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Insights on the laccase extraction and activity in ionic-liquid-based aqueous biphasic systems
... One of the interesting examples for various biotechnological and environmental uses is laccases, which are flexible multi-copper oxidases [57]. The high cost of downstream processing in synthesizing pure and stable biocatalysts is one of their major limitations when evaluating the economic sustainability of the process on an industrial scale [58]. Research on ILs has been conducted to overcome these limitations and develop efficient and green enzyme downstream processes, especially when the water solubility of substrates is low [59]. ...
... It has been suggested that ABSs based on ILs, salts, or polymers are an efficient extraction and recovery technique for several biomolecules, including proteins and enzymes [13]. Additionally, it has been claimed that IL-ABSs are efficient at maintaining and extracting enzyme activity [58]. Understanding the function of ILs as phase-forming elements and their impact on enzyme extraction and activity is essential to utilizing the full capacity of IL-ABSs [60]. ...
... Capela, Valente, Nunes, Magalhães, Rodríguez, Soto, Freire and Tavares [58] extracted and enhanced laccase activity using IL-ABSs [58]. To achieve this, a comprehensive assessment of ILs based on imidazolium, pyridinium, pyrrolidinium, piperidinium, tetraalkylphosphonium, and tetraalkylammonium as phase-forming components of ABSs had been performed. ...
... The extraction percentage of pectinase was found to be greater than 90%. Additionally, pectinase activity was found to increase by A. flavipes when it was scaled up to 5 L from 1 L. Jian et al. [89] examined the capacity of ABS constituted by [C 4 MIM][BF 4 ] IL and NaH 2 PO 4 at pH 4.8 for the partitioning and purification of wheat esterase enzyme. It was shown that wheat esterase distributed into the phase containing hydrophilic IL [84]. ...
... Based on these characteristics, they can be regarded as excellent biological catalysts. Most importantly, laccases are multi-Cu oxidases with outstanding potential for an array of uses; both in the environment and in biotechnology [89]. Several industrial processes have utilized laccases over the past few decades based on the reports made by some authors [98,99]. ...
... ILs were also employed as adjuvants in polymer-based systems at pH 8. Laccase was found to show a preference for the phase of the ABS with the most hydrophilic nature regardless of the systems involved. Out of all the examined ILs, cholinium-based ILs proved to be the best candidates for the extraction of laccase and they availed the highest enzyme activity values [89]. ...
... ABS consist of two immiscible aqueous-rich phases formed when water-soluble components are mixed above given concentrations (Freire et al., 2012;Pereira et al., 2020). Recently, ABS systems have been investigated as reaction media and liquid supports for enzymes since they provide a suitable and friendly environment for the maintenance of the enzymatic activity and its reuse (Ferreira et al., 2018;Capela et al., 2020;Ferreira et al., 2021;Muñiz-Mouro et al., 2021). Another advantage and application of this approach is the possibility of simultaneous extract and concentrate the reaction product by manipulating the volume of the ABS phases, using the tie-line of binodal curves (Dinis et al., 2018), while maintaining the enzyme in the opposite phase. ...
... The results depicted in Figure 3A show a selective partition of laccase for the bottom phase (IL-, K 2 HPO 4 -or PEG-rich phase) of all ABS, with an EE Laccase of 100%. These results are in accordance with previous works demonstrating that proteins and enzymes have a high affinity for the more hydrophilic phase (Ferreira et al., 2018;Capela et al., 2020;Ferreira et al., 2021). For example, the study carried out by Capela et al. (2020) proved that using ABS composed of cholinium-based ILs or PEG 400 + PPG 400, laccase complete partitioned for the more hydrophilic phase namely IL-or PEG 400-rich phase with an EE Laccase of 100%. ...
... These results are in accordance with previous works demonstrating that proteins and enzymes have a high affinity for the more hydrophilic phase (Ferreira et al., 2018;Capela et al., 2020;Ferreira et al., 2021). For example, the study carried out by Capela et al. (2020) proved that using ABS composed of cholinium-based ILs or PEG 400 + PPG 400, laccase complete partitioned for the more hydrophilic phase namely IL-or PEG 400-rich phase with an EE Laccase of 100%. ...
Polydopamine (PDA), a bioinspired polymer from mussel adhesive proteins, has attracted impressive attention as a novel coating for (nano) materials with an adequate conformal layer and adjustable thickness. Currently, PDA is obtained from dopamine chemical oxidation under alkaline conditions, limiting its use in materials sensible to alkaline environments. Envisaging a widespread use of PDA, the polymerization of dopamine by enzymatic catalysis allows the dopamine polymerization in a large range of pHs, overcoming thus the limitations of conventional chemical oxidation. Moreover, the conventional method of polymerization is a time-consuming process and produces PDA films with poor stability, which restricts its applications. On the other hand, the main bottleneck of enzyme-based biocatalytic processes is the high cost of the single use of the enzyme. In this work, laccase was used to catalyse dopamine polymerization. To improve its performance, a liquid support for integrating the laccase and its reuse together with the PDA production and recovery was developed using aqueous biphasic systems (ABS). Firstly, dopamine polymerization by laccase was optimized in terms of pH, temperature and initial dopamine concentration. It was demonstrated that the highest enzymatic polymerization of dopamine was achieved at pH 5.5, 30°C and 2 mg ml ⁻¹ of dopamine. Then, ABS composed of polymers, salts and ionic liquids were evaluated to optimize the laccase confinement in one phase while PDA is recovered in the opposite phase. The most promising ABS allowing the separation of laccase from the reaction product is composed of polypropylene glycol (400 g mol ⁻¹ ) and K 2 HPO 4 . The polymerization of dopamine in ABS leads to a remarkable improvement of polymerization of 3.9-fold in comparison to the conventional chemical PDA polymerization. The phase containing the confined laccase was reused for four consecutive reaction cycles, with a relative polymerization of 68.9% in the last cycle. The results of this work proved that ABS are a promising approach to create a liquid support for enzyme reuse allowing the process intensification efforts. The use of biocatalysts in ABS emerges as sustainable and alternative platforms from environmental and techno-economic points of view.
... The tertiary structure of biomolecules, such as proteins is the one that needs to be stabilized against the disrupting forces [4]. During protein storage, thermal denaturation must also be accounted for, as it is the main source of denaturation [5]. As proteins are only marginally stable at room temperature, all types of molecular interactions are to be accounted for, even small interactions can contribute significantly to the stability of proteins [5]. ...
... During protein storage, thermal denaturation must also be accounted for, as it is the main source of denaturation [5]. As proteins are only marginally stable at room temperature, all types of molecular interactions are to be accounted for, even small interactions can contribute significantly to the stability of proteins [5]. The main type of interactions that contribute to maintaining the three-dimensional structure of proteins is hydrogen bonding, hydrophobic, and ionic interactions [6]. ...
... ILs are constituted by an organic cation combined with an organic or inorganic anion that remains liquid at temperatures below 100 • C [3]. These non-aqueous solvents present a wide range of desired properties, such as no vapor pressure, low flammability, improved thermal and ionic conductivity, high capacity for dissolution of many compounds, and high thermal and chemical stability [3][4][5]. ILs are known as designer solvents due to the different combinations of cations and anions; thus, ILs can be specifically designed for different purposes and applications [3][4][5]. Various applications of ILs have been reported in the literature [19]. ...
Currently, the use of alternative solvents is increasing, namely ionic liquids (ILs) and deep eutectic solvents (DESs) in diverse fields of knowledge, such as biochemistry, chemistry, chemical engineering, biotechnology and biomedicine. Particularly, when compared to traditional solvents, these alternative solvents have great importance for biomolecules due to the enhanced solubility, structure stability and the biological activity of biomolecules, such as protein and enzymes. Thus, in this review article, the recent developments and efforts on the technological developments carried out with ILs and DESs for the stabilization and activation of proteins and enzymes are provided. The most studied IL- and DES-based formulations for proteins and enzymes are discussed and the molecular mechanisms and interactions related to the increased stability promoted by these alternative solvents are disclosed, while emphasizing their main advantages.
... Lai et al. [29] proved that ammonium ILs can offer potential advantages in facilitating enzyme functioning and that IL cations may play a main role over their counter-anions in affecting the enzyme behavior. In addition, Capela et al. [30] demonstrated that quaternary alkyl ammoniums with short alkyl side chains have beneficial effects on the stability of laccase [30], while Attri et al. [31] showed that triethyl ammonium cations with small alkyl chains of ILs (triethyl ammonium acetate and triethyl ammonium phosphate) are strong stabilizers for α-chymotrypsin. ...
... Lai et al. [29] proved that ammonium ILs can offer potential advantages in facilitating enzyme functioning and that IL cations may play a main role over their counter-anions in affecting the enzyme behavior. In addition, Capela et al. [30] demonstrated that quaternary alkyl ammoniums with short alkyl side chains have beneficial effects on the stability of laccase [30], while Attri et al. [31] showed that triethyl ammonium cations with small alkyl chains of ILs (triethyl ammonium acetate and triethyl ammonium phosphate) are strong stabilizers for α-chymotrypsin. ...
... Figure 6. Activity of immobilized ASNase (AIA) (symbols, line) and immobilized activity yield(ηIA) (bars) on the supports: [Si][N3114]Cl, [Si][N3116]Cl, [Si][N3118]Cl, [Si][N3222]Cl, [Si][N3444]Cl, [Si][N3666]Cl, and [Si][N3888]Cl for different ASNase immobilization contact times(30,45, 60, 90, and 120 min). Error bars correspond to standard deviation. ...
L-asparaginase (ASNase) is an amidohydrolase that can be used as a biopharmaceutical, as an agent for acrylamide reduction, and as an active molecule for L-asparagine detection. However, its free form displays some limitations, such as the enzyme’s single use and low stability. Hence, immobilization is one of the most effective tools for enzyme recovery and reuse. Silica is a promising material due to its low-cost, biological compatibility, and tunable physicochemical characteristics if properly functionalized. Ionic liquids (ILs) are designer compounds that allow the tailoring of their physicochemical properties for a given task. If properly designed, bioconjugates combine the features of the selected ILs with those of the support used, enabling the simple recovery and reuse of the enzyme. In this work, silica-based supported ionic liquid-like phase (SSILLP) materials with quaternary ammoniums and chloride as the counterion were studied as novel supports for ASNase immobilization since it has been reported that ammonium ILs have beneficial effects on enzyme stability. SSILLP materials were characterized by elemental analysis and zeta potential. The immobilization process was studied and the pH effect, enzyme/support ratio, and contact time were optimized regarding the ASNase enzymatic activity. ASNase–SSILLP bioconjugates were characterized by ATR-FTIR. The bioconjugates displayed promising potential since [Si][N3444]Cl, [Si][N3666]Cl, and [Si][N3888]Cl recovered more than 92% of the initial ASNase activity under the optimized immobilization conditions (pH 8, 6 × 10⁻³ mg of ASNase per mg of SSILLP material, and 60 min). The ASNase–SSILLP bioconjugates showed more enhanced enzyme reuse than reported for other materials and immobilization methods, allowing five cycles of reaction while keeping more than 75% of the initial immobilized ASNase activity. According to molecular docking studies, the main interactions established between ASNase and SSILLP materials correspond to hydrophobic interactions. Overall, it is here demonstrated that SSILLP materials are efficient supports for ASNase, paving the way for their use in the pharmaceutical and food industries.
