Laccase-catalyzed Reactions Research Articles

Structural properties and antioxidant capacity of different aminated starch-phenolic acid conjugates

Different aminated starch (AS) [EEAS (introducing ethylenediamine into starch using cross-linking-etherification-amination method (CEA)), EPAS (introducing o-phenylenediamine using CEA), OEAS (introducing ethylenediamine using cross-linking-oxidation-amination method (COA)), and OPAS (introducing o-phenylenediamine using COA)] were synthesized. The AS-phenolic acids [gallic acid (GA), syringic acid (SA), and vanillic acid (VA)] conjugates were prepared by laccase-catalyzed reaction. The grafting efficiency of EEAS on GA, SA, and VA was 36.59%, 69.71%, and 68.85%, respectively. SA reduced the maximum depolymerization rate of EEAS. The relative crystallinity of EEAS and EPAS grafted phenolic acid increased, and their particles showed severe breakage in appearance. OEAS-phenolic acid conjugates lost its granular structure and behaved as flakes and lumps, while the surface of OPAS-phenolic acid conjugates remained smooth after grafting phenolic acid. GA increased the DPPH· scavenging efficiency of EEAS from 16.12% to 79.92%. The increased antioxidant capacity of the conjugates suggested that AS-phenolic acids conjugates have high potential for applications.

Eutectic Mixtures As Green Solvents for Laccase-Catalyzed Reactions

Eutectic Mixtures As Green Solvents for Laccase-Catalyzed Reactions

Application of Laccase Catalysis in Bond Formation and Breakage: A Review

Laccase belongs to the superfamily of multicopper oxidases and has been widely investigated in recent decades. Due to its mild and efficient oxidation of substrates, laccase has been successfully applied in organic catalytic synthesis, the degradation of harmful substances, and other green catalytic fields. Nevertheless, there are few reports on the green catalysis with laccase. This review focuses on reporting and collating some of the latest interesting laccase-catalyzed bond formation and breakage research. This is discussed with a focus on the effects of the medium system on the laccase-catalyzed reaction, as well as the formation and the breakage of C–N, C–C, and C–O bonds catalyzed by laccase. It provides abundant references and novel insights for furthering the industrial applications of laccase.

Study on the Polymerization and Mechanism of Amino Acid Catalyzed by Laccase

ABSTRACT Laccase-catalyzed coloration toward wool was carried out and the K/S of wool fabric dyed was much higher than raw wool, presenting a claybank color. On this basis, the reaction mechanism is elucidated using tyrosine, tryptophan and the mixture as substrates. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and LC-MS as well as FT-IR measurements were used to study the reactions. The results indicate that the laccase-catalyzed reaction of tyrosine monomer, tyrosine monomer and the mixture, respectively, generates free radicals, including phenoxy radical and benzene radicals, pyrrolidine radical. The coupling reaction can occur between two free radicals, and further the Michael addition reaction occurs between benzene radicals with monomer another tyrosine. In view of the tan polytyrosine and rufous polytryptophan, as well as the higher content of tyrosine than tryptophan, the oxidation coupling reaction of tyrosine residue predominates over that of tryptophan residue in the process of laccase-catalyzed coloration toward wool.

Enzymatic Dyeing of Wool Fibers with Enhancement of Strength Using Laccase as Catalyst

ABSTRACT Laccase-catalyzed polymerization of catechol on wool yarns was explored in this study, their dyeing performance and physical properties were also analyzed. Wool yarns were pretreated by chemical (Na2CO3) and plasma treatment. Catechol oligomers were then deposited or cross-linked on wool fibers through laccase-catalyzed reaction. It was shown that both chemical (Na2CO3) and plasma pretreatment could promote the subsequent dyeing process. Besides, the capacity of laccase to oxidize catechol was observed by UV-vis spectra. The FTIR spectra and SEM observation confirmed the coating of oligomers on wool. The results show that laccase-catalyzed reaction could be an efficient methodology for the in-situ coloration and performance enhancement in tensile strength, abrasion resistance, alkali solubility and antioxidant activity of wool.

Laccase-Catalyzed Derivatization of Aminoglycoside Antibiotics and Glucosamine.

