Novel multienzyme oxidative biocatalyst for lignin bioprocessing
Introduction
Today the rising energy consumption and the depletion of fossil fuel feedstocks have focused the attention on the use of alternative renewable materials and on the development of environmentally friendly processes that operate in mild reaction conditions.
Lignin is the second most abundant organic polymer in plant kingdom and constitutes up to 30% of wood.1 It constitutes to date the bottleneck to the development of integrated biorefinery since it is the residue of modern saccharification processes. Current bioethanol production from wood originate about 500 g of lignin each liter of bioethanol. From this viewpoint the development of processes of lignin upgrade through oxidative depolymerization or functionalization is mandatory.2, 3, 4
In Nature the selective oxidation of lignin is carried out by white-rot basidiomycetes fungi that produce a pool of extracellular ligninolytic enzymes such as laccases and peroxidases.5, 6 In particular laccases can easily oxidise phenolic groups7 and in presence of radical mediator, such as 1-hydroxybenzotriazole, their reactivity can be extended towards other functional groups as phenyl–aryl ethers.8 Mn-peroxidase and lignin peroxidases are able to oxidize lignin at the phenolic and non-phenolic aryl–ether positions respectively.9, 10, 11, 12, 13, 14, 15, 16
Laccases and peroxidases constitute an interesting tool for the development of alternative oxidative processes due to their low substrate specificity and relatively wide pH of action. In the last years both laccases and peroxidases have been used in several biotechnological applications such as oxidation of organic pollutants, pulp delignification, bleaching and development of biosensors or biofuel cells. Unfortunately, the exploitation of their potentiality is prevented, especially in the case of peroxidases, by their low stability.17
There are a number of constraints in the use of the enzymes; the common perception is that enzymes are sensitive, unstable and have to be used in water, features that are not ideal for a catalyst and undesirable in most syntheses.12
Several approaches have been proposed to overcome these limitations; among them immobilization is generally considered favorable for industrial scale applications since it allows for continuous processes.13 The basic requirement for the development of economically sustainable enzymatic processes are the possible recycle of the catalyst and a high stability of the enzyme. As such a number of different immobilization techniques have been developed.17, 18, 19
The layer-by-layer (LbL) adsorption technique, introduced by Decher et al.20 is a general and versatile tool for the controlled fabrication of multimaterial surface coatings on a large variety of surfaces.21 By means of this technique the construction of multilayer films is possible by the consecutive deposition of alternatively charged polyelectrolytes on a solid surface.21 The LbL technique has been demonstrated to be an effective means for the immobilization of enzymes.22 In fact, polyelectrolyte coatings have the ability to protect immobilized proteins from high-molecular-weight denaturing agents or bacteria and allow regulation of the permeability towards small substrates, which can enter and leave the protecting layers to react with the biomolecules in the interior.23
Another key aspect of enzyme immobilization is the possibility to perform multi-enzyme immobilization with the development of multi-enzyme or chemoenzymatic cascade processes.24
Biocatalysis is becoming a transformational technology for chemical synthesis as a result of recent advances in large-scale DNA sequencing, structural biology, protein expression, high throughput screening, and enzyme evolution technologies. To truly impact chemical synthesis at the industrial scale, enzyme discovery, biocatalyst optimization, process design and development must be integrated in order to deliver cost-effective and green chemistry solutions for processes. Domino or cascade reactions involve the transformation of materials through several non-separable steps in a concurrent fashion, which often proceed via highly reactive intermediates. These processes show a remarkable synthetic advantage despite the fact that the cascade is proceeding through one (or more) highly unstable intermediate(s), which are prone to decomposition reactions, the final product can often be isolated in good yields, because undesired side reactions of the reactive intermediate are largely avoided since the intermediate is transformed in the same instant as it appears.25
Recently, significant efforts have been made to develop organo-catalytic cascade reactions with the objective to mimic the biosynthetic strategy, but to date the innovative potential of biocatalysis by promoting the multistep catalytic concept by multienzyme systems has not been fully explored.
Most of the multienzyme systems synthesized to date were targeted towards sensors development26, 27, 28, 29, 30, 31, 32, 33 however, examples dealing with preparative cascade processes have been described.34, 35, 36 Many immobilised HRP and laccase have been reported in literature.37, 38, 39, 40, 41, 42, 43, 44, 45, 46
Recently both laccase47, 48 and horseradish peroxidase49 were immobilised onto alumina supports and coated by the LbL technique with polyelectrolytes. The stability of such systems was found significantly enhanced upon coating. As a consequence the reactivity toward lignin oxidation was found increased, probably due to the enhanced stability.
In this work, novel multienzyme biocatalysts were developed for bioprocessing applications.
More specifically, the present study describes a new process for the co-immobilization of oxidative enzymes by cross linking and layer by layer coating and their application to the development of an oxidative cascade process on lignin. The efficiency and specificity of the new multienzyme system, the occurrence of a synergy of the co-immobilizsed enzymes, the lignin oxidation pathway and the nature of the structural modifications occurred in treated lignins have been investigated in detail.
Section snippets
Enzymes immobilization and coating
Laccase and HRP were immobilized onto 2 mm alumina particles suitably silanized and activated by glutaraldehyde treatment. The two enzymes were both singularly and co-immobilized in order to subsequently point out the possibility of different behavior of the LbL multienzyme biocatalyst from the separately immobilized enzymes (Fig. 1). At pH 6 the enzymes are both negatively charged; it was thus possible to coat them by use of a first layer of polyallyl amine hydrochloride (PAA). Overall three
Conclusions
Chemical co-immobilization of laccase and HRP onto alumina particles followed by layer-by-layer coating with alternatively charged polyelectrolytes was found to be a valuable approach for the design and synthesis of a novel LbL-multienzyme oxidative biocatalyst. The co-immobilized enzymes maintained their activity and the LbL coating improved enzyme stability. The oxidative cascade processes were studied on wheat straw lignin. It was thus possible to draw a detailed picture of soluble and
Reagents
All solvents and chemicals were of analytical grade and high purity. Laccase from Trametes versicolor, 1-hydroxybenzotriazole (HBT), Horserhadish Peroxidase from Armoracia rusticana G. (type VI), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), H202 solution (35% w/v), poly(allylamine hydrochloride) (PAH, Mw = 70,000), poly(sodium 4-styrenesulphonate) (PSS, Mw = 70,000), alumina (Al2O3) spherical pellets (3 mm diameter), γ-aminopropyltriethoxysilane (γ-APTS), glutaraldehyde and
Acknowledgments
Dr. Luciano Pilloni and Dr. Marzia Pentimalli (ENEA FIMMATTEC CR Casaccia, Rome, Italy) are gratefully acknowledged of their skillful help in SEM studies.
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