... Ionic liquids (ILs) are molten salts that appear in liquid state at room temperature with numerous unique features including non-volatility, non-flammability, low melting temperature, high thermal and electrochemical stabilities [1,2]. The IL with great solvation power has been widely used to recover and purify enzymes with enhanced catalytic activity, stability and selectivity [3,4]. Moreover, IL can be recycled after the extraction process through dialysis, absorption, membrane separation and crystallization and reused for subsequent extraction process, resulting in a lower processing cost required for the processes [5]. ...
... Moreover, IL can be recycled after the extraction process through dialysis, absorption, membrane separation and crystallization and reused for subsequent extraction process, resulting in a lower processing cost required for the processes [5]. The IL can alter the polarity of the aqueous phases of the biphasic system because of the tunable cations and anions, thereby improving the extraction efficiency of the target biomolecules [4]. ...
... At a later stage, the feasibility of IL-based ABES on the recovery of K. sedentarius TWHKC01 keratinase was assessed by investigating the effects of the operation duration and operation voltage of the voltage supplier for the keratinase partition and recovery. 4 ] with two different types of salt including carbonate and phosphate. Both carbonate and phosphate ions are kosmotropes, which possess a strong intermolecular interaction with water molecules, thereby reducing the amount of free water molecules for hydration of IL and inducing the phase separation [22]. ...
Aqueous biphasic electrophoresis system (ABES) incorporates electric fields into the biphasic system to separate the target biomolecules from crude feedstock. Ionic liquid (IL) is regarded as an excellent candidate as the phase-forming components for ABES because of the great electrical conductivity, which can promote the electromigration of biomolecules in ABES, and thereby enhances the separation efficiency of the target biomolecules from crude feedstock. The application of electric fields to the conventional biphasic system expedites the phase settling time of the biphasic system, which eases the subsequent scaling-up steps and reduces the overall processing time of the recovery process. Alkyl sulphate-based IL is a green and economical halide-free surfactant when compared to the other halide-containing IL. The feasibility of halide-free IL-based ABES to recover Kytococcus sedentarius TWHK01 keratinase was studied. Optimum partition coefficient (Ke = 7.53 ± 0.35) and yield (YT = 80.36% ± 0.71) were recorded with IL-ABES comprised of 15.0% (w/w) [EMIM][ESO4], 20.0% (w/w) sodium carbonate and 15% (w/w) crude feedstock. Selectivity (S) of 5.75 ± 0.27 was obtained with the IL-ABES operated at operation time of 5 min with 10 V voltage supplied. Halide-free IL is proven to be a potential phase-forming component of IL-ABES for large-scale recovery of keratinase.
... Because of their homogeneity in aqueous media, it is challenging to recover them after the reaction, thus impeding their recycling in the production process. [6][7][8] Another issue is related to their stability, as they are sensitive to extreme conditions of temperature, pH, and substrate concentration, which can induce changes in their chemical structures, thereby deactivating their catalytic activity in harder conditions. [7,[9][10][11][12][13] Immobilization of several enzymes, including the oxidoreductases, on solid supports through covalent bonding is an important alternative for its protection against structural changes caused by denaturing conditions, as well as to prevent their leaching, thereby enabling recycling. ...
... [6][7][8] Another issue is related to their stability, as they are sensitive to extreme conditions of temperature, pH, and substrate concentration, which can induce changes in their chemical structures, thereby deactivating their catalytic activity in harder conditions. [7,[9][10][11][12][13] Immobilization of several enzymes, including the oxidoreductases, on solid supports through covalent bonding is an important alternative for its protection against structural changes caused by denaturing conditions, as well as to prevent their leaching, thereby enabling recycling. [14][15][16] Covalent bonding techniques are widely employed to immobilize enzymes as they establish stable and permanent chemical bonds that anchor the enzyme onto a solid polymer surface. ...
... ILs are commonly used as ABS phase-forming agents owing to their outstanding ability to solvate compounds with a wide range of polarities and to increase the extraction efficiency of these compounds while decreasing the viscosity and separation time of the phases [8,[10][11][12]. The addition of small amounts of ILs as adjuvants in polymer-polymer ABS not only allows the phase separation at much lower polymer concentrations and with lower molecular weights but also allows the partitioning tailoring [7,[14][15][16][17][18][19][20]. Within this framework, recent studies have highlighted cholinium chloride as a highly advantageous adjuvant in ABS, surpassing other ILs due to its exceptional biocompatibility, lower toxicity, superior ability to tailor phase behavior, and ability to enhance the extraction of biomolecules while maintaining an environmentally friendly nature [5,9,12,18,19,21]. ...
... The addition of small amounts of ILs as adjuvants in polymer-polymer ABS not only allows the phase separation at much lower polymer concentrations and with lower molecular weights but also allows the partitioning tailoring [7,[14][15][16][17][18][19][20]. Within this framework, recent studies have highlighted cholinium chloride as a highly advantageous adjuvant in ABS, surpassing other ILs due to its exceptional biocompatibility, lower toxicity, superior ability to tailor phase behavior, and ability to enhance the extraction of biomolecules while maintaining an environmentally friendly nature [5,9,12,18,19,21]. ...
A series of polymeric aqueous biphasic systems (ABS) were determined using polyethylene glycol (PEG) and sodium polyacrylate (NaPA) with choline chloride ([Ch]Cl) as an adjuvant. The effect of (i) PEG and NaPA molecular weights, (ii) PEG functionalization, (iii) [Ch]Cl addition (at different concentrations), and (iv) temperature (25, 37 and 50 °C) was evaluated through their ability to promote the two-phase separation. The results showed that the polymerization degree and functionalization of PEG polymers exhibit a large influence on the ABS formation, with high molecular weight PEG inducing an increase in the biphasic region. Furthermore, the addition of small amount (1–5 wt%) of [Ch]Cl also increased the liquid–liquid demixing. Temperature and the increase in the NaPA molecular weight did not influence the ABS formation ability. Finally, the partition performance of PEG/NaPA + [Ch]Cl ABS was evaluated using caffeine as a model compound. Unlike the ABS formation trend, NaPAs molecular weight significantly influenced the partitioning, which was strengthened when using NaPA-8000. Moreover, the incorporation of [Ch]Cl facilitated an inversion in the partitioning behavior of caffeine, thereby emphasizing the remarkable partitioning tailoring potential exhibited by these systems. Overall, all systems seem to be promising alternatives for the effective extraction, purification and/or concentration of different value-added biomolecules.
... 17 When studying laccase purification, Silveŕio et al. 16 analyzed different polymer−polymer and polymer−salt-based ABS, observing that polymer−salt ABS led to better results concerning both laccase extraction and stability and showing that ABS based on poly(ethylene glycol) (PEG) and phosphate salt combinations could lead to promising results and hence should be further investigated in the future. Moreover, Capela et al. 18 analyzed laccase purification and activity in IL-based ABS, identifying cholinium-based ILs as promising candidates for ABS formulation regarding both enzyme extraction and activity. ...
... According to the results (Table S6 and Figure S6), rutin conversion, laccase activity, and reaction kinetics are improved when the reaction takes place in the [Ch][DHph]-rich phase, which can be explained by the positive effect of cholinium cations over both laccase stability and activity, as shown in the literature. 18 After 24 h of reaction, the final enzyme activity was 10% higher in this phase than in the PEG-rich phase, reaching the complete oligomerization of rutin, while only 80% yield was achieved when the reaction was performed in the PEG-rich phase. Moreover, when the oligomerization was performed in the monophasic region of the ABS, the concentration of each ABS constituent was 26 (Table S5) show an 88% rutin extraction efficiency toward the PEG-rich phase, higher than that observed for oligorutin (∼70%). ...
... In this sense, aqueous two-phase systems (ATPS) have been studied as biocompatible and environmentally friendly alternatives for the recovery and purification of different bioproducts [16,17]. Selective separation is based on partitioning the target enzyme between the two aqueous phases, governed by its relative affinity for the phase-forming components [18][19][20]. ATPS are formed by two compounds dissolved in aqueous media, which above given concentrations undergo liquid-liquid demixing [21,22]. These are highly versatile extraction systems since many constituents can be used in their formation [23][24][25][26]. ...
Aqueous two-phase systems (ATPS) are considered efficient and sustainable downstream processing techniques in extracting and separating enzymes. In this work, we evaluated the use of ATPS based on quaternary ammonium (tetrabutylammonium bromide ([N4444]Br), tetrabutylammonium chloride ([N4444]Cl), choline chloride (ChCl), and betaine) + potassium phosphate buffer (pH 7) to separate selective lipase and protease from the fermentation broth with Y. lipolytica. Cultivation in YPD medium supplemented with 200 mM potassium phosphate buffer (pH 7) in the preparation of the culture medium positively influenced the production of lipase and protease, whereby led to lesser pH variations and better values of biomass production (15.29 g p.s. L-1), lipolytic (455.96 U L-1), and proteolytic activity (23.70 U L-1). The presence of the culture medium enhanced the two-phase region, and the phase separation followed the order of ammonium compounds hydrophobicity: [N4444]Br > [N4444]Cl > betaine > ChCl. Lipase was partitioned mainly to the ammonium-rich phase and protease migrates preferentially to the salt-rich phase in all systems studied. Remarkable extraction efficiencies of 100% for lipase and 96.87% for protease were achieved in a single step for [N4444]Cl-based ATPS. Furthermore, a high level of purification was achieved with values of 10.55 and 1.69 for lipase and protease, respectively. According to the remarkable results, quaternary ammonium-based ATPS can be considered an alternative and efficient platform to separate lipase from protease, obtaining two high-value-added compounds selectively.
... In this sense, aqueous two-phase systems (ATPS) have been studied as biocompatible and environmentally friendly alternatives for the recovery and purification of different bioproducts [16,17]. Selective separation is based on partitioning the target enzyme between the two aqueous phases, governed by its relative affinity for the phase-forming components [18][19][20]. ATPS are formed by two compounds dissolved in aqueous media, which above given concentrations undergo liquid-liquid demixing [21,22]. These are highly versatile extraction systems since many constituents can be used in their formation [23][24][25][26]. ...
Aqueous two-phase systems (ATPS) are considered efficient and sustainable downstream processing techniques in extracting and separating enzymes. In this work, we evaluated the use of ATPS based on quaternary ammo-nium (tetrabutylammonium bromide ([N 4444 ]Br), tetrabutylammonium chloride ([N 4444 ]Cl), choline chloride (ChCl), and betaine) + potassium phosphate buffer (pH 7) to separate selective lipase and protease from the fermentation broth with Y. lipolytica. Cultivation in YPD medium supplemented with 200 mM potassium phosphate buffer (pH 7) in the preparation of the culture medium positively influenced the production of lipase and protease, whereby led to lesser pH variations and better values of biomass production (15.29 g p.s. L-1), lipolytic (455.96 U L-1), and proteolytic activity (23.70 U L-1). The presence of the culture medium enhanced the two-phase region, and the phase separation followed the order of ammonium compounds hydrophobicity: [N 4444 ]Br > [N 4444 ]Cl > betaine > ChCl. Lipase was partitioned mainly to the ammonium-rich phase and pro-tease migrates preferentially to the salt-rich phase in all systems studied. Remarkable extraction efficiencies of 100% for lipase and 96.87% for protease were achieved in a single step for [N 4444 ]Cl-based ATPS. Furthermore, a high level of purification was achieved with values of 10.55 and 1.69 for lipase and protease, respectively. According to the remarkable results, quaternary ammonium-based ATPS can be considered an alternative and efficient platform to separate lipase from protease, obtaining two high-value-added compounds selectively.
... This drawback may be overcome by employing ionic liquids (ILs) as phase-forming components of ABS [14]. In this way, ABS can be tailored by taking advantage of ILs as "designer solvents", allowing low viscosity of the phases, rapid phase separation and high extraction efficiencies and selectivity [14][15][16][17]. ILs are becoming increasingly popular as alternative solvents in extraction processes, with several startups, such as Calagua Innovations, 525 Solutions, Wyonics and Marisignum, already using them at a large scale. ...
... Enzymes are fundamental in several fields, as they present excellent catalytic properties, selectivity, efficiency, low toxicity, and biodegradability. Laccases, for instance, are of special interest due to their capacity of oxidizing a wide range of molecules and thus, being able to catalyze several processes [65][66][67]. Without recycling, working with enzymes is expensive, so promoting their reuse with small compound losses or activity is crucial, albeit very challenging. ...
Aqueous biphasic systems are widely known for their enhanced biocompatibility and selectivity in downstream processes. Hence, being a key tool for the extraction/separation of innumerous biomolecules. This is particularly important for the food industry considering the higher consumers’ awareness for the health hazards associated with chemicals used in food processing and applications as well as the replacement of synthetic food additives such as pigments and preservatives. Therefore, this mini-review offers a critical perspective on the progress done over the last 3 years regarding the application of aqueous biphasic systems i) for the extraction of food related biomolecules, ii) as analytical tools in food processing, iii) as in-situ bio-based platforms, iv) for the valorization of food waste and v) to shed some light on how molecular simulation approaches may be a key element in this framework.
... On the other hand, ILs comprised of imidazolium-based cations and triflate showed better performance in terms of improving the substrate solubility and and activity of the biocatalyst. Moreover, the combination of ILs and microwave ited synergistic effects for enzymatic synthesis of biodiesel production, improv Notably, ionic liquids (ILs), as a greener alternative to organic solvents [91,92], are ideal solvents for enzyme-catalyzed reactions under microwave radiation because of their excellent microwave-absorbing ability, high boiling point, and low vapor pressure [93]. Currently, a lipase-mediated, microwave-assisted anthocyanin lipophilization with ILs, which can improve the biomedical properties of anthocyanins, including the anti-inflammatory and antioxidant properties, has been reported for the first time [94,95]. ...
Enzyme activation is a powerful means of achieving biotransformation function, aiming to intensify the reaction processes with a higher yield of product in a short time, and can be exploited for diverse applications. However, conventional activation strategies such as genetic engineering and chemical modification are generally irreversible for enzyme activity, and they also have many limitations, including complex processes and unpredictable results. Recently, near-infrared (NIR), alternating magnetic field (AMF), microwave and ultrasound irradiation, as real-time and precise activation strategies for enzyme analysis, can address many limitations due to their deep penetrability, sustainability, low invasiveness, and sustainability and have been applied in many fields, such as biomedical and industrial applications and chemical synthesis. These spatiotemporal and controllable activation strategies can transfer light, electromagnetic, or ultrasound energy to enzymes, leading to favorable conformational changes and improving the thermal stability, stereoselectivity, and kinetics of enzymes. Furthermore, the different mechanisms of activation strategies have determined the type of applicable enzymes and manipulated protocol designs that either immobilize enzymes on nanomaterials responsive to light or magnetic fields or directly influence enzymatic properties. To employ these effects to finely and efficiently activate enzyme activity, the physicochemical features of nanomaterials and parameters, including the frequency and intensity of activation methods, must be optimized. Therefore, this review offers a comprehensive overview related to emerging technologies for achieving real-time enzyme activation and summarizes their characteristics and advanced applications.
... As mentioned above, MIER is provided with numerous preponderance in performing chemical reactions over traditional technologies. In order to improve the working efficiency of the microfluidic reactor and meet the needs of some special reactions, the following auxiliary methods are used, including microparticles, ionic liquid, ultrasound, microwave, etc. [95][96][97][98]. In addition, some special forms of microfluidic reactors have also been developed and applied. ...
Biocatalysts toward multi-step reactions, though they make the construction of versatile molecules possible, struggle to achieve a rapid expansion due to many challenges to be addressed. Continuous flow technique has been used extensively in synthetic organic chemistry over the last decades, and the development of biocatalytic processes in continuous flow, however, has been only recently become increasingly popular. The integration of continuous flow with biocatalysts toward multi-step reactions was endowed with distinguished advantages that cannot be achieved in traditional batch mode. Within this mini-review, we will mainly summarize the recent applications in the area of biocatalysts toward multi-step continuous flow reactions, covering chemo-enzymatic and multi-enzymatic systems, with particular focus on the main challenges and strategies.
... Compared to polymer-based ABS, IL-rich systems show a set of important advantages, such as low viscosity, fast phase separation, and high extraction efficiency and selectivity achieved by the proper tailoring of the IL chemical structure (Ferreira et al., 2014;Freire et al., 2012). Furthermore, ILs, if properly selected, lead to improved activity and stability of a wide number of enzymes and improved selectivity in biocatalysis (Bisht et al., 2017;Capela et al., 2020;Dossantos et al., 2018;Santos et al., 2018;Schindl et al., 2019). ...
Novel liquid supports for enzyme immobilization and reuse based on aqueous biphasic systems (ABS) constituted by cholinium‐based ionic liquids (ILs) and polymers for the degradation of dyes are here proposed. The biocatalytic reaction for dye decolorization using laccase occured in the biphasic medium, with the enzyme being “supported” in the IL‐rich phase and the dye and degradation products being enriched in the polymer‐rich phase. An initial screening of the laccase activity in aqueous solutions of ABS constituents, namely cholinium dihydrogen citrate ([Ch][DHC]), cholinium dihydrogen phosphate ([Ch][DHP]), cholinium acetate ([Ch][Acet]), polypropylene glycol 400 (PPG 400), polyethylene glycol 400 (PEG 400) and K2HPO4 was carried out. Compared to the buffered control, a relative laccase activity of up to 170%, 257%, and 530% was observed with PEG 400, [Ch][DHP], and [Ch][DHC], respectively. These ABS constituents were then investigated for the in situ enzymatic biodegradation of the Remazol Brilliant Blue R (RBBR) dye. At the optimized conditions, the ABS constituted by PPG 400 at 46 wt% and [Ch][DHC] at 16 wt% leads to the complete degradation of the RBBR dye, further maintaining the enzyme activity. This ABS also allows an easy immobilization, recovery, and reuse of the biocatalyst for six consecutive reaction cycles, achieving a degradation yield of the dye of 96% in the last cycle. In summary, if properly designed, high enzymatic activities and reaction yields are obtained with ABS as liquid supports, while simultaneously overcoming the safety and environmental concerns of conventional organic solvents used in liquid–liquid heterogeneous reactions, thus representing more sustainable biocatalytic processes.
... In addition, ILs can impact the biocatalytic activity of the laccase. For instance, aqueous biphasic systems containing IL cholinium dihydrogen citrate ([Chol][DHCit]) have been shown to enhance the extraction efficiency of the enzyme and increase the biocatalytic activity by 50% [164]. ...
In the past decade, innovative protein therapies and bio-similar industries have grown rapidly. Additionally, ionic liquids (ILs) have been an area of great interest and rapid development in industrial processes over a similar timeline. Therefore, there is a pressing need to understand the structure and function of proteins in novel environments with ILs. Understanding the short-term and long-term stability of protein molecules in IL formulations will be key to using ILs for protein technologies. Similarly, ILs have been investigated as part of therapeutic delivery systems and implicated in numerous studies in which ILs impact the activity and/or stability of protein molecules. Notably, many of the proteins used in industrial applications are involved in redox chemistry, and thus often contain metal ions or metal-associated cofactors. In this review article, we focus on the current understanding of protein structure-function relationship in the presence of ILs, specifically focusing on the effect of ILs on metal containing proteins.
... As mentioned above, MIER is provided with numerous preponderance in performing chemical reactions over traditional technologies. In order to improve the working efficiency of the microfluidic reactor and meet the needs of some special reactions, the following auxiliary methods are used, including microparticles, ionic liquid, ultrasound, microwave, etc. [95][96][97][98]. In addition, some special forms of microfluidic reactors have also been developed and applied. ...
Microfluidic, as the systems for using microchannel (micron- or sub-micron scale) to process or manipulate microflow, is being widely applied in enzyme biotechnology and biocatalysis. Microfluidic immobilized enzyme reactor (MIER) is a tool with great value for the study of catalytic property and optimal reaction parameter in a flourishing and highly producing manner. In view of its advantages in efficiency, economy, and addressable recognition especially, MIER occupies an important position in the investigation of life science, including molecular biology, bioanalysis and biosensing, biocatalysis etc. Immobilization of enzymes can generally improve their stability, and upon most occasions, the immobilized enzyme is endowed with recyclability. In this review, the enzyme immobilization techniques applied in MIER will be discussed, followed by summarizing the novel developments in the field of MIER for biocatalysis, bioconversion and bioanalysis. The preponderances and deficiencies of the current state-of-the-art preparation ways of MIER are peculiarly discussed. In addition, the prospects of its future study are outlined.
Condensable gases are the sum of condensable and volatile steam or organic compounds, including water vapor, which are discharged into the atmosphere in gaseous form at atmospheric pressure and room temperature. Condensable toxic and harmful gases emitted from petrochemical, chemical, packaging and printing, industrial coatings, and mineral mining activities seriously pollute the atmospheric environment and endanger human health. Meanwhile, these gases are necessary chemical raw materials; therefore, developing green and efficient capture technology is significant for efficiently utilizing condensed gas resources. To overcome the problems of pollution and corrosion existing in traditional organic solvent and alkali absorption methods, ionic liquids (ILs), known as "liquid molecular sieves", have received unprecedented attention thanks to their excellent separation and regeneration performance and have gradually become green solvents used by scholars to replace traditional absorbents. This work reviews the research progress of ILs in separating condensate gas. As the basis of chemical engineering, this review first provides a detailed discussion of the origin of predictive molecular thermodynamics and its broad application in theory and industry. Afterward, this review focuses on the latest research results of ILs in the capture of several important typical condensable gases, including water vapor, aromatic VOCs (i.e., BTEX), chlorinated VOC, fluorinated refrigerant gas, low-carbon alcohols, ketones, ethers, ester vapors, etc. Using pure IL, mixed ILs, and IL + organic solvent mixtures as absorbents also briefly expanded the related reports of porous materials loaded with an IL as adsorbents. Finally, future development and research directions in this exciting field are remarked.
Efforts have been concentrated on developing alternative methods of enzyme purification that are less costly and highly efficient. In this work, we evaluated three different methods for lipase purification from Yarrowia lipolytica, such as precipitation using ammonium sulfate, ethanol, or acetone; aqueous two-phase systems (ATPS) based on polyethylene glycol (PEG) and potassium phosphate; and direct immobilization. It was impossible to obtain stable precipitates of the enzyme due to the low concentration of total protein and the presence of biosurfactant produced by the microorganism. Different mixture compositions were selected for the partitioning study. Three ATPS showed selective partitioning of the target enzymes, i.e., lipase and protease migrated to opposite phases. In the ATPS composed of 13 wt% PEG-4000 and 10 wt% salts, it was possible to achieve a purification factor for lipase of 4.2. Purification by immobilization performed by lipase-lipase interactions showed three lipases of distinct sizes in the crude extract. In the immobilization method by hydrophobic supports, phenyl-agarose and butyl‑agarose were more selective in immobilizing than octyl-agarose. In the ion exchange immobilization method, only the lipases identified at 66 kDa and 41 kDa have an attraction for DEAE-agarose (anionic) and sulfopropyl-agarose (cationic) matrices.
Biocatalysis is an important area in the context of green chemistry and sustainable development, competing with traditional chemical catalytic processes. However, to be a sustainable bioprocess, the proper choice of the reaction medium plays a significant role, combined with better performance and reduced costs. Volatile organic solvents are commonly used as solvents or cosolvents in certain enzymatic reactions that need to be performed in hydrophobic media. Within biocatalysis, ionic liquids have emerged as promising alternative solvents, particularly to replace less benign organic solvents in nonaqueous reactions, and have been widely investigated over recent decades. In addition, ionic liquids have been used as surface modifiers of solid materials to act as supports of biocatalysts in order to improve the catalytic performance. This chapter provides an overview and outlook of the recent and emerging applications of ionic liquids in nonaqueous media biocatalysis, with emphasis on the pretreatment of biomass, lignin valorization, biofuels production, biosensors, and bioremediation. Final considerations and challenges in this field are also provided.
In this work, N-1 substituted 1,2,4-triazolium-based cation and trifluoromethanesulfonate anion based IL is synthesized and used as a solvent system to stabilize haemoglobin (Hb). The conformational stability of Hb at different concentrations of developed IL was inspected through spectroscopic techniques like UV-Vis, steady-state fluorescence, fluorescence time-correlated single photon counting (TCSPC), temperature dependent UV-Vis, and fluorescence, and circular dichroism (CD). Hb structure is stabilized in the IL concentration range 0.001 to 0.01 M; Hb structure starts opening up in the IL concentration range 0.05 to 0.1 M; at 0.2 M IL concentration, denaturation of Hb takes place. Further, the nature of the interaction between IL and Hb was verified through a competitive hydrophobic fluorescence probe, 1-Anilino-8-Naphthalene Sulfonate (ANS), and observed that ANS does not interact with IL till 0.01 M IL concentration. Finally, the experiments illustrate that the IL concentration plays a significant role in stabilizing/destabilizing a particular protein.
In this work, phase behaviors and pigment extraction performance of the ionic liquid-based aqueous two-phase system (IL-ATPS) formed by 1-ethylpiperazinium tertrafluoroborate ([C2pi][BF4]) + sodium dodecyl sulfonate (SDS) and 1-phenylpiperazinium tertrafluoroborate ([Phpi][BF4]) + sodium dodecyl benzene sulfonate (SDBS) are investigated. Phase composition of ATPS is determined, revealing ion exchange between conjugated phases. Mixing the piperazinium-based IL with the anionic surfactant produces a weakly acidic environment of ATPS which is suitable for the separation of curcumin. The influences of surfactant/IL mass ratio, pigment concentration and temperature on extraction efficiency and distribution coefficient are evaluated. The separation mechanism of ATPS is further explored by DLS, TEM and FTIR. The packing effect induced by the re-assembly of micelles formed mainly by IL cations and surfactant anions are the driving force for the preferable separation of curcumin, while carmine distributes in both phases with similar concentrations due to its high water solubility.
The concept of supercritical fluids and their potential applications in biotechnology and related areas has attracted particular attention in the past decade. The most appealing feature, making supercritical solvents an attractive candidate for enzyme-catalyzed processes, is that they are “tunable solvent”; their physical characteristics significantly depend on the operating conditions. Therefore, for each specific reaction, it is easy to obtain the desired efficiency by the manipulation of the operating parameters. Among potentially appealing supercritical fluids for enzymatic reactions, however, carbon dioxide has attracted considerable attention because of its low cost, nontoxicity, nonflammability, and eco-friendly feature. Herein, in this research, we aim to review the typical biocatalytic reactions carried out in scCO2 medium including carboxylation, oxidation, reduction, and hydrolysis/esterification catalyzed by decarboxylase, oxidase, alcohol dehydrogenase, and lipases. Considerations behind the selection of operating parameters to avoid or slow catalyst deactivation are also discussed elaborately. Finally, the major obstacles that may hinder their progress including the issues of low solubility in scCO2 and negative impact of pressure on enzyme activity are broadly presented throughout this study, providing a roadmap for future research opportunities.
Organic carbonates are important classes of organic compounds and chemical intermediates, which are renowned for their great versatility, low toxicity, and high biodegradability. Therefore, they are widely acknowledged as green reagents and benign alternatives to conventional organic solvents. Today, most of the industrial technologies are compelled to implement the green processes to address both sustainability and eco-friendliness. In this context, the deployment of biocatalysis leading to high chemo-, regio-, and stereo-selectivities of the target products turns out to be an attractive solution. In this chapter, we will focus on a multiple of attempts to integrate the remarkable features of carbonates as green solvents and reagents in the biocatalyst-mediated reactions.
This chapter aims to give some insights into supercritical fluid (SCF) applications in reaction systems in view of chemical engineering applications. In chemical reactions, the reaction medium is usually a fluid solvent that must have specific characteristics that can be regulated through temperature and pressure of the system to above its critical point. The supercritical technology can favorably modify the solvent properties as required for reactions. Some applications of SCFs and specifically supercritical water (SCW) are discussed in this chapter including waste destruction, biomass processing, and chemical synthesis. Furthermore, important factors influencing SCF-biocatalytic reaction systems (enzyme-catalyzed reactions) including the effect of water content on enzyme operation in the presence of SCFs, the effect of operation conditions, and the effect of solvent characteristics are also elaborated. A discussion on the challenges and limitations of SCFs for industrial applications is also provided.
The global rise in urbanization and industrial activity has led to the production and incorporation of foreign contaminant molecules into ecosystems, distorting them and impacting human and animal health. Physical, chemical, and biological strategies have been adopted to eliminate these contaminants from water bodies under anthropogenic stress. Biotechnological processes involving microorganisms and enzymes have been used for this purpose; specifically, laccases, which are broad spectrum biocatalysts, have been used to degrade several compounds, such as those that can be found in the effluents from industries and hospitals. Laccases have shown high potential in the biotransformation of diverse pollutants using crude enzyme extracts or free enzymes. However, their application in bioremediation and water treatment at a large scale is limited by the complex composition and high salt concentration and pH values of contaminated media that affect protein stability, recovery and recycling. These issues are also associated with operational problems and the necessity of large-scale production of laccase. Hence, more knowledge on the molecular characteristics of water bodies is required to identify and develop new laccases that can be used under complex conditions and to develop novel strategies and processes to achieve their efficient application in treating contaminated water. Recently, stability, efficiency, separation and reuse issues have been overcome by the immobilization of enzymes and development of novel biocatalytic materials. This review provides recent information on laccases from different sources, their structures and biochemical properties, mechanisms of action, and application in the bioremediation and biotransformation of contaminant molecules in water. Moreover, we discuss a series of improvements that have been attempted for better organic solvent tolerance, thermo-tolerance, and operational stability of laccases, as per process requirements.
The application of enzymes offers an enormous potential in the improvement of existing industrial procedures and in the establishment of new processes for obtaining high‐added value products. Enzymes provide cleaner and more efficient industrial processes and contribute to the sustainability concept. In this sense, laccases are very versatile biocatalysts currently used in food, textile and pulp and paper sectors among others. During the last years, scientific efforts have been diverted to the exploitation of such interesting enzymes in novel fields like lignocellulosic biorefineries, biosensors or enzymatic biofuel cells. This review provides a general vision of the use of laccase enzymes describing their main characteristics and mode of action. Furthermore, their current uses in industrial processes are summarized and the most novel potential application of laccases are revealed. The increasing interest on laccases is also demonstrated by the research efforts on enzyme engineering as it is detailed in this review. This article is protected by copyright. All rights reserved.
Laccases have received the attention of researchers in the last few decades due to their ability to degrade phenolic and lignin-related compounds. This study aimed at obtaining the highest possible laccase activity and evaluating the methods of its purification. The crude laccase from bioreactor cultivation of Cerrena unicolor fungus was purified using ultrafiltration, aqueous two-phase extraction (ATPE) and foam fractionation (FF), which allowed for the assessment of these three downstream processing (DSP) methods. The repeated fed-batch cultivation mode applied for the enzyme production resulted in a high laccase specific activity in fermentation broth of 204.1 U/mg. The use of a specially constructed spin filter inside the bioreactor enabled the integration of enzyme biosynthesis and biomass filtration in one apparatus. Other methods of laccase concentration and purification, namely ATPE and FF, proved to be useful for laccase separation; however, the efficiency of FF was rather low (recovery yield of 24.9% and purification fold of 1.4). Surprisingly, the recovery yield after ATPE in a PEG 6000-phosphate system in salt phase was higher (97.4%) than after two-step ultrafiltration (73.7%). Furthermore, it was demonstrated that a simple, two-step purification procedure resulted in separation of two laccase isoforms with specific activity of 2349 and 3374 U/mg. All in all, a compact integrated system for the production, concentration and separation of fungal laccases was proposed.
Laccase enzyme has many important applications as bioremediation and removal of environmental wastes and also in industrial processes. Therefore, large-scale of novel producers of laccase is demanded to use cheap and low cost substrates. Hence, solid state fermentation is the best strategy to achieve this proposes. The present study is designed to optimize laccase production by Streptomyces mutabilis A17 using agro-wastes including rice bran, castor seed cake, wheat bran, wheat straw, soybeans cake, peanut cake, cotton seed cake and chicken feathers. Cotton seed cake significantly enhanced S. mutabilis A17 laccase production when it was incubated for 6 days at 37°C and 70% moisture content in presence of yeast extract and glucose as the best nitrogen and carbon sources, respectively. After purification steps, the purified laccase showed maximum activity at 40°C and pH 8.0 within stability pH range of 7.0 to 9.0. Laccase activity was highly increased to 195, 180 and 166% by the addition of Ba ⁺² , Cu ⁺² and Mn ⁺² , respectively. Sulfa drugs and synthetic dyes cause various pollutants due to their toxic effects in different environments. Therefore, a purified laccase was utilized for removal of sulfa drugs as sulfadiazine (SDZ) and sulfathiazole (STZ) and also synthetic dyes. The results showed that the maximal enzymatic removal of SDZ and STZ was attained at 50°C and pH 6.0 for 1 h at presence of 1mM HBT(1-hydroxybenzotriazole) as a laccase mediator by removal percentage of 73% and 90%, respectively. Detoxification effects of laccase-treated sulfa drugs and two synthetic dyes; congo red and malachite green solutions, were investigated using microbial toxicity test. Our results indicated that the toxicity of these laccase-treated samples against tested bacterial strains were significantly decreased. It was concluded from this study that SSF laccase is playing an important role for decreasing the toxic effects of pharmaceutical wastes and organic pollutants.
Background
Beer is the most popular alcoholic beverage worldwide. In the manufacture of beer, various by-products and residues are generated, and the most abundant (85% of total by-products) are spent grains. Thanks to its high (hemi)cellulose content (about 50% w/w dry weight), this secondary raw material is attractive for the production of second-generation biofuels as butanol through fermentation processes.
Results
This study reports the ability of two laccase preparations from Pleurotus ostreatus to delignify and detoxify milled brewer’s spent grains (BSG). Up to 94% of phenols reduction was achieved. Moreover, thanks to the mild conditions of enzymatic pretreatment, the formation of other inhibitory compounds was avoided allowing to apply the sequential enzymatic pretreatment and hydrolysis process (no filtration and washing steps between the two phases). As expected, the high detoxification and delignification yields achieved by laccase pretreatment resulted in great saccharification. As a fact, no loss of carbohydrates was observed thanks to the novel sequential strategy, and thus the totality of polysaccharides was hydrolysed into fermentable sugars. The enzymatic hydrolysate was fermented to acetone-butanol-ethanol (ABE) by Clostridium acetobutilycum obtaining about 12.6 g/L ABE and 7.83 g/L butanol within 190 h.
Conclusions
The applied sequential pretreatment and hydrolysis process resulted to be very effective for the milled BSG, allowing reduction of inhibitory compounds and lignin content with a consequent efficient saccharification. C. acetobutilycum was able to ferment the BSG hydrolysate with ABE yields similar to those obtained by using synthetic media. The proposed strategy reduces the amount of wastewater and the cost of the overall process. Based on the reported results, the potential production of butanol from the fermentation of BSG hydrolysate can be envisaged.
BACKGROUND - Self-buffered aqueous biphasic systems (ABS) are prepared with ionic liquids (ILs) based in Good's buffers (GB-ILs) and used to purify proteins.
RESULTS - A set of new GB-ILs based on the tetrabutylphosphonium cation ([P4444]+) was here synthesized and characterized. The self-buffering behaviour of the GB-ILs was asserted by measuring their protonation constants by potentiometry. These ionic liquids display self-buffering characteristics as well as a low toxicity towards the luminescent bacteria Vibrio fischeri. The ability of the GB-ILs to form ABS with the potassium citrate salt was investigated. These systems were then evaluated to extract and purify BSA from bovine serum samples. Extraction efficiencies of 100 ± 5 % of BSA to the GB-IL-rich phase were obtained in a single-step. In addition, good recovery yields (59.4 ± 0.8 % to 80.1 ± 0.7 %), and purity levels of BSA (75.0 ± 0.3 % to 92 ± 1 %) were also achieved. The BSA secondary structure in the aqueous IL–rich solutions was evaluated through infrared spectroscopic studies revealing the protein-friendly nature of the synthesized GB-ILs. Dynamic light scattering (DLS), “COnductor-like Screening MOdel for Real Solvents” (COSMO-RS), and molecular docking studies were finally carried to better understand the main driving forces of the extraction process.
CONCLUSION - The results obtained show that GB-IL based ABS can be prepared and used for protein purifications, with the GB-ILs showing a stabilizing effect on the protein.
Aqueous two-phase system (ATPS) is a liquid-liquid fractionation technique and has gained an interest because of great potential for the extraction, separation, purification and enrichment of proteins, membranes, viruses, enzymes, nucleic acids and other biomolecules both in industry and academia. Although, the partition behavior involved in the method is complex and difficult to predict. Current research shows that it has also been successfully used in the detection of veterinary drug residues in food, separation of precious metals, sewage treatment and a variety of other purposes. The ATPS is able to give high recovery yield and is easily to scale up. It is also very economic and environment friendly method. The aim of this review is to overview the basics of ATPS, optimization and its applications.
Monitoring of phenolic compounds in the food industry and for environmental and medical applications has become more relevant in recent years. Conventional methods for detection and quantification of these compounds, such as spectrophotometry and chromatography, are time consuming and expensive. However, laccase biosensors represent a fast method for on-line and in situ monitoring of these compounds. We discuss the main transduction principles. We divide the electrochemical principle into amperometric, voltammetric, potentiometric and conductometric sensors. We divide optical transducers into fluorescence and absorption. The amperometric transducer method is the most widely studied and used for laccase biosensors. Optical biosensors present higher sensitivity than the other biosensors. Laccase production is dominated by a few fungus genera: Trametes, Aspergillus, and Ganoderma. We present an overview of laccase biosensors used for the determination of phenolic compounds in industrial applications.
The breakdown of plant lignin modifies the structure of lignocelluloses, thus making carbohydrates accessible for efficient bioconversion. White-rot fungi produce ligninolytic enzymes such as lignin peroxidase, manganese peroxidase, laccases and various peroxidases, which mineralize lignin efficiently. We review here applications of ligninolytic enzymes for the delignification of lignocellulosic materials, the removal of recalcitrant organic pollutants, wastewater treatment, decolorization of dyes, soil treatment, conversion of high molecular weight coal fractions to low molecular weight coal fractions, which could be used as a feed stock for the production of commodity chemicals, biopulping and biobleaching in paper industries and enzymatic polymerization in polymer industries.
Over the past decade, a variety of ionic liquids have emerged as greener solvents for use in the chemical manufacturing industries. Their unique properties have attracted the interest of chemists worldwide to employ them as replacement for conventional solvents in a diverse range of chemical transformations including biotransformations. Biocatalysts are often regarded as green catalysts compared to conventional chemical catalysts in organic synthesis owing to their properties of low toxicity, biodegradability, excellent selectivity and good catalytic performance under mild reaction conditions. Similarly, a selected number of specific ionic liquids can be considered as greener solvents superior to organic solvents owing to their negligible vapor pressure, low flammability, low toxicity and ability to dissolve a wide range of organic and biological substances, including proteins. A combination of biocatalysts and ionic liquids thus appears to be a logical and promising opportunity for industrial use as an alternative to conventional organic chemistry processes employing organic solvents. This article provides an overview of recent developments in this field with special emphasis on the application of more sustainable enzyme-catalyzed reactions and separation processes employing ionic liquids, driven by advances in fundamental knowledge, process optimization and industrial deployment.
Over the last years, aqueous two-phase systems (ATPS) regained an increasing interest due to their potential in the downstream processing of biomolecules. After many years with only a few articles published, a lot of effort and work has been put into studying these systems for the partitioning of a range of compounds including proteins, organic low-molecular weight molecules or metal ions. Although several research and review articles appeared, a background review on ATPS partitioning fundamentals is needed. In this article, partitioning theories and main effects of several important factors for partitioning, such as molecular weight of the polymer, effect of added salts, pH, electrical charges, and temperature on phase diagrams, tie-line lengths, interfacial tension and settling time of the two aqueous phases are extensively reviewed. The trend in ATPS research is given compiling the recent 2008-2013 research articles published in the field.
Aqueous biphasic systems (ABS) composed of ionic liquids (ILs) are promising platforms for the extraction and purification of proteins. In this work, a series of alternative and biocompatible ABS composed of cholinium-based ILs and polypropylene glycol were investigated. The respective ternary phase diagrams, tie-lines, tie-line lengths and critical points were determined at 25°C. The extraction performance of these systems for commercial bovine serum albumin (BSA) was then evaluated. The stability of BSA at the IL-rich phase was ascertained by size exclusion high-performance liquid chromatography (SE-HPLC) and Fourier transform infrared (FT-IR) spectroscopy. Appropriate ILs lead to the complete extraction of BSA for the IL-rich phase, in a single-step, while maintaining the protein's native conformation. Furthermore, to evaluate the performance of these systems when applied to real matrices, the extraction of BSA from bovine serum was additionally carried out, revealing that the complete extraction of BSA was maintained and achieved in a single-step. The remarkable extraction efficiencies obtained are far superior to those observed with typical polymer-based ABS. Therefore, the proposed ABS may be envisaged as a more effective and biocompatible approach for the separation and puri?cation of other value-added proteins.
Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
The use of a wide range of water miscible and immiscible ionic liquids (ILs) as reaction media for ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) oxidation by Trametes versicolor laccase was studied. Thirteen ILs were shown to be suitable media for the laccase oxidation reaction, increasing the activity with respect to conventional media. Among them, the water-miscible IL choline dihydrogen phosphate [Chol][H2PO4] allowed over-laccase activity with an enhancement rate of 451% at 25°C and pH 7.0. This ionic liquid improved the stability of the enzyme in the face of high temperature and high pH, while storage at room temperature in aqueous medium was increased up to 4.5 times. Moreover, it was found that its use in the reaction medium for decolourizing dyes (antraquinonic and azoic) using laccase increased the decolourization rate by up to 216% and 137% for the azoic dyes Acid Black 1 and Remazol Brillant Blue R, respectively. A high decolorization rate was also obtained for a mix of dyes (80% within 8 h). To understand the effect of [Chol][H2PO4] on the secondary protein structure of the laccase, several spectroscopic techniques were used such as Circular Dichroism (CD), Fourier transform infrared (FT-IR) and Fluorescence, all of which demonstrated that the β sheets structure was affected. A shift to α-helix structure [Chol][H2PO4] could be responsible of the enhancement of the enzyme activity observed at 300 mM.
An analysis of the scientific literature published in the last 10 years reveals a constant growth of laccase applicative research in several industrial fields followed by the publication of a great number of patents. The Green Chemistry journal devoted the cover of its September 2014 issue to a laccase as greener alternative for chemical oxidation. This indicates that laccase "never-ending story" has found a new promising trend within the constant search for efficient (bio)catalysts able to meet the 12 green chemistry principles. A survey of ancient and cutting-edge uses of laccase in different industrial sectors is offered in this review with the aim both to underline their potential and to provide inspiration for new ones. Applications in textile and food fields have been deeply described, as well as examples concerning polymer synthesis and laccase-catalysed grafting. Recent applications in pharmaceutical and cosmetic industry have also been reviewed.
Next to xylanases, laccases from fungi and alkali-tolerant bacteria are the most important biocatalysts that can be employed for eco-friendly biobleaching of hard and soft wood pulps in the paper industry. Laccases offer a potential alternative to conventional, environmental-polluting chlorine and chlorine-based bleaching and has no reductive effect on the final yield of pulp as compared to hemicellulases (xylanases and mannanases). In the last decade, reports on biobleaching with laccases are based on laboratory observations only. There are several critical challenges before this enzyme can be implemented for pulp bleaching at the industrial scale. This review discusses significant factors like redox potential, laccase mediator system (LMS)-synthetic or natural, pH, temperature, stability of enzyme, unwanted grafting reactions of laccase, and cost-intensive production at large scale which constitute a great hitch for the successful implementation of laccases at industrial level.
The activity and stability of commercial peroxidase was investigated in the presence of five 1-alkyl-3-methylimidazolium-based ionic liquids (ILs) with either bromide or chloride anions: [Cxmim][X]. The peroxidase activity and stability were better for the shorter alkyl chain lengths of the ILs and peroxidase was more stable in the presence of the bromide anion, rather than chloride. The thermal inactivation profile was studied from 45 to 60 °C in [C4mim][Cl] and [C4mim][Br]. The activation energy was also determined. Kinetic analysis of the enzyme in the presence of the [C4mim][Br] or control (buffer solution) showed that the KM value increased 5-fold and Vm decreased 13-fold in the presence of the IL. The increase in KM indicates that this IL can reduce the binding affinity between substrate and enzyme.
In nature, the biodegradation of lignin is a challenging process since lignin is highly cross-linked and poorly water-soluble. Laccases (EC 1.10.3.2, benzenediol: oxygen oxidoreductase) play a key role in the enzymatic degradation of lignin and ionic liquids (ILs) have been used successfully to dissolve lignin. One limitation in lignin degradation using laccases is their low activity/resistance in the presence of ILs. In order to improve the resistance of laccase in IL, a directed evolution protocol based on the ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid))-screening assay in 96-well microtiter plate format was developed. 1-Ethyl-3-methylimidazolium ethylsulfate ([EMIM] [EtSO4]) can dissolve lignin efficiently and its anion does not inhibit laccase. The stability of the ABTS radical cation was not affected in the presence of [EMIM] [EtSO4]. Therefore, ([EMIM] [EtSO4]) is a suitable cosolvent for directed laccase evolution. Four laccases (lcc1_2005, lcc1_1997, lcc2 and CVLG1) from T. versicolor (Trametes versicolor) were expressed in Saccharomyces cerevisiae and finally lcc2 was selected as the starting point due to its superior resistance and activity in presence of [EMIM] [EtSO4]. After two rounds of directed evolution, the lcc2 variant M3 (Phe265Ser/Ala318Val) displayed about 4.5-fold higher activity than the lcc2 wild type (WT) in the presence of 15% (v/v) [EMIM] [EtSO4] and a 3.5-fold higher activity than lcc2 WT in buffer. The IC50 value of [EMIM] [EtSO4] towards M3 increases from 392 mM (lcc2 WT) to 497 mM.
A successful process to extract lipolytic enzymes based on an aqueous biphasic system (ABS), which uses both ionic liquids (ILs) and a high charge-density inorganic salt (K 2 CO 3), is proposed in this work. The activity of a model Thermomyces lanuginosus lipase (TlL) in some of the most common hydrophilic ILs, based on the 1-alkyl-3-methylimidazolium cation, combined with chloride, alkylsulfate, alkylsulfonate and acetate, was investigated. Several operating conditions influencing lipase activity and ABS formation were investigated. Parameters such as temperature, pH, deactivation kinetics and water content were evaluated in order to propose a viable extraction process. A deeper analysis in terms of enzyme deactivation kinetics was carried out, and the data were modelled through a series-type deactivation equation. ATR-FTIR studies aimed at identifying the TlL structure in selected ILs have also provided an insight into the enzyme deactivation behaviour.
The activity and stability of horseradish peroxidase (HRP) were investigated in a hydrophilic room temperature ionic liquid 1-butyl-3-methylimidazolium tetrafluroborate ([bmim][BF4]) by electrochemical methods. Although no detectable activity exhibited in anhydrous [bmim][BF4], HRP was active in the presence of a small amount of water (4.53%, v/v). And its activity can be improved by immobilization in agarose hydrogel. The immobilized HRP possesses excellent activity at 65 °C. It remained 80.2% of its initial activity after being immersed for 10.5 h in an aqueous mixture of [bmim][BF4] with some hydrogen peroxide (H2O2) under room temperature, implying extremely high stability. Moreover, the immobilized HRP was found to be very sensitive and stable in H2O-containing [bmim][BF4] for the detection of H2O2, with a wide linear range of 6.10 × 10−7 to 1.32 × 10−4 mol l−1 and low detection limit of 1.0 × 10−7 mol l−1.
The intensive use of water containing dyes by the textile industry, and consequently the contamination of soils and water, represents serious environmental concerns. Amongst the several processes applied in the treatment of textile effluents, biological-based processes, if designed to be cost-effective and ecofriendly, are promising alternatives to decolorize textile effluents. In this work we investigate and propose the novel use of ionic liquids (ILs) with surfactant characteristics to improve the degradation of the largely used and highly hydrophobic textile dye indigo carmine (IC) by laccase. An initial screening on the activity of laccase in aqueous solutions of twelve surfactant-based ILs from three different families, namely tetraalkylammonium- and imidazolium-based cationic surfactants and cholinium-based anionic surfactants, at different concentrations, was carried out. Significant improvements in the activity of laccase were observed with decyltrimethylammonium bromide, [N10111]Br, and 1-decyl-3-methylimidazolium chloride, [C10mim]Cl, at 75 mM (above the critical micellar concentration of each IL). These ILs were then investigated in aqueous solutions to simultaneously encapsulate laccase and IC for the in situ enzymatic biodegradation of the dye. The use of ILs remarkably increases the degradation rate of the dye and decolorization efficiency; a degradation efficiency of IC of 82% is attained in 0.5 h using aqueous solutions of [N10111]Br, whereas without IL only 6% of IC is degraded. Furthermore, 93% of the dye decolorization was achieved with [N10111]Br. The overall gathered results show that it is possible to significantly improve the degradation of hydrophobic dyes by enzymes using appropriate surfactant-based ILs, while foreseeing the use of the treated water by the same textile industries in new dyeing steps and thus contributing to a substantial decrease of the economic input and environmental footprint of these industries.
Monoclonal antibodies (mAbs) are of crucial interest for therapeutic purposes, particularly in vaccination and immunization, and in the treatment of life-threatening diseases. However, their downstream processing from the complex cell culture media in which they are produced still requires multiple steps, making mAbs extremely high-cost products. Therefore, the development of cost-effective, sustainable and biocompatible purification strategies for mAbs is in high demand to decrease the associated economic, environmental and health burdens. Herein, novel aqueous biphasic systems (ABS) composed of glycine–betaine analogue ionic liquids (AGB-ILs) and K2HPO4/KH2PO4 at pH 7.0, the respective three-phase partitioning (TPP) systems, and hybrid processes combined with ultrafiltration were investigated and compared in terms of performance as alternative strategies for the purification and recovery of anti-human interleukin-8 (anti-IL-8) mAbs, which are specific therapeutics in the treatment of inflammatory diseases, from Chinese Hamster Ovary (CHO) cell culture supernatants. With the studied ABS, mAbs preferentially partition to the IL-rich phase, with recovery yields up to 100% and purification factors up to 1.6. The best systems were optimized in what concerns the IL concentration, allowing to take advantage of IL-based three-phase partitioning approaches where a precipitate enriched in mAbs is obtained at the ABS interface, yielding 41.0% of IgG with a purification factor of 2.7 (purity of 60.9%). Hybrid processes combining the two previous techniques and an ultrafiltration step were finally applied, allowing the recovery of mAbs from the different fractions in an appropriate buffer solution for further biopharmaceutical formulations, while allowing the simultaneous IL removal and reuse. The best results were obtained with the hybrid process combining TPP and ultrafiltration, allowing to obtain mAbs with a purity higher than 60%. The recyclability of the IL was additionally demonstrated, revealing no losses in the purification and recovery performance of these systems for mAbs. The biological activity of anti-IL-8 mAbs is maintained after the several purification and recovery steps, indicating that the novel ABS, three-phase partitioning and hybrid processes comprising AGB-ILs are promising and sustainable strategies in mAbs downstream processing.
Laccase was immobilized on a glassy carbon electrode layered with multi-walled carbon nanotubes using a film of botryosphaeran, a fungal (1→3)(1→6)-β-D-glucan. This novel biosensing platform was characterized by electrochemical impedance spectroscopy and scanning electron microscopy, and applied for the determination
of dopamine. Experimental variables such as enzyme concentration, pH value and operational parameters of the electroanalytical technique were optimized. Using square-wave voltammetry, there was a linear dependence of peak current and dopamine concentration within the range of 2.99–38.5 μmol L−1 with a limit of detection of
0.127 μmol L−1. The biosensor was successfully applied in the determination of dopamine in pharmaceutical injection and synthetic biological samples, and presented good selectivity even in the presence of uric acid and ascorbic acid, as well as other phenolic compounds. The different aspects regarding the operational stability of the laccase biosensor were evaluated, demonstrating good intra-day and inter-day repeatability, and long-storage stability. Furthermore, this biosensor was evaluated in the indirect determination of spironolactone by using the analytical signal of dopamine, presenting a limit of detection of 0.94 μmol L−1. The results obtained in the analysis of spironolactone in commercial pharmaceutical samples were satisfactory.
Laccase was immobilized on a glassy carbon electrode layered with multi-walled carbon nanotubes using a film of botryosphaeran, a fungal (1→3)(1→6)-β-D-glucan. This novel biosensing platform was characterized by electrochemical impedance spectroscopy and scanning electron microscopy, and applied for the determination of dopamine. Experimental variables such as enzyme concentration, pH value and operational parameters of the electroanalytical technique were optimized. Using square-wave voltammetry, there was a linear dependence of peak current and dopamine concentration within the range of 2.99–38.5 μmol L−1 with a limit of detection of 0.127 μmol L−1. The biosensor was successfully applied in the determination of dopamine in pharmaceutical
injection and synthetic biological samples, and presented good selectivity even in the presence of uric acid and ascorbic acid, as well as other phenolic compounds. The different aspects regarding the operational stability of the laccase biosensor were evaluated, demonstrating good intra-day and inter-day repeatability, and long-storage
stability. Furthermore, this biosensor was evaluated in the indirect determination of spironolactone by using the analytical signal of dopamine, presenting a limit of detection of 0.94 μmol L−1. The results obtained in the analysis of spironolactone in commercial pharmaceutical samples were satisfactory.
The research on alternative solvents and co-solvents is a relevant aspect when envisioning the improvement of biocatalytic reactions. Among these solvents and co-solvents, deep eutectic solvents (DES) may be considered as customizable new reaction media for biocatalysis. Accordingly, in this work, sixteen DES aqueous solutions, as well as of the individual DES components at the same conditions have been investigated in laccase-catalyzed reactions. Cholinium- and betaine-based DES formed with polyols at different molar ratio and concentrations were evaluated. The results reported show that in presence of most DES the laccase activity is preserved and, with a particular DES, enhanced up to 200%. Molecular docking studies demonstrated that while most DES components establish hydrogen-bonds with the enzyme amino acids, those that establish stronger interactions with the enzyme (expressed by absolute values of docking affinity energies) lead to an enhanced laccase activity. Finally, the laccase stability was evaluated in additional tests under extreme storage temperatures (60 ºC and -80 ºC). Although no significant protection to high temperatures was afforded by DES, an enhanced laccase activity when stored at low temperatures was found, at least up to 20 days. Combining experimental results and molecular docking this work shows that DES can be designed as co-solvents to improve biocatalytic reactions.
Coloured and conductive fabrics were obtained through “in situ” laccase polymerization of catechol and p-phenylenediamine under high-pressure homogenization. Both monomers, catechol and p-phenylenediamine, were polymerized by different laccase forms, namely native, PEGylated and Epoxy-PEGylated. All the catalysts were placed inside a textile fabric bag which served simultaneously as enzyme support and as substrate for coating with the newly produced polymers. The PEGylated laccase forms gave rise to a higher amount of oligomers/polymers and higher colouration level of polyethylene terephthalate (PET), cotton and wool fabrics compared to native laccase. Both functional polymers were able to confer conductivity to the substrates however in a different extent. Fabrics coated with poly(p-phenylenediamine) present higher conductivity, rather due to its polymerized structure than to the amount of polymer produced by enzyme catalysis. Herein a green approach was presented to produce polyphenols with increased fixation onto different textile substrates. These substrates reach high levels of colouration and good fastness behaviour after washing.
The absence of gluten in gluten-free flours presents a challenge to their application in baking. Enzymatic modification of the protein and polysaccharides may result in a network that mimics gluten. In the current study, the effects of laccase on the rheological properties of amadumbe dough were investigated. Thiol and total phenolic contents of dough decreased by up to 28% and 93%, respectively, as laccase activity was increased (0–3 U/g flour). Both G′ and G″ of laccase-treated dough increased significantly due to laccase-catalysed cross-linking of proteins and polysaccharides esterified with phenolics, as demonstrated by relevant model reactions. Tan δ decreased with increase in laccase activity indicating an increase in the elastic character of the dough. The improvement in dough viscoelasticity may enable the retention of adequate carbon dioxide during proofing and production of more acceptable gluten-free bread.
Background:
The paper and pulp industry is a capital and resource-intensive industry that contributes to ecosystem toxicity and affects human beings.
Objective:
The study aimed to appraise the potential of xylanases, laccases and manganese peroxidase for the bio-bleaching of paper pulp and to highlight the role of these enzymes as a promising substitute for chlorine-based chemical methods in the bleaching process.
Methods:
The ligninolytic enzymes including xylanase, laccase and manganese peroxidase isolated from white rot fungi were used for pre-bleaching and bleaching of oven-dried wheat straw pulp.
Results:
During the sequential enzymatic treatment of oven-dried pulp the brightness was improved and kappa number was reduced by 3.1% and 3.1 points respectively after xylanase treatment, 0.3% and 0.4 points after laccase treatment and 3% and 0.2 points after MnP treatment. During separate treatment of pulp samples with individual enzymes, brightness and kappa number improved by 8% and 3 points respectively after xylanase treatment, by 5% and 1.7 points after laccase treatment and 5% and 1.8 points after treatment with MnP. During subsequent treatment with 4% sodium hypochlorite, the brightness was further improved by 27.9 % for xylanase treated pulp and 29% for the laccase and MnP treated pulp. The xylanase was found most efficient in comparison to laccase and MnP in reduction of kappa number and improvement of brightness.
Conclusion:
These results clearly indicate the role of laccase, MnP and xylanase from white rot fungi as effective bio-bleaching agents. Therefore, these enzymes can facilitate the bleaching process without threat to environment.
Ionic liquids (ILs) have been proposed as promising media for the extraction and separation of bioactive compounds from the most diverse origins. This critical review offers a compilation on the main results achieved by the use of ionic-liquid-based processes in the extraction and separation/purification of a large range of bioactive compounds (including small organic extractable compounds from biomass, lipids, and other hydrophobic compounds, proteins, amino acids, nucleic acids, and pharmaceuticals). ILs have been studied as solvents, cosolvents, cosurfactants, electrolytes, and adjuvants, as well as used in the creation of IL-supported materials for separation purposes. The IL-based processes hitherto reported, such as IL-based solid-liquid extractions, IL-based liquid-liquid extractions, IL-modified materials, and IL-based crystallization approaches, are here reviewed and compared in terms of extraction and separation performance. The key accomplishments and future challenges to the field are discussed, with particular emphasis on the major lacunas found within the IL community dedicated to separation processes and by suggesting some steps to overcome the current limitations.
Based on the particular ability of aliphatic amino acids to form aqueous biphasic systems with ionic liquids, it is here shown how these systems can be used to selectively and efficiently separate mixtures of aliphatic and aromatic amino acids usually present in protein hydrolysates or fermentation broths.
Room temperature ionic liquids (RTILs) and biomolecules are both paradigmatic classes of organic molecules, each consisting of a prodigious number of distinct chemical species, organized into large families of homologous compounds. Their combination is set to open new avenues for discoveries and for applications in biochemistry, biomedicine and pharmacology, food science, and nanotechnology. We provide a survey of past and current investigations of the chemical physics properties of systems made of RTILs and biomolecules, focusing on the most microscopic scales of their structure and dynamics. The primary goal of our discussion is to identify the basic principles able to organize and rationalize the vast variety of properties and phenomena displayed by these systems. We consider in turn RTILs combinations with phospholipids, with proteins and peptides, with nucleic acids (mainly DNA) and with carbohydrates from simple sugars to large polysaccharides. These basic components have a clear electrostatic signature, and Coulomb interactions represent the first ordering principle. However, because of size and complexity of both RTILs and biomolecules, dispersion interactions, steric effects, and hydrogen bonding play a secondary but certainly non-negligible role that is reflected in the sensitive dependence of RTIL/biomolecule properties on the RTIL choice. Our overview of the available results highlights both the need and the difficulty of devising general approaches able to predict properties of at least extended families of RTIL/biomolecules combinations, without having to consider them one by one in turn.
Aiming at gathering further evidence about the molecular-level mechanisms of the polymer–polymer aqueous biphasic systems (ABSs), systematic studies on the vapor–liquid and liquid–liquid equilibria of several ternary aqueous solutions of two water soluble polymers capable or not of inducing phase separation were performed at 298.15 K. It was found that the hydrophilicity of the investigated polymers follows the order PEGs ≈ PEGDMEs > PVP >> PPG400 and the aqueous solutions of PPG400 form ABS with PEG, PEGDME, and PVP however other investigated systems don't form ABS. The ability of PPG400-polymer solutions to form ABS increases by increasing temperature and polymer molar mass. It was also found that, the constant water activity lines of the ternary systems which capable and not of inducing phase separation respectively have the concave (or plane) and convex slopes. Therefore, from the slopes of the isopiestic constant water activity lines one can predict the phase forming abilities of the corresponding systems.
Enzymes are the most proficient catalysts, offering much more competitive processes compared to chemical catalysts. The number of industrial applications for enzymes has exploded in recent years, mainly owing to advances in protein engineering technology and environmental and economic necessities. Herein, we review recent progress in enzyme biocatalysis, and discuss the trends and strategies that are leading to broader industrial enzyme applications. The challenges and opportunities in developing biocatalytic processes are also discussed.
Copyright © 2015. Published by Elsevier Inc.
In the past few years, ionic liquid-based aqueous biphasic systems have become the subject of considerable interest as a promising technique for the extraction and purification of several macro/biomolecules. Aiming at developing guidelines for more benign and efficient extraction processes, phase diagrams for aqueous biphasic systems composed of ionic liquids and inorganic/organic salts are here reported. Several combinations of ionic liquid families (imidazolium, pyridinium, phosphonium, quaternary ammonium and cholinium) and salts [ potassium phosphate buffer (KH2PO4/K2HPO4 at pH 7), potassium citrate buffer (C6H5K3O7/C6H8O7 at pH 5, 6, 7 and 8) and potassium carbonate (K2CO3 at pH similar to 13)] were evaluated to highlight the influence of the ionic liquid structure (cation core, anion and alkyl chain length), the pH and the salt nature on the formation of aqueous biphasic systems. The binodal curves and respective tie-lines reported for these systems were experimentally determined at (298 +/- 1) K. In general, the ability to promote the aqueous biphasic systems formation increases with the pH and alkyl chain length. While the influence of the cation core and anion nature of the ionic liquids on their ability to form aqueous biphasic systems closely correlates with ionic liquids capacity to be hydrated by water, the effect of the different salts depends of the ionic liquid nature and salt valency.
Laccase, a blue multicopper oxidase, has recently received considerable attention because of its usefulness in oxidizing phenolic and non‐phenolic compounds, as well as its suitability for organic synthesis, environmental pollutant treatment, and other biotechnological applications. This review covers recent studies on the structural properties, occurrence, reaction mechnisms, redox mediators of laccases and their application in organic synthesis procedures, such as dimerization, polymerization, oxidation, and amination. We also present a brief discussion on laccase activity in non‐aqueous media. Given that the development of green protocols for the synthesis of pure compounds is one of the main goals of sustainable chemistry, the exploitation of laccases is expected to remain one of the most popular directions in future biocatalysis research.
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This work reveals, for the first time, that polymer-ionic-liquid-based aqueous biphasic systems (ABS) exhibit a much wider hydrophilic-hydrophobic range than conventional systems reported to date. Three probe dyes were used to demonstrate that either the polymer-rich or the ionic-liquid-rich layer can serve as the most hydrophobic phase. It was found that the phase polarities can be easily tuned by the choice of an appropriate ionic liquid.
In this work, the potential of several aqueous two-phase systems (ATPS) for laccase recovery was evaluated. For this purpose, different types of ATPS were prepared and the partition coefficient of pure commercial laccase was determined in each one, at 23 °C. A total of 21 ATPS were investigated: 12 composed by a polymer and a salt and 9 composed by two different polymers. For polymer–salt ATPS, different compositions of the same biphasic system were also studied. Laccase partition coefficients (K) obtained were used to conclude about ATPS effectiveness for this enzyme recovery. According to the results, it was found that UCON-phosphate salts (K ≤ 0.604), PEG-Li2SO4 (K = 2.081) and PES-dextran (K = 1.911) ATPS can be interesting options for laccase extraction, with laccase partitioning toward opposite directions. However, the most effective ATPS for laccase extraction was UCON-K2HPO4, presenting K values from 0.272 up to 0.306. UCON is a thermo-separating polymer which facilitates its recovery and reutilization. Additionally, for the first time, laccase stability in different ATPS was investigated by incubating the enzyme in each equilibrium phase during a week, at room temperature. The results obtained showed that UCON-sulfate salts, UCON-NaH2PO4 and UCON-KH2PO4 ATPS are not recommended for laccase recovery since a high loss of activity was observed: approximately 88% for sulphates and 80% for both dihydrogen phosphates. The best stability results were obtained with PEG-sulfate salts. For these ATPS, laccase stability remained similar or improved over time.
Albeit ionic-liquid-based aqueous biphasic systems (ABS) have been largely explored as liquid–liquid extractive approaches for a large array of (bio)molecules, the application of biodegradable and nontoxic salts as phase constituents of these systems has been seldom investigated. In this work 15 ionic liquids were evaluated toward their ability to form ABS in the presence of a common biodegradable organic salt: potassium citrate. The ternary phase diagrams, tie-lines, and respective tie-line lengths, were determined at 25 °C. The gathered data allowed the evaluation of the effects of the ionic liquid cation core, of the cation side alkyl chain length, and of the anion nature, to form two-phase systems. It is shown that the ionic liquids aptitude to undergo liquid–liquid demixing is mainly controlled by their hydrophobicity. The large differences observed between the phase diagrams behavior suggest the possibility of tailoring the aqueous phases’ polarities for a specific extraction. Therefore, the partitioning of a hydrophobic amino acid produced by bacteria fermentation, l-tryptophan, was also addressed aiming at exploring the applicability of the proposed systems in the biotechnology field. Single-step extraction efficiencies of l-tryptophan for the ionic-liquid-rich phase range between 72% and 99%.
The ability of alternative aqueous biphasic systems (ABS) composed of polyethylene glycol and imidazolium-based ionic liquids (ILs) to selectively separate similar biomolecules was here investigated. The preferential partitioning of three alkaloids (caffeine, xanthine and nicotine) was addressed by means of their partition coefficients and selectivity values. Aiming at optimizing the selectivity of the studied ABS, factors such as the chemical structure of the IL (cation side alkyl chain length, number of aliphatic moieties or their functionalization, and the anion nature) and the temperature of equilibrium were experimentally addressed. In almost all examples it was observed a preferential concentration of caffeine in the polymer-rich phase whereas nicotine and xanthine preferentially migrate to the (opposite) IL-rich phase. In spite of the alkaloids chemical similarities, the studied ABS presented selectivity values of xanthine vs. caffeine as large as 19. The gathered results show that polymer-IL-based ABS allow the selective separation of similar structures by an adequate manipulation of the IL chemical structure and temperature of equilibrium, and can be envisaged as potential platforms to be applied in countercurrent chromatography.
As a group of novel green solvents, ionic liquids have attracted extensive attention and gained popularity in various applications including protein assays. Ionic liquids are not only an excellent reaction medium to replace the conventional organic solvents, but are also an efficient participant to achieve better performances. The aim of the present mini-review is to illustrate the state-of-the-art progress of implementing ionic liquids in protein assays, focusing on the investigations of protein stability/activity, proteinextraction and isolation/purification, proteincrystallization, separation of protein species and their detections.
Laccases from different sources have been used, for the first time, for the selective oxidation of the stilbenic phytoalexin trans-resveratrol (3,5,4′-trihydroxystilbene, 1a) on a preparative scale. Specifically, the enzymes from Myceliophtora thermophyla and from Trametes pubescens gave the dehydrodimer 2a in 31 and 18% isolated yields, respectively. These results compare favorably with the reported data for the chemically catalyzed dimerization of 1a (18% yields). The antioxidant properties of 2a have also been investigated.
A series of choline carboxylate ionic liquids (ILs) have been synthesized by neutralization of choline hydroxide solution with acetic, propanoic, butanoic, pivalic, and hexanoic acids. The salts so obtained were characterized by NMR spectroscopy, thermal methods, and elemental analysis. Key physical properties (density, viscosity, and refractive index) were measured for the propanoate, butanoate, and hexanoate salts at temperatures from (293.15 to 353.15) K. The densities were used to estimate the molecular volumes, standard entropies, crystal lattice energies, and thermal expansion coefficients. All five choline carboxylates were found to have cytotoxicities (IC50 values) above 10 mM toward the human breast cancer cell line, MCF-7, indicating they are much less toxic than common imidazolium-based ILs.
BACKGROUND: The use of enzymes has attracted much interest for the decolourization of dyes from wastewater as an alternative to conventional treatments, which have limitations. Laccase can be used to decolourize dyes and its substrate range can be extended by inclusion of mediators.
RESULTS: Sequential decolourization of reactive dyes was carried out by a laccase mediator system using as mediators violuric acid and 1‐hydroxybenzotriazole. Violuric acid resulted in a high level of decolourization on the first and second cycles for reactive blue 114 (≥95%), reactive yellow 15 and reactive red 239 (≥80%), while for reactive black 5 a slightly lower value was observed (70%) on the second cycle. The degree of reactive blue decolourization remained at 90% after the third cycle, and at about 60% after seven cycles. With the other dyes decolourization decreased to 50–70% on the third cycle and further up to the final cycle (lower than 20%). When using 1‐hydroxybenzotriazole as mediator a slight decrease in decolourization efficiency was obtained. Correlation between dye oxidation peak potential and dye degradation after 10 min reaction was obtained for both mediators.
CONCLUSIONS: These results show that sequencing batch decolourization by laccase mediator system is a useful methodology for treating textile dying wastewaters with a high potential for application at industrial level. Copyright © 2008 Society of Chemical Industry
BACKGROUND: In the textile industry, large quantities of intensely coloured and toxic effluents are released, causing serious environmental pollution. Several biotechnological approaches have been suggested to eliminate this pollution source in an eco-efficient manner. Laccase can be used to decolourise dyes and its substrate range can be extended by inclusion of a mediator.
RESULTS: A screening using several laccase mediators (2,2-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), 1-hydroxybenzotriazole (HBT), N-hydroxyacetanilide (NHA), polioxometalates, violuric acid (VA) and (2,2,6,6-tetramethylpiperidin-1-yloxy) (TEMPO)) was performed on the degradation of six reactive textile dyes. ABTS was the most effective mediator leading to higher decolourisation. The efficiency of ABTS depends on the type of dye, pH, temperature and dye concentration. The optimum temperature and pH values were 35 °C and 5.0, respectively, for maximum decolourisation (above 70%) of reactive black 5, reactive blue 114 and reactive yellow 15. For reactive red 239 the optimum conditions were found to be a temperature of 40 °C and pH of 4.5 (above 56% decolourisation). ABTS has no effect at low concentrations, except for reactive blue 114, where it resulted in the best decolourisation (93%). A comparison of decolourisation based on the percentage absorbance reduction at the maximum absorbance wavelength of each dye and throughout the visible spectrum was made.
CONCLUSIONS: These results suggest that the laccase mediator system could be used to treat textile dying wastewaters. Copyright
Separation and Purification Technology j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / s e p p u r Partitioning and recovery of Canavalia brasiliensis lectin by aqueous two-phase systems using design of experiments methodology: Canavalia brasilienses lectin Aqueous two-phase systems (ATPS) Design of experiments (DoE) Optimization Downstream processing a b s t r a c t The performance of aqueous two-phase systems (ATPS) composed by poly(ethylene glycol) (PEG)-and different salts (sodium citrate, potassium phosphate, sodium sulfate and ammonium sulfate) has been evaluated and compared for the selective isolation of Canavalia brasilienses lectin (ConBr) from crude extracts of C. brasilienses seeds. Among the systems studied, PEG-phosphate was the most efficient for the purification of ConBr. A central composite design was applied to study the effects of different factors such as, PEG concentration, phosphate buffer concentration, sodium chloride concentration and pH on ConBr extraction and to optimize its isolation from a plant extract. ATPS comprising PEG 600–phosphate buffer provided a means for the recovery of proteins from crude extracts of C. brasilienses seeds. The best conditions of purification were achieved using an ATPS composed by 16.5% (w/w) PEG 600, 15.0% (w/w) phosphate pH 7.5, 4.5% (w/w) NaCl. The maximum percentage yield of protein extracted was about 100% with a final purity of 73.04%.
A method to measure laccase and lignin peroxidase (LiP) activity at 408 nm (402–410 nm) using 1-hydroxybenzotriazole (HBT) was developed. The assay can be performed either as a kinetic measurement or as a stopped reaction using 5 mM Na-azide which improves the spectrum. Only white-rot fungal laccases and LiP were found to oxidize HBT to give shoulders or peaks at 402-410 nm. Phanerochaete and Phlebia manganese peroxidases did not give absorbance increase at 402–410 nm. Rapid Science Ltd. 1998
During the past decade, ionic-liquid-based Aqueous Biphasic Systems (ABS) have been the focus of a significant amount of research. Based on a compilation and analysis of the data hitherto reported, this critical review provides a judicious assessment of the available literature on the subject. We evaluate the quality of the data and establish the main drawbacks found in the literature. We discuss the main issues which govern the phase behaviour of ionic-liquid-based ABS, and we highlight future challenges to the field. In particular, the effect of the ionic liquid structure and the various types of salting-out agents (inorganic or organic salts, amino acids and carbohydrates) on the phase equilibria of ABS is discussed, as well as the influence of secondary parameters such as temperature and pH. More recent approaches using ionic liquids as additives or as replacements for common salts in polymer-based ABS are also presented and discussed to emphasize the expanding number of aqueous two-phase systems that can actually be obtained. Finally, we address two of the main applications of ionic liquid-based ABS: extraction of biomolecules and other added-value compounds, and their use as alternative approaches for removing and recovering ionic liquids from aqueous media.
Aqueous solutions that undergo sol-to-gel transition as the temperature increases have been extensively studied during the last decade. The material can be designed by controlling the hydrophilic and hydrophobic balance of the material. Basically, the molecular weight of the hydrophilic block and hydrophobic block of a compound should be fine-tuned from the synthetic point of view. In addition, stereochemistry, microsequence, topology, and nanostructures of the compound also affect the transition temperature, gel window, phase diagram, and modulus of the gel. From a practical point of view, biodegradability, biocompatibility, and interactions between the material and drug or cell should be considered in designing a thermogelling material. The interactions are particularly important in that they control drug release profile and initial burst release of the drug in the drug delivery system, and affect cell proliferation, differentiation, and biomarker expression in three-dimensional cell culture and tissue engineering application. This review provides an in-depth summary of the recent progress of thermogelling systems including polymers, low molecular compounds, and nanoemulsions. Their biomedical applications were also comparatively discussed. In addition, perspectives on future material design of a new thermogelling material and its application are suggested.
Improvement of laccase production by Trametes versicolor was made by employing different operational strategies. In the cell growth medium, various glucose concentrations were compared for improving laccase production. A clear and significant stimulation of enzyme production under carbon limitation was obtained. Copper, 2,5-xylidine, and a phenolic mixture were also used as laccase inducers. A cooperative effect between the inducers on laccase production was identified. Mixtures of inducers produced higher laccase activities, reaching values of 5500 U dm−3. Further productivity enhancement can be obtained using the inducers along with the carbon limitation strategy. It is shown that low laccase concentrations are obtained by a primary metabolism of T versicolor, and that phenolic compounds and carbon limitation induce a secondary metabolism, providing higher laccase concentrations. A mathematical model for laccase production based on a direct experimental measure of biomass, along with substrate consumption and enzymatic activity over time is proposed for non-homogeneous fermentations of T versicolor. Copyright © 2005 Society of Chemical Industry
Laccase isolated from Trametes versicolor was immobilized in polyacrylamide gel and used for the conversion of 4-methyl-3-hydroxyanthranilic acid to 2-amino-4,6-dimethyl-3-phenoxazinone-1,9-carboxylic acid (actinocin) which is a phenoxazinone chromophore occuring in actinomycins. The reaction proceeded successfully in aqueous media and in 60% acetonitrile.