The increasing demand for new and effective antibiotics requires intelligent strategies to obtain a wide range of potential candidates. Laccase-catalyzed reactions have been successfully applied to synthesize new β-lactam antibiotics and other antibiotics. In this work, laccases from three different origins were used to produce new aminoglycoside antibiotics. Kanamycin, tobramycin and gentamicin were coupled with the laccase substrate 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide. The products were isolated, structurally characterized and tested in vitro for antibacterial activity against various strains of Staphylococci, including multidrug-resistant strains. The cytotoxicity of these products was tested using FL cells. The coupling products showed comparable and, in some cases, better antibacterial activity than the parent antibiotics in the agar diffusion assay, and they were not cytotoxic. The products protected mice against infection with Staphylococcus aureus, which was lethal to the control animals. The results underline the great potential of laccases in obtaining new biologically active compounds, in this case new antibiotic candidates from the class of aminoglycosides.

Fractionated Lignosulfonates for Laccase-Catalyzed Oxygen-Scavenging Films and Coatings.

Lignin derivatives have potential as antioxidants in advanced packaging materials through their ability to scavenge oxygen in reactions catalyzed by phenol-oxidizing enzymes, such as laccase. The effects of size fractionation of lignosulfonates on laccase-catalyzed reactions were investigated in experiments with aqueous solutions, films, and coated paperboard. Four industrial lignosulfonate preparations were compared: Feed (unfractionated), Prod (5–60 kDa enriched), Conc (≥60 kDa enriched), and Perm (≤60 kDa enriched). Extraction of lignosulfonates from films showed that the enzymic reaction increased the average molecular weight from <10,000 to up to 66,000. The enzymatic reaction resulted in an increase in the water contact angle of the films from the range 25–49° to 56–81°. The four preparations showed relatively small differences with regard to their ability to scavenge oxygen in aqueous solution and in experiments with coated paperboards in sealed chambers. Coatings with lignosulfonates where the contents of low-molecular weight material had been reduced (i.e., Prod and Conc) showed improved water resistance after the enzymic reaction. Thus, in both aqueous and solid media, fractionation of lignosulfonates had little effect on oxygen scavenging, but fractionation was beneficial for other reasons, such as improved cross-linking resulting in higher molecular weight and superior water resistance.

Natural Syringyl Mediators Accelerate Laccase-Catalyzed β-O-4 Cleavage and Cα-Oxidation of a Guaiacyl Model Substrate via an Aggregation Mechanism.

Laccase–mediator systems (LMSs) have been intensively investigated in lignin degradation. Although only natural metabolites are available for fungal lignin degradation, mediator molecules from metabolites have received substantially less attention than artificial organic–synthetic compounds. It remains unclear which metabolites can accelerate laccase-catalyzed reactions and how those natural mediators influence lignin degradation. In this work, we evaluated Trametes versicolor laccase-catalyzed reaction kinetics on a lignin guaiacyl subunit model (guaiacylglycerol-β-guaiacyl ether, G-β-GE) in the presence of a group of lignin syringyl subunit molecules: syringaldehyde, acetosyringone, and methyl syringate. We then compare their performance to a well-known synthetic mediator ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Time-resolved UPLC-TOF-MS revealed that the syringyl mediators were more effective in accelerating the β-O-4 cleavage and Cα-oxidation of G-β-GE than ABTS under laccase-catalysis, despite the syringyl compounds possessing slower individual oxidation rates. In addition, the product profile of polymerization was also promoted dramatically, compared to that of the ABTS/laccase system. The LMS kinetic modeling suggested that mediator–substrate aggregation played a critical role in the laccase–mediator system; in which, the lignin syringyl and guaiacyl subunits likely form a π–π stacking van der Waals complex that can be oxidized faster than the syringyl or guaiacyl monomers by themselves. This syringyl–guaiacyl aggregation hypothesis postulates that the weak interactions in lignin biopolymers are able to accelerate the laccase-catalyzed biodegradation.

Modulation of laccase catalysed oxidations at the surface of magnetic nanoparticles

We explored the coupling of laccases to magnetic nanoparticles (MNPs) with different surface chemical coating. Two laccase variants offering two opposite and precise orientations of the substrate oxidation site were immobilised onto core-shell MNPs presenting either aliphatic aldehyde, aromatic aldehyde or azide functional groups at the particles surface. Oxidation capabilities of the six-resulting laccase-MNP hybrids were compared on ABTS and coniferyl alcohol. Herein, we show that the original interfaces created differ substantially in their reactivities with an amplitude from 1 to > 4 folds depending on the nature of the substrate. Taking enzyme orientation into account in the design of surface modification represents a way to introduce selectivity in laccase catalysed reactions.

