Review
Chemical and physical Chitosan modification for designing enzymatic industrial biocatalysts: How to choose the best strategy?

https://doi.org/10.1016/j.ijbiomac.2021.04.004Get rights and content

Highlights

  • Different methods for using Chitosan as a support for enzymatic immobilization are discussed.

  • Chemical and physical modifications can improve Chitosan properties.

  • Key factors to consider are the magnitude of the enzyme-support interaction and the operating conditions.

  • The choice of modification route will mainly depend on the properties that need improving.

  • Biocatalysts based on Chitosan supports can be used for industrial applications.

Abstract

Chitosan is one of the most abundant natural polymer worldwide, and due to its inherent characteristics, its use in industrial processes has been extensively explored. Because it is biodegradable, biocompatible, non-toxic, hydrophilic, cheap, and has good physical-chemical stability, it is seen as an excellent alternative for the replacement of synthetic materials in the search for more sustainable production methodologies. Thus being, a possible biotechnological application of Chitosan is as a direct support for enzyme immobilization. However, its applicability is quite specific, and to overcome this issue, alternative pretreatments are required, such as chemical and physical modifications to its structure, enabling its use in a wider array of applications. This review aims to present the topic in detail, by exploring and discussing methods of employment of Chitosan in enzymatic immobilization processes with various enzymes, presenting its advantages and disadvantages, as well as listing possible chemical modifications and combinations with other compounds for formulating an ideal support for this purpose. First, we will present Chitosan emphasizing its characteristics that allow its use as enzyme support. Furthermore, we will discuss possible physicochemical modifications that can be made to Chitosan, mentioning the improvements obtained in each process. These discussions will enable a comprehensive comparison between, and an informed choice of, the best technologies concerning enzyme immobilization and the application conditions of the biocatalyst.

Introduction

Millions of tons of fishing waste are produced yearly worldwide [1]. The reprocessing of this biomass has excellent potential to be used a raw material in the preparation of added-value biomaterials, such as biopolymers, biodiesel, and biolubricants [[1], [2], [3]]. The search for biomaterials with feasible applications in industrial processes where synthetic materials are usually employed is one of the current priorities of the scientific community [[4], [5], [6]]. Among the most studied biomaterials, Chitosan can be highlighted, as shown by the significant increase over the years in the number of works published annually using the material (Fig. 1).

Chitosan is a polyaminosaccharide mainly obtained through total or partial deacetylation of Chitin, the second most common organic compound in nature, as well as the main component of the exoskeleton of crustaceans [[7], [8], [9]]. Structurally, Chitosan is a copolymer of N-acetyl-d-glucosamine and d-glucosamine [10]. As a natural polymer, it presents several attractive properties that enable its use in several applications, such as its non-toxicity, biodegradability, biocompatibility, ease of structural modification, thermal and chemical stability, and low cost [2,7,9]. The foremost applications of Chitosan include medicine [5,11], water and waste treatment [12], agriculture [13], and biotechnology [14]. Also, the most current biotechnological application of Chitosan has been as primary support for enzyme immobilization [15,16] due to its unique characteristics such as being biodegradable, water-insoluble, and to possess multiple reactive functional groups [8,[17], [18], [19]].

In enzymatic catalysis, enzymes are able to optimize reactions due to their high specificity and stereoselectivity [20,21], in addition to being able to work under mild conditions of pH and temperature, which contributes strongly to the reduction of the mass and energy costs of chemical processes [[22], [23], [24], [25], [26], [27], [28], [29]]. These factors render enzymes strong potential catalysts to be used in industrial settings [29]. However, the ideal operating conditions of enzymes (pH, temperature, water activity, substrate class, etc.) are quite specific, hindering their use in free form unviable for industrial processes [22,[30], [31], [32], [33]]. To mitigate these limitations, a widely-used technique is enzymatic immobilization, a procedure in which a soluble enzyme is attached to a support to form an insoluble and reusable enzyme-support complex [[34], [35], [36]]. It has been shown that enzymatic immobilization allows for the enzyme recovery and reuse, while also improving other properties, such as activity, specificity, selectivity, and resistance to inhibitors, deeming the technique ideal for the use of enzymatic catalysis in industry [37,38]. The most common methods for carrying out enzyme immobilization on supports include covalent attachment, adsorption, cross-linking, and capture [39]; each technique having its respective advantages and challenges [[40], [41], [42]]. The most suitable immobilization method is usually determined by the type of enzyme, type of support, and catalyst application [30,[43], [44], [45]]. While physical methods (entrapment and adsorption) are known to maintain the catalytic activity of enzymes better, chemical methods (covalent bonding and cross-linking) are known to maintain their stability for longer periods [46,47].

Ideal materials to be used as supports for the immobilization of enzymes must have important properties, such as high surface area, permeability, resistance and chemical and thermal stability, apart from also being insoluble in water, low cost, and having functional groups able to bind to enzymes under different conditions [30,48]. Hence, Chitosan is a biomaterial that exhibits great potential to be used as a catalyst supporter [14]. An efficient and robust interaction between enzymes and Chitosan have been observed across various methodologies, including adsorption and covalent bonding [14,19,49].

Due to its hydroxyls and primary amino groups, structural modification of Chitosan is usually needed, with existing methods using chemical, enzymatic and radiation routes to achieve this [5]. These changes aim to improve properties essential for enabling optimized performance and industrial applications, such as mechanical and thermal stability, high moisture absorption capacity, hydrophilicity, acidic pH tolerance, and the control of the interaction between Chitosan and other elements, such as metal ions, drugs, and organic compounds [17,50]. To improve Chitosan-enzyme interactions, chemical modifications such as functionalization, activation by other reagents, or even the formulation of hybrid supports, can be performed [22,51,52] [8,14,18]. Nowadays, several classes of enzymes are immobilized on Chitosan-based supports for applications in various reactions of industrial interest, such as oxidoreductases [53,54], transferases [55], hydrolases [16,56], lyases [57,58] and isomerases [[59], [60], [61]].

In this scenario, the purpose of this review article is to discuss the roles and the mechanisms of action of Chitosan as a support for enzymatic immobilization, aiming at obtaining several biocatalysts for different industrial applications. Modification techniques will be presented, along with their advantages and disadvantages, which allow Chitosan to be used both as a single support and also in the production of hybrid composites via the employment of other efficient materials for enzymatic immobilization. These modifications have been shown to improve Chitosan properties, streamlining the implementation of this low-cost biomaterial to the formulation of new biocatalysts and its use for the development of sustainable industrial processes by enzymatic catalysis.

Section snippets

Enzyme immobilization

Enzymes in their free state are generally unstable and very sensitive to changes in process conditions, such as pH, temperature, and substrate concentration, in addition to showing limitations on their recovery and reuse [[62], [63], [64], [65]]. To overcome these problems, enzymatic immobilization emerged, allowing the obtainment of reusable biocatalysts, with improved activity and stability, resistance to solvents, high potential for industrial-scale applications, and reduced operational

Chitosan

As previously mentioned, Chitosan is a natural polymer with unique properties such as high bioactivity, non-toxicity, biocompatibility, and biodegradability [[191], [192], [193], [194]]. Chitosan has become a biomaterial potentially attractive for several uses due to its extreme economic and environmental importance [195]. As it is a renewable and low-cost material, the versatility of Chitosan is considered multidimensional [196], with a vast array of possibilities in many areas such as in

Chitosan modification for use as supports

In addition to the advantages previously cited, Chitosan also shows excellent potential for use in enzyme immobilization due to its ability to increase enzyme activity and stability [[321], [322], [323]]. In 4.1 Epoxy group, 4.2 Carboxyl group, 4.3 Divinyl sulfone, 4.4 Glutaraldehyde, 4.5 Glyoxyl group, 4.6 Cysteine, 4.7 Genipin, 4.8 Cyclodextrins, 4.9 Sulfonate, 4.10 Tripolyphosphate (TPP), some activation agents used in Chitosan biopolymers are presented. These agents mainly aim to modify

Chitosan-alginate

The organic-organic hybrid materials deriving from Chitosan are promising supports in the field of enzymatic engineering because they exhibit unique properties in relation to their non-hybrid counterparts, such as a higher number of functional groups that can act as active sites, natural abundance, non-toxicity, and improved biodegradability [[546], [547], [548]]. Alginate is another promising biopolymer in the field of enzymatic catalysis due to its excellent biocompatibility, thermostability,

Oxidoreductase

Oxidoreductases (Enzyme Commission, EC 1) is a substantial and essential class of enzymes, including peroxidases, oxygenases, dehydrogenases, and oxidases [131,761,762]; an example of its structure is shown in Fig. 24. These enzymes are used in the catalysis of reactions involving hybrid transfers, the inclusion of oxygen, the removal of protons, and other essential stages [761,763,764]. During the reaction, the reduced substrate is considered