Kinetic and thermodynamic study of laccase cross-linked onto glyoxyl Immobead 150P carrier: Characterization and application for beechwood biografting

Highlights

• The surface epoxy groups of Immobead 150P carrier were modified to produce active glyoxyl groups.

• The animated laccase was immobilized on the modified Immobead 150P surface via cross-linking.

• Optimum conditions, stability, and kinetics of free and cross-linked laccases were assessed.

• A highly stable bonded hydrophobic compound was grafted on wood samples using immobilized laccase.

Abstract

In this study, we cross-linked aminated Thermothelomyces thermophilus laccase onto Immobead 150P epoxy carrier, and achieved an immobilization yield of 99.84 %. The optimum temperature and pH values for the oxidation of ABTS by laccase were determined to be 70 °C and pH 3.0. After 6 h at 50 °C, laccase activity was diminished by about 13 % in the free form and 28 %, in the immobilized form. K_m values for both free and cross-linked laccase were 0.051 and 0.567 mM, whereas V_max values were 2.027 and 0.854 μmol. min⁻¹, respectively. The immobilized laccase was able to preserve its full activity for 6 weeks, retaining approximately 95 % and 78 % of its initial activity after 8 and 20 weeks, respectively. The contact angles were two-fold higher when the laccase enzyme was occupied in the biografting reaction, revealing that the hydrophobic compound bonded stably onto beechwood samples.

Introduction

Enzyme catalysis is a “green” process, providing proficient substrate conversion, low energy expenditure, and environmental responsiveness. The reusability and stability of these enzymes are vital to their practical use [1,2]. Enzyme reusability and stability may be affected by unexpected alterations of ambient conditions, like pH, temperature, solvent, and inhibitors. One way to overcome this problem is to create a controlled microenvironment, using an approach such as enzyme immobilization [3]. In a “free state,” enzymes have many-sided operation processes and are not easy to reuse and regenerate [4]. Consequently, a combination of enzymes and carriers may facilitate the proficient recovery of catalytic capacity [2]. New methods and materials for immobilizing enzymes have been the subject of considerable research. However, further improvements are still essential, to permit broader applications of the enzymes [5]. Numerous studies have investigated different physical and chemical immobilization techniques using a range of immobilization carriers [1,6]. Enzyme immobilization helps to improving the performance of enzymes as industrial biocatalysts [7]. The catalytic properties of the enzymes may be reduced in many enzyme immobilization systems, because the immobilization conditions can block the active center of the enzyme [1,5].

One of the recent marketable carriers is Immobead 150, which is composed of methacrylate polymers with a particle size of 0.15−0.30 mm, and which has epoxy linkers able to interact with the amino groups of lysines present on the enzyme molecule. The use of Immobead 150 to immobilize different enzymes produced strong binding and retention of the efficiency of catalytic enzymes [8]. Epoxy-activated carriers appear to be perfect systems for developing effective enzyme immobilization protocols [9]. Epoxy carriers can act in response to diverse nucleophilic groups, including thiol, hydroxyl, and amino moieties, present on the surfaces of protein molecules, forming strong linkages by ether bonds, secondary amino bonds, and thioether bonds, with minimal chemical alteration of the protein. Conversely, epoxy groups are poorly interactive for enzyme immobilization outside of gentle experimental circumstances [10]. Enzyme immobilization using this sort of carrier occurs in two steps. First, the protein is adsorbed on the exterior of the carrier, and second, the adsorbed protein undergoes further covalent attachment to the carrier surface via the epoxy groups present on the surface [9,11]. Substitution of epoxy groups by aldehyde groups on the epoxy carrier surface is a possible strategy to conquer the negative effects of the high glycosylation content of some enzymes, where the polysaccharide moieties shield the active groups of the enzyme [12]. This strategy is feasible because of the absence of steric hindrance between enzyme amine groups and aldehyde groups [13]. The short distances between residues occupied in the immobilization process allows the maximum possible number of amino acid residues on the enzyme to react with the immobilization carrier, leading to reduced conformational changes and the formation of strong attachments [8].

Laccases (EC 1.10.3.2, benzenediol: oxygen oxidoreductases) are copper-glycoprotein enzymes that contain four copper ions, and are able to catalyze the oxidation of a large number of compounds. This process is accompanied by the molecular reduction of oxygen to water, with a four-electron reduction [14,15]. Laccases do not necessitate or generate poisonous H₂O₂, so they are deemed to be green catalysts, and are used for numerous biotechnological applications [16]. Unfortunately, the laccases potential applications are limited by high price, difficult reuse, low stability, and short service life [17]. For that, laccase immobilization was considered to solve these drawbacks and improve the laccase performance in various biotechnological applications [7]. Immobilized laccase has several potential applications of great significance in fields of environmental sustainability and remediation, water purification, dye decolorization [14], micro-pollutants removal (like endocrine disruptors, herbicides, pesticides, pharmaceuticals and personal care products), phenolic compounds removal, [17], biosensors [18], polycyclic hydrocarbons degradation [19], xenobiotics degradation, biofuel cells [20], pulp and paper industry, and textile industry [21].

Wood-based materials are attractive since they originate from renewable assets and can have an eco-friendly end of life, being biodegradable and recyclable. Humidity and bio-deterioration are the main factors affecting wood lifespan [22]. Research has therefore focused on producing biological treatments to reduce these effects and improve the hydrophobicity of the wood, without negative impacts on wood properties [23]. The ability of laccases to operate on lignocellulosic materials offers new ecologically-friendly strategies to graft phenolic compounds onto the wood surface. The aim of these treatments is to produce wood with new characters, by bonding hydrophobic compounds such as lauryl gallate (LG) to the wood surface and preventing their washout from the wood, which can produce polluted effluent [24]. LG (3,4,5-tri hydroxybenzoate) is antibacterial, antioxidant, and has hydrophobic properties, caused by carrying an elongated aliphatic chain [25]. LG and free laccase amalgamation have been developed for the hydrophobization of wool fibers [26], paper sheets [27], paper pulp [28], thermo-mechanical pulp fibers [29], and wood [23,24].

In this study, we conducted beechwood hydrophobization through the reaction of cross-linked laccase with Immobead 150 for LG biografting. The surface groups of the Immobead 150P epoxy carrier underwent modification via cross-linking immobilization techniques, to produce glyoxyl modified groups, which can react with the amino groups existing on the exterior of aminated Thermothelomyces thermophilus laccase. The immobilized enzyme was compared to the free laccase with respect to its optimum operating pH and temperature conditions for activity, reusability, stability, thermodynamics, kinetic constants, and storage stability. Finally, the cross-linked laccase was used to produce a competent eco-friendly protocol for the hydrophobization of beechwood surfaces using LG laccase-assisted grafting.