Combining phenolic grafting and laccase-catalyzed cross-linking: Effects on structures, technofunctional properties and human immunoglobulin E binding capacity of egg white proteins

The efficiency of laccase-catalyzed protein cross-linking can be impacted by substrate protein structure and competing reactions. In this study, chemical grafting of ferulic acid (FA) on protein surface was applied to modulate the cross-linking of two inflexible globular proteins, lysozyme (LZM) and ovalbumin (OVA). The extent of FA-grafting was positively correlated with protein cross-linking extent, and determined the molecular weight profile and structures of the cross-linked product. While laccase-catalyzed reactions (with or without free FA mediator) did not lead to evident cross-linking of the native proteins, oligomeric (up to 16.4%), polymeric (up to 30.6%) FA-LZMs and oligomeric FA-OVA (5.1–31.1%) were obtained upon the enzymatic treatments. The cross-linking on the grafted FA sites occurred mainly through the formation of 8–5′-noncyclic-dehydro-diferulic linkages. The effects of investigated cross-linking approach on the emulsifying, foaming properties and the immunoglobulin E (IgE) binding capacity of LZM and OVA were also evaluated in relation to the structural properties of cross-linked proteins.

Laccase-Catalyzed Reactions in Ionic Liquids for Green Sustainable Chemistry

Biocatalysis is an important and enabling tool in organic synthesis, degradation of chemical compounds, and biosensors. Laccases are copper ions containing oxidase and catalyze the conversion of po...

The Study of the Oxidation of N,N,N',N'-Tetramethyl-p-Phenylenediamine for the Clarification of the Differences in the Mechanisms of Biocatalytic and Chemical Polymerization of Aniline

The oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) using ammonium peroxydisulfate and the high redox potential laccase from Trametes hirsuta is studied in order to clarify the significant differences in the chemical and enzymatic polymerization reactions of aniline. As opposed to the laccase-catalyzed reaction, the chemical oxidation of TMPD proceeds in two stages. The radical cation formed at the first stage is oxidized by the peroxydisulfate ion to quinoiddiimine that can interact with the initial TMPD. The kinetics of the formation of the radical cation $${\text{TMP}}{{{\text{D}}}^{{\centerdot + }}}$$ is studied by ultraviolet–visible (UV–Vis) and electron paramagnetic resonance spectroscopy. The obtained data allow making an assumption about the difference in the mechanisms of chemical and enzymatic polymerization of aniline.

The Effects of Adding Different HALS on the Curing Process, Film Properties and Lightfastness of Refined Oriental Lacquer.

Oriental lacquer is a natural polymeric coating with a satiny texture and excellent characteristics, such as chemical resistance and durability. However, the poor lightfastness resulted in the natural aromatic structures of the urushiol structures limited its suitability for outdoor application. This study aimed at the improvement of the lightfastness by adding the different hindered amine light stafbilizers (HALS) with 2 phr addition as well as the effects on the coating and film property of the refined oriental lacquers (RL). The Cryptomeria japonica plate, glass sheets, and the other substrates were used for finishing. The results showed that the lightfastness of RL film was obviously improved by adding 2 phr HALS of Bis(2,2,6,6-tetramethyl-4-piperidinyl) sebacate (H90) containing -NH group and Bis (1,2,2,6,6-pentamethyl-4-piperidinyl)-[[3,5-bis(1,1-dimethyl ethyl) -4-hydroxyphenyl] methyl] butylmalonate (H60) containing -NCH3 groups. The HALS additions increased the pH value of RL and improved the activities of laccase-catalyzed reaction. Meanwhile, the oxidative polymerization of the side chains of RL was inhibited, caused by a radical scavenging ability of HALS. The changes in the drying process affected not only the curing time, but also the film properties. Among the 2 phr additions of different HALS, the film containing H90 had the best lightfastness. Meanwhile, it kept the most similar properties with RL and shortened the drying time of RL, and it was selected as the best HALS addition under 2 phr in this study.

Biomedical and Pharmaceutical-Related Applications of Laccases.

The oxidation of a vast range of phenolic and non-phenolic substrates has been catalyzed by laccases. Given a wide range of substrates, laccases can be applied in different biotechnological applications. The present review was conducted to provide a broad context in pharmaceutical- and biomedical- related applications of laccases for academic and industrial researchers. First, an overview of biological roles of laccases was presented. Furthermore, laccase-mediated strategies for imparting antimicrobial and antioxidant properties to different surfaces were discussed. In this review, laccase-mediated mechanisms for endowing antimicrobial properties were divided into laccase-mediated bio-grafting of phenolic compounds on lignocellulosic fiber, chitosan and catheters, and laccase-catalyzed iodination. Accordingly, a special emphasis was placed on laccase-mediated functionalization for creating antimicrobials, particularly chitosan-based wound dressings. Additionally, oxidative bio-grafting and oxidative polymerization were described as the two main laccase-catalyzed reactions for imparting antioxidant properties. Recent laccase-related studies were also summarized regarding the synthesis of antibacterial and antiproliferative agents and the degradation of pharmaceuticals and personal care products.

