Enzymatic ring opening polymerization of 2-methoxycarbonyl-3-(3,4-dibenzyloxyphenyl)oxirane (MDBPO) using lipase from Candida rugosa leads to formation of poly[2-methoxycarbonyl-3-(3,4-dibenzyloxyphenyl)oxirane] (PMDBPO), with a degree of polymerization up to 5. Catalytic debenzylation of PMDBPO using H2 on Pd/C yields poly[2-methoxycarbonyl-3-(3,4-dihydroxyphenyl)oxirane] (PMDHPO) without loss in molecular mass. Antibacterial assessment of natural polyethers from different species of Boraginaceae family Symhytum asperum, S. caucasicum,S. grandiflorum, Anchusa italica, Cynoglossum officinale, and synthetic polymers, poly[2-methoxycarbonyl-3-(3,4-dimethoxyphenyl)oxirane (PMDMPO) and PMDHPO, reveals that only the synthetic analogue produced in this study (PMDHPO) exhibits a promising antimicrobial activity against pathogenic strains S.aureus ATCC 25923 and E.coli ATCC 25922 the minimum inhibitory concentration (MIC) being 100 µg/mL.

Post-Translational Modification Mimicry for Programmable Assembly of Elastin-Based Protein Polymers.

Post-translational modification (PTM) of protein polymers is emerging as a powerful bioinspired strategy to create protein-based hybrid materials with molecularly encoded assembly and function for applications in nanobiotechnology and medicine. While these modifications can be accomplished by harnessing native biological machinery (i.e., enzymes), the evolutionarily programmed specificity of these enzymes (recognition of select substrates and the limited repertoire of ligation chemistries catalyzed by these enzymes) can limit the type and linkage of PTMs appended to proteins. One approach to overcome this limitation is to leverage advances in site-selective biomolecular modification to prepare synthetic mimics of naturally occurring PTMs that are absent in nature. As a proof of concept, we used scalable bio-orthogonal reactions to prepare synthetic mimics of lipidated proteins with tunable assembly and disassembly. Additionally, we demonstrated that our PTM mimicry regulates the stimuli-responsive phase behavior of intrinsically disordered biopolymers, modulates their self-assembly at the nanoscale, and can be used for programmable disassembly of these materials in acidic environments. Synthetic PTM mimicry opens a path to encode new assembly and disassembly capabilities into hybrid materials that cannot be produced via biosynthesis.

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

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 ( Mw = 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.

Polymer-Assisted Biocatalysis: Effects of Macromolecular Architectures on the Stability and Catalytic Activity of Immobilized Enzymes toward Water-Soluble and Water-Insoluble Substrates.

The aim of this study is to develop efficient enzyme immobilization media that will enable the reuse of the biocatalysts over multiple cycles, increase their thermal stability, and attenuate their activity toward hydrophobic substrates for "green" transformations in aqueous media. For this purpose, amphiphilic AB and ABA block copolymers were synthesized and tested with laccase (a multicopper oxidase). In all cases, the hydrophilic B block consisted of poly(ethylene glycol), PEG, with molecular masses of 3, 5, 13, 20, or 13 kDa poly(ethylene oxide). The hydrophobic A blocks were made of linear poly(styrene), PS; hyperbranched poly(p-chloromethyl styrene); or dendritic poly(benzyl ether)s of generations 2, 3, and 4 (G2, G3, and G4) with molecular masses ranging from 1 to 24 kDa. A total of 23 different copolymers (self-assembling into micelles or physical networks) were evaluated. Notable activity enhancements were achieved with both micelles (up to 253%) and hydrogels (up to 408%). The highest enzymatic activity and thermal stability were observed with laccase immobilized in hydrogels consisting of the linear ABA block copolymer PS2.7k-PEG3k-PS2.7k (13 290 µkat/L, 65 °C, ABTS test). This represents a 1245% improvement over native laccase at the same conditions. At 25 °C, the same complex showed a 1236% higher activity than the enzyme. The highest polymerization yield for a water-insoluble monomer was achieved with laccase immobilized in hydrogels composed of linear-dendritic ABA copolymer G3-PEG5k-G3 (85.5%, 45 °C, tyrosine monomer). The broad substrate specificity and reusability of the immobilized laccase were also demonstrated by the successful discoloration of bromophenol blue, methyl orange, and rhodamine B over eight repetitive cycles.