Genipin and glutaraldehyde based laccase two-layers immobilization with improved properties: new biocatalysts with high potential for enzymatic removal of trace organic contaminants

Current research carried out on bioprocess engineering topics has focused heavily on the design of green and environmentally sustainable biocatalysts [1], [2], [3]. To achieve this, new enzymatic biocatalysts have been studied and developed over the last years [4]. Among them, laccase-based biocatalysts have gained attention due to their great biotechnological potential [5], [6]. Laccases (EC. 1.10.3.2) are glycoproteins widely used in the degradation of micropollutants from water due to their ability to catalyze a wide variety of phenolic substrates[7] with concomitant reduction of oxygen to water. Laccases belong to a large group of enzymes termed the blue-multicopper oxidase family that can be found in bacteria, insects, plants, and, mainly, fungi [8]. Among these varieties of enzymes, Novozym 51003 (Laccase from Aspergillus sp.) has been widely used in immobilization studies [6] and its immobilized form has been applied for biodegradation of phenolic compounds [9] and pollutants degradation [10], among others. Nevertheless, inherent issues such as loss of stability, high production costs, and water solubility can limit laccase applications[11] for bioenvironmental bioprocess development. Thus, enzyme immobilization is an important step in overcoming these limitations since this process can improve the native characteristics of the biocatalyst and, consequently, its applicability[12]. If immobilization strategies are well-designed and well-implemented, they can provide the reaction systems with many interesting advantages, such as increased enzymatic activity and stability, better control in product formation, as well as improved process efficiency, selectivity, and enzyme recovery and reuse [13], [14], [15], [16].

Layer-by-layer immobilization is a method that focuses on the preparation of heterogeneous biocatalysts by alternating layers of enzyme and bonding materials to create a stack of enzyme layers[17], [18]. These multilayer biocatalysts can be formed by electrostatic or hydrophobic interactions, hydrogen or covalent bonding, or a combination of these[19], [20]. This strategy is applied to enhance the loading capacity of the support [17], [21] by the addition of an increased amount of the desired enzyme, consequently improving the overall activity of the biocatalyst.

Genipin was chosen as a crosslinker due to its low toxicity compared to others, such as glutaraldehyde[22]. Glutaraldehyde was also tested in the production of laccase multilayers, since it is a bifunctional agent that has been widely used over the last few years to immobilize enzymes owing to its versatility [23], [24]. However, due to its native toxicity and poor stability under alkaline conditions[23], alternative strategies to produce new heterofuncional supports have been developed [25]. Genipin, for example, emerges as an important replacement for glutaraldehyde due to its lower degradation rate, lower toxicity, and improved biocompatibility compared to materials cross-linked by glutaraldehyde [22]. Genipin is a hydrolytic product of geniposide, which is one of the primary active principles of the Gardenia jasminoides fruit extract, showing high stability and biocompatibility[25], [26], [27]. Genipin can react with primary and secondary amine groups between the support and proteins [22], [28], being an excellent reagent for cross-linking processes [25]

It has been shown that the characteristics of the support used in the immobilization of enzymes greatly influence the performance of the biocatalyst [29]. Chitosan is a natural, low-cost, non-toxic, and biocompatible polymer with chemical resistance, relative thermal stability, and convenient mechanical properties[30]. As a support for enzyme immobilization, it is a versatile polymer due to its active amino groups that act as a reactive site for new groups[31]. Moreover, chemical modification is required if the enzyme is to be covalently immobilized onto the support [32]. Chitosan activation with genipin or glutaraldehyde provides a heterofuncional support [33]. The reagents react with the amino groups in the chitosan and can form covalent bonds with the protein. In addition, these crosslinkers agents show a strong pH dependency that is linked to the degree of chitosan protonation[34].

Over the past decades, the rapidly increasing population growth has caused, among other consequences, the worsening of the water pollution issue, which affects the health of both humans and aquatic organisms[35]. Most of the pharmaceutical compounds found in wastewater are composed of drug molecules that are frequently prescribed or purchased over the counter, such as acetaminophen and mefenamic acid (Fig. 1) [36], [37], [38]. The molecular structure of acetaminophen includes a phenol ring, which renders this compound stable and difficult to degrade (Fig. 1A) [36]. The mefenamic acid molecule, in turn, possesses amine, carboxylic, and benzylic rings, and also methyl groups that present low aqueous solubility and high permeability through biological membranes (Fig. 1B) [39]. An alternative for removing these chemicals is the application of laccase-based biocatalysts.

In this research, we propose a strategy based on a layer-by-layer immobilization of laccase to produce a heterogeneous biocatalyst with improved properties. The novelty of this study involves the preparation of these two-layer biocatalysts using only genipin or glutaraldehyde, or both. To our knowledge, no previous research has been conducted on layer-by-layer immobilization of laccase using genipin or a combination of genipin and glutaraldehyde. One of the objectives of this research is to produce two-layer biocatalysts using laccases, which were previously immobilized onto chitosan. Genipin and glutaraldehyde were used as cross-linking agents. The immobilization parameters and stability of the final biocatalysts will be determined, and their potential for removing the aforementioned trace organic contaminants from aqueous solutions will also be assessed.

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