Phenolic and non-phenolic substrates oxidation by laccase at variable oxygen concentrations: Selection of bisubstrate kinetic models from polarographic data

Using laccase as a case study, an alternative methodology for discrimination of bisubstrate mechanism types, useful for Bi-Bi reactions when water is one of the products, is presented. A purified enzyme from fungal cultures of Ganoderma resinaceum and two reducing substrates: 2,2´-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and 2,6-dimethoxyphenol (DMP) are used. Kinetic assays of ABTS or DMP oxidation using variable initial concentrations of dioxygen are performed in a closed system and depletion rates of dioxygen are measured using polarography. Seven bisubstrate models based on Michaelis-Menten Bi-Bi mechanisms are applied to the experimental data. Akaike information criterion, a penalized likelihood analysis, and Akaike weights are used for model selection. Results show that the more likely bisubstrate models for ABTS and DMP oxidation are ping-pong and Theorell-Chance, respectively. However, the catalytic efficiency of oxygen reduction to water exhibits similar values, and is compatible with a process relatively independent of reducing substrates and the bisubstrate mechanism type. Finally, Michaelis-Menten constants towards dioxygen, 233 μM with ABTS and 700 μM with DMP, indicate that the soluble electron acceptor available for laccase-catalyzed reactions, is far removed from saturation.

Transformation of atenolol by a laccase-mediator system: Efficiencies, effect of water constituents, and transformation pathways

In this study, we investigated the transformation of atenolol (ATL) by the naturally occurring laccase from Trametes versicolor in aqueous solution. Removal efficiency of ATL via laccase-catalyzed reaction in the presence of various laccase mediators was examined, and found that only the mediator 2, 2, 6, 6-tetramethyl-1-piperidinyloxy (TEMPO) was able to greatly promote ATL transformation. The influences of TEMPO concentration, laccase dosage, as well as solution pH and temperature on ATL transformation efficiency were tested. As TEMPO concentrations was increased from 0 to 2000 μM, ATL transformation efficiency first increased and then decreased, and the optimal TEMPO concentration was determined as 500 μM. ATL transformation efficiency was gradually increased with increasing laccase dosage. ATL transformation was highly pH-dependent with an optimum pH of 7.0, and it was almost constant over a temperature range of 25–50 °C. Humic acid inhibited ATL transformation through competition reaction with laccase. The presence of anions HCO3− and CO32− reduced ATL transformation due to both anions enhanced solution pHs, while Cl−, SO42−, and NO3− at 10 mM showed no obvious influence. The main transformation products were identified, and the potential transformation pathways were proposed. After enzymatic treatment, the toxicity of ATL and TEMPO mixtures was greatly reduced. The results of this study might present an alternative clean strategy for the remediation of ATL contaminated water matrix.

Graphene enhanced transformation of lignin in laccase-ABTS system by accelerating electron transfer

The degradation of lignin has attracted much attention since it represents approximately 30% of all non-fossil carbon sources and constitutes a sustainable bio-resource for fuels and aromatic derivatives. Here we investigated the degradation of lignin by laccase-catalysed reactions using 2,2′-Azino-bis(3-ethybenzothiazoline-6-sulfonic acid) (ABTS) as a mediator coupled with the carbon material graphene. Results indicated that there was a significant, two-fold, increase in the catalytic activity of lignin degradation in laccase-ABTS systems in the presence of graphene. Analysis suggested that the enhancement of lignin degradation could be attributed to graphene acting as an electron transfer conductor, thereby accelerating electron transfer, which facilitated the formation of intermediate oxidation states of ABTS and rendered the reactions between lignin and ABTS intermediates more efficient. This study could promote the development of novel enzymatic lignin degradation systems coupled with the carbon-based material graphene.

