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.

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This study reports a new strategy for enzyme immobilization based on passive immobilization in neat and magnetically responsive polyamide 4 (PA4) highly porous particles. The microsized particulate supports were synthesized by low-temperature activated anionic ring-opening polymerization. The enzyme of choice was laccase from Trametes versicolor and was immobilized by either adsorption on prefabricated PA4 microparticles (PA4@iL) or by physical in situ entrapment during the PA4 synthesis (PA4@eL). The surface topography of all PA4 particulate supports and laccase conjugates, as well as their chemical and physical structure, were studied by microscopic, spectral, thermal, and synchrotron WAXS/SAXS methods. The laccase content and activity in each conjugate were determined by complementary spectral and enzyme activity measurements. PA4@eL samples displayed >93% enzyme retention after five incubation cycles in an aqueous medium, and the PA4@iL series retained ca. 60% of the laccase. The newly synthesized PA4-laccase complexes were successfully used in dyestuff decolorization aiming at potential applications in effluent remediation. All of them displayed excellent decolorization of positively charged dyestuffs reaching ~100% in 15 min. With negative dyes after 24 h the decolorization reached 55% for PA4@iL and 85% for PA4@eL. A second consecutive decolorization test revealed only a 5–10% decrease in effectiveness indicating the reusability potential of the laccase-PA4 conjugates.

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.

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.

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.

This study describes a unique "quasi-living" block copolymerization method based on an initiation by a single enzyme. We use this term to describe a process where a preformed polymer chain can be reactivated to continue propagating with a second or third comonomer without addition of new catalyst. The presented strategy involves a laccase (oxidoreductase) mediated initial polymerization of 4-hydroxyphenylacetic acid to a homopolymer containing phenolic terminal units, which in turn can be easily reactivated by the same enzyme in the same reaction vessel to continue propagation with a second monomer (tyramine). Increased copolymer yield (up to 26.0%) and polymer molecular mass (up to M w = 116 000 Da) are achieved through the addition of previously developed micellar and hydrogel enzyme complexing agents. The produced poly(tyramine)-b-poly(4-hydroxyphenylacetic acid)-b-poly(tyramine) is water-soluble and able to self-assemble in aqueous solution. Both tyramine blocks were successfully modified with ibuprofen moieties (up to 24.6% w/w load) as an example for potential polymer drug conjugation. The copolymerization could be further extended with addition of a third (fluorescent) comonomer in the same reaction vessel to yield a fluorescent pentablock copolymer. The successful modifications and advantageous solution behavior of the produced copolymers demonstrate their viability as versatile drug delivery and/or bioimaging agents, as confirmed by cytotoxicity and cellular uptake studies.

Micropipette-based thermocouples provide the advantage of a high tip diameter-to-length aspect ratio allowing the maintenance of a reference temperature crucial for accurate thermal sensing in microdomains. The research efforts in this field strive to achieve high thermoelectric power (voltage change per unit temperature change) while minimizing the sensing area, a pair of tasks that is by nature contradictory and thus, challenging. Herein, the design and fabrication of a carbon-based micropipette thermal sensor are described. A novel manufacturing method and set of materials are used to overcome the reduction in thermoelectric performance associated with small sensor sizes. A glass micropipette is utilized as a template in a chemical vapor deposition process to form a carbon layer in the lumen of the pipette. This carbon micropipette then serves as a scaffold on which gold and nickel are deposited, enabling the device to function as a thermocouple. This low-cost fabrication process results in a thermocouple with a sub-500 nm tip. The response of the thermocouple was characterized and demonstrated good repeatability in a temperature range of 0 to 60 °C. The unique material selection provided a thermoelectric power of 14.9 µV·K-1, a significant improvement (68%) relative to other micropipette-based thermocouples.