Alternative lignopolymer-based composites useful as enhanced functionalized support for enzymes immobilization
Graphical abstract
Introduction
Modern polymer industry makes it possible to produce wide range of polymeric materials with highly tunable properties (e.g. stiff/ soft, transparent/ opaque, conducting/ insulating, permeable/ impermeable, stable/ degradable) and broad range of applications (e.g. composites, construction materials, engineered plastics, coatings, adhesives, implants, artificial hearts, drug delivery etc) [1]. Polymers are the unique class of materials with such diverse properties and versatile applications. As an important consequence, the modern life is dependent on the polymer industry providing high quality of life for our society [2]. Nevertheless, the future of this industry is unclear because it is heavily relied on fossil resources which represent the global warming and littering problems of nowadays [3].
Biomass and biomass derivatives have been pointed out as the most promising resource alternatives for polymeric industry perfect equivalent to petroleum [4]. It is considered the only sustainable source of renewable carbon for the production of fuels and fine chemicals without carbon emission [5,6]. In this context, lignocellulosic biomass has a critical importance mostly due to large abundance and bio-renewable capacity on earth [5]. The literature started to be flooded with publications reporting the involvement of lignocellulose to production of biofuels and biomaterials [7,8], especially the lignin fraction due to a generous content of phenolic units.
Lignin can be defined as a natural polymer with irregular oxygenated structure based on p-propylphenol units placed in the plant cell wall providing plant rigidity as well as resistance to microbial attack [9]. The polymeric structure of lignin consists three building blocks (p-coumaryl alcohol p-CA, coniferyl alcohol CA and sinapyl alcohol SA) called monolignols, different in the amount of methoxy groups on phenolic nucleus [10]. However, monolignols are phenolic hydroxy structure which are the most reactive functionalities and their presence in the lignin molecule can significantly affect the chemical reactivity of this material. Relative distribution of monolignols in lignin makes the difference between plant species. Therefore, softwood lignin (e.g. pine, spruce) abounds in CA, hardwood lignin (birch, polar, eucalyptus) consists both CA and SA, and herbaceous lignin is composed from all three monolignols (p-CA, CA and SA) with usually low content of p-CA [9]. Beside these, the lignin incorporates other compounds such as hydroxycinnamates, p-hydroxybenzoate, tricin, acetate and intermediators from biosynthesis of monolignols. [11]
Biosynthesis of lignin involves the oxidation of monolignols at the phenolic −OH group leading to phenoxy radicals which combine together through the polymerization mechanism. The process is catalyzed by peroxidase and/or laccase enzymes [12]. The alkyl unsaturated chain of the monolignols enables the formation of carbon-carbon bonds together with the ether bonds (e.g. α-O-4, β-O-4) [12]. Lignin consists substantial fraction of carbon-carbon bonds, such as 5-5, β-5, β-1, β-β connections. Monolignols distribution can affect the formation of carbon-carbon binding in lignin structure. Therefore, low fraction of carbon-carbon units corresponds to high content of SA compared with CA since the presence of methoxy substitute in ortho position (e.g. for SA) avoids the formation of 5-5 and β-5 carbon-carbon bounds [10,12,13]. All of these ether and carbon-carbon bound-units are not equally distributed in the lignin structure. So that, lignin samples from the same plant species can have similar but not identical composition. This is an important issue related to lignin and is often mentioned as a high heterogeneity of lignin. On the other hand, lignin can form large aggregates based on intermolecular association through physico-chemical interactions (hydrogen bonding, between various ether oxygens and hydroxyl groups, van der Waals attraction of polymer chains, and π-π stacking of aromatic groups) [13]. The chemical structure as well as the molecular weight of native lignin are difficult to appoint and preserve for further use due to the changes occurring during lignin isolation. Additionally, lignin properties can be affected during the isolation and purification steps.