Laccase-catalyzed poly(ethylene glycol)-templated ‘zip’ polymerization of caffeic acid for functionalization of wool fabrics

Abstract A cleaner approach for functionalization of wool fabrics via laccase-catalyzed polymerization of caffeic acid was developed. Caffeic acid was first polymerized to poly(caffeic acid) in the system where poly(ethylene glycol) was used as a template under the catalysis of laccase/O2. Ultraviolet–Visible spectroscopic measurements verified that poly(ethylene glycol) formed a complex with caffeic acid though hydrogen bonds based on the ‘zip’ mechanism. During the catalysis of caffeic acid by laccase, poly(ethylene glycol) template promoted the enzymatic polymerization in term of the quantity of poly(caffeic acid) and the degree of polymerization. The data obtained from Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry and High Performance Liquid Chromatography-Electro Spray Ionization confirmed that poly(ethylene glycol) template aided the arrangement of the caffeic acid monomer, resulting in the increased degree of polymerization of caffeic acid in the laccase-catalyzed reaction. The poly(caffeic acid)/poly(ethylene glycol)-treated wool fabrics were prepared by laccase-catalyzed in-situ polymerization of caffeic acid under the same conditions. The treated wool fabrics showed good wettability and electroactivity. The results from fibre conductivity test and cyclic voltammetry showed that the wool fabric treated with poly(caffeic acid)/poly(ethylene glycol) complex was found to have better electrical conductivity. These data showed that the presence of poly(ethylene glycol) as a template in the laccase-catalyzed polymerization reaction greatly improved the characteristic of poly(caffeic acid). Moreover, higher color depth of poly(caffeic acid)/poly(ethylene glycol)-treated wool fabrics further validated the important role of the poly(ethylene glycol) as a template.

Selective control for the laccase-catalyzed synthesis of dimers from 2,6-dimethoxyphenol: Optimization of 3,3’,5,5’-tetramethoxy-biphenyl-4,4’-diol synthesis using factorial design, and evaluation of its antioxidant action in biodiesel

The synthesis of transformed products from 2,6-dimethoxyphenol (2,6-DMP) can be selectively obtained through controlled oxidative coupling in laccase-catalyzed reactions, and the products formed are dependent upon the enzyme source, as well as, upon the reactions conditions applied. The laccase from Botryosphaeria rhodina MAMB-05 was investigated for its ability to catalyze the oxidative transformation of 2,6-DMP. Studies conducted in aqueous medium at pH 3.0 and 6.5, for reaction times of 48, 96 and 144 h, led to the formation of three dimers (I, II, III) via carbon-carbon and carbon-oxygen coupling reactions depending upon the pH and reaction time. Dimer I was identified as 3,3′,5,5′-tetramethoxydiphenylquinone by 13C NMR, while dimer II was identified by 1H NMR as 3,3′,5,5′-tetramethoxybiphenyl-4,4′-diol (TMBP). Dimer III was synthesized at pH 3.0 and was influenced by the reaction time (96h), and is predicted to be formed via carbon-oxygen coupling. The chemical structure of III (4-(2,6-dimethoxy-phenoxy)-2,6-dimethoxyphenol) was identified by ESI-Q-TOF HRMS. TMBP (II) was the only product obtained at pH 6.5 independent of the reaction times. TMBP synthesis at pH 6.5 was optimized by design-matrix using a three-factor Box-Behnken incomplete factorial-design that defined the parameters: 2,6-DMP concentration (2.6–3.4 mmol) laccase activity (1.35–3.38 U) and reaction time (120 h) to give a yield of 11.93 ± 0.49%, which agreed with the mathematical model value of 12%. TMBP was evaluated as an antioxidant to stabilize biodiesel, and showed an efficacy similar to the commercial antioxidant standard, butyl hydroxytoluene, indicating that TMBP could serve as an alternative antioxidant to stabilize biodiesel.

Laccase Activity in CTAB-Based Water-in-Oil Microemulsions.

The aim of this study was to develop a microemulsion system as a medium for laccase-catalyzed reactions. Phase behavior studies were conducted by constructing partial pseudo-ternary phase diagrams for systems comprising of cetyltrimethylammonium bromide (CTAB), various organic solvents as the oil phase (i.e., hexane, cyclohexane, heptane, octane, isooctane, toluene, isopropyl myristate), two co-surfactants (i.e., 1-butanol and 1-hexanol) and citrate buffer solution, at various surfactant/co-surfactant weight ratios (Rsm). A monophasic, transparent, non-birefringent area (designated as microemulsion domain) was seen to occur in some phase diagrams along the surfactant/organic solvent axis, the extent of which was dependent mainly upon the nature of co-surfactant and Rsm. On each phase diagram, three different water-in-oil (w/o) microemulsion systems with less than 50 wt% surfactant mixture and less than 20 wt% of aqueous phase were selected for laccase loading and activity measurements. Results revealed that the catalytic activity of laccase in CTAB-based w/o microemulsions decreased considerably, compared with its activity in the buffer solution, the extent of which depended upon the type of component and their compositions in the microemulsions. It was suggested that the conformational changes due to the electrostatic interactions between the cationic head group of CTAB and the negative enzyme might be the reason for the reduction of laccase activity, once entrapped in the microemulsion.