Oxidative functionalisation of lignin by layer-by-layer immobilised laccases and laccase microcapsules
Graphical abstract
Laccase was either supported onto alumina particles or physically entrapped inside microcapsules. In both cases, the enzyme was protected by coating with oppositely charged polyelectrolytes by means of the layer-by-layer technique. The activities of these laccase particles and microcapsules were studied on softwood milled wood and kraft residual lignins, in the presence and absence of different mediators. Compared with the native enzyme, the laccase particles and microcapsules were found to be more reactive, showing higher conversions.
Introduction
Laccase, EC 1.10.3.2, p-diphenol:dioxygen oxidoreductase, is a part of a larger group of enzymes termed the multicopper oxidases, an important class found in many organisms, including plants, fungi, bacteria and humans [1], [2]. This enzyme catalyses the reduction of molecular oxygen by various organic compounds to water without the step of hydrogen peroxide formation [3]. Its active site structure is highly conserved and consists of four copper atoms, classically divided into three types based upon their different spectral features: type 1 (T1) or blue copper; type 2 (T2) or normal copper; type 3 (T3) or coupled binuclear copper centres. The T2 and T3 sites form a trinuclear cluster onto which dioxygen is reduced [1]. The function of the T1 site is intramolecular electron transfer, shuttling electrons from the substrate to the trinuclear cluster [4].
The industrial interest in the application of this enzyme lies in the low substrate specificity, high value of catalytic constants, use of air oxygen as the primary oxidant and high thermal resistance [5], [6], [7]. However, its efficiency in bleaching pulps is poor, while when used in the presence of radical (natural or synthetic) chemical mediators, which are low molecular weight phenols, or N-hydroxy derivatives such as 1-hydroxybenzotriazole (HBT), a universal increase in laccase reactivity has been shown [5], [6], [7].
Extensive efforts have been made to understand the mechanism of the laccase-mediator system [5], [6], [7]. Oxidation mediators are able to increase the enzyme reactivity by the modification of the reaction mechanism from the one electron oxidation to a hydrogen atom abstraction process [8]. In recent years, the laccase-mediator system has been used for many different biotechnological applications such as pulp delignification, oxidation of organic pollutants and development of biosensors or biofuel cells [9], [10]. Nevertheless, the industrial application of laccases is limited since their stability and catalytic activity are considerably affected by a wide variety of environmental conditions [11]. One approach to overcome these constraints is the use of immobilised laccases. In fact, immobilised enzymes allow easy recovery of products, multiple reuse of the biocatalyst, plug flow processes, rapid termination of reactions and a greater variety of bioreactor designs. Immobilised laccases have been extensively reported in the archival scientific literature [12], [13], [14], [15], [16]. In principle, there are several strategies that can be used for the immobilisation of laccases on a solid support. Among them, chemical immobilisation, where covalent bonds are formed, and physical immobilisation, where weak interactions between the support and the enzyme exist. These have been applied with varying degrees of success, mainly due to denaturation processes and restricted permeability of the substrate [12], [13], [14], [15], [16].
The layer-by-layer (LbL) technique, first introduced by Decher et al. allows multilayer assemblies to be obtained by the alternate deposition of polycations and polyanions in a cyclic way on a solid surface [17], [18]. Encapsulation is an effective means for the immobilisation of enzymes since the polyelectrolyte films have the ability to protect proteins from high-molecular-weight denaturing agents or bacteria and to allow regulation of the permeability towards small substrates, which can enter the multilayer and react with the catalytic site [17]. More specifically, immobilisation of laccases based on the LbL technique and the LbL stabilisation has recently been reported [18], [19], [20]. There are also reports on the preparation of laccase immobilised in microcapsules for sensor development [21], [22].
To date, the continuous reduction of fossil fuel feedstocks as well as the requirement of environmentally friendly processes have prompted the development of novel processes based on alternative renewable materials. In this context, lignin, the second most abundant natural polymer on the planet, represents an invaluable resource. This product is a three-dimensional phenylpropanoid polyphenol mainly linked by arylglycerol ether bonds between monomeric phenolic units, most of which are not readily hydrolysable even under severe experimental conditions [23]. Oxidative enzymes, such as laccases, are potential tools for the selective modification of lignin as well as for the simplification of its structure, but the industrial application is still limited to processes with free enzymes. Moreover, the mechanism of action of oxidation of lignin with these enzymes is still only partially understood [24].
In this regard, we describe here the design and development of novel immobilised laccases into polystyrene sulphonate and polyallylamine microcapsules based on the LbL technology as compared to chemically immobilised laccases coated by the same polyelectrolytes, the evaluation of their reaction pathway with respect to the native enzyme and their use in the oxidative functionalisation of lignin. More specifically, we applied the LbL procedure to improve the stability of laccase, to achieve higher resistance in the lignin oxidation reaction conditions and to develop processes suitable for eventual industrial scale-up. By means of quantitative 31P NMR spectroscopy, a detailed study of the chemical selectivity of differently immobilised laccases was performed.
Section snippets
Reagents
All solvents and chemicals were of analytical grade and high purity. Laccase from Trametes versicolor, HBT, violuric acid (VA), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), poly(allylamine hydrochloride) (PAH, Mw = 70,000), poly(sodium 4-styrenesulphonate) (PSS, Mw = 70,000), alumina (Al2O3) spherical pellets (3 mm diameter), γ-aminopropyltriethoxysilane, glutaraldehyde and 2-chloro-4,4′,5,5′-tetramethyl-1,3,2-dioxaphospholane were purchased from Sigma–Aldrich The polyelectrolyte
Preparation of cLbL-laccase
We applied the LbL technology in two different biocatalytic systems. In the first case, laccase from T. versicolor was chemically bonded onto functionalised alumina pellets, which were selected as the carrier material due to their known mechanical resistance at high values of pH and reaction temperature [40]. The yield of immobilised laccase was evaluated by analysing the residual enzymatic activity in the waste waters after the reaction of the enzyme with activated alumina particles and
Conclusion
The results obtained in this study show that cLbL-laccase and mLbL-laccase are more effective in the oxidation of MWL and RKL than native laccase. The highest reactivity of immobilised laccases might be explained by the stabilisation effect of coating polyelectrolytes towards enzyme deactivation processes. Moreover, kinetic barriers for the approach of substrate to the active site of the enzyme are not operative. The strategy used for the laccase immobilisation is also an efficient tool to tune
Acknowledgements
The European Cooperation in the field of Scientific and Technical Research (COST Action FP0602, Biotechnology for Lignocellulose Biorefineries) is acknowledged.
Dr. Luciano Pilloni and Dr. Marzia Pentimalli (ENEA FIM MATTEC CR Casaccia, Rome, Italy) are gratefully acknowledged for their skillful help in SEM studies.
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