In order to avoid all these drawbacks, we developed in our previous studies the biocatalytic process mimicking the nature for providing lignopolymeric materials [14]. Oxi-polymerization process of monolignols (CA or SA) was designed as a biocatalytic route where a peroxidase enzyme assisted the oxidation of the substrate by means of H2O2 [14]. The developed system allowed to provide polymeric products with similar/ improved structure and properties of native lignin. The building of the lignopolymer from the monolignols instead of whole lignin molecule allowed to produce homogeneous polymeric materials with better control on its composition. Furthermore, one-pot approach has been set up combining the oxi-(co)polymerization of CA/SA and the attachment of the resulted lignopolymer on the support surface (methacrylate) functionalized with phenolic derivatives (caffeic acid) [15]. In this way, lignin-composites with controlled and reproducible composition were prepared.
In this study, we investigated the production of lignocomposites functionalized with amine groups (ADL-composite). The polymeric materials developed in the previous study (P1) was enhanced by insertion of -NH2 functional groups leading to an amino-derivatized polymer (P2) with increased chemical reactivity. The one-pot biocatalytic approach allowed to construct and also to attach the polymer on the support surface. CA monolignol and aniline were oxi-copolymerized directly on the support surface (SC2/SC6) pre-functioned with amino-phenolic derivatives (p-phenylenediamine (p-Ph-2-NH2) or p-amino-2-hydroxybenzoic acid (p-NH2-SalA)). The oxidation process was catalyzed by peroxidase enzyme (horseradish peroxidase, HRP) using H2O2 as oxidation agent. Therefore, the prepared composites (ADL-composites) were designed as an amino-derivatized lignopolymeric (P2) layer covering the support surface. Production of ADL-composites was monitored using spectrophotometric analysis. Different techniques (e.g. FTIR, TPD, TGA, static contact angle, elemental analysis, and size exclusion chromatography) were used for detailed characterization of the resulted composites. Additionally, the investigation of the ADL-composites as support for enzyme immobilization (lipase) has been performed.
Section snippets
Chemicals and solutions
Peroxidase from horseradish (HRP, type VI, essentially salt-free, lyophilized powder, 950–2000 units/mg solid) was used for oxi-(co)polymerization process. Also, lipase from Candida antartica (lyophilized, powder, beige, ⁓0.3 U/mg) was immobilized on the prepared composites. Both enzymes were purchased from Sigma-Aldrich. Coniferyl alcohol (CA), aniline, p-phenylenediamine (p-Ph-2-NH2), p-aminosalicylic acid (p-NH2-SalA), methanol (MeOH), ethanol (EtOH), tetrahydrofuran (THF),
Oxi-copolymerization of CA and aniline for the construction of ADL-composite
The oxi-copolymerization of CA and aniline was performed directly on the supports surface previously functionalized with amino phenolic derivatives (SC2/ SC6-p-Ph-2-NH2 and SC2/ SC6- p-NH2-SalA) leading to ADL-composites (lignopolymer-based composites) designed as functionalized support covered with P2 polymeric layer. Scheme 1 presents a general view on the preparation of lignopolymer-based composites.
The reaction was catalyzed by HRP enzyme using H2O2 as oxidation reagent. Peroxidase enzyme
Conclusions
Amino-derivatized lignopolymeric-composites (ADL-composites) have been prepared using one-pot biocatalytic approach. CA monolignol and aniline was successfully oxi-copolymerized directly on the surface of a solid support based on an enzymatic process with HRP catalyzing the oxidation of the substrates and H2O2 as oxidation reagent. The biocatalytic transformation allowed to produce a homogeneous polymer P2 with large volume (MW = 9 × 106) ready to be attached on a support surface.
CRediT author statement
Cristina Lite – Participation to the preparation the original draft, Performing the experimental part, Data analysis/interpretation.
Sabina Ion – Methodology, Performing the experiments.
Madalina Tudorache – Conceptualization, Supervision, Preparation of the manuscript, reviewing and editing.
Irina Zgura – Performing contact angle analysis and data interpretation.
Aurelian C. Galca – Contact angle analysis.
Madalina Enache – Investigation based on SEM analysis.
Gabriel-Mihai Maria – Performing SEM
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was financially supported by The Education, Scholarship, Apprenticeships and Youth Entrepreneurship Programmer – EEA Grants 2014-2021, Project No. 18-Cop-0041. We thank Purolite Life Science Company for supports SC2 (amino C2 methacrylate particles - ECR8309F) and SC6 (amino C6 methacrylate particles - ECR8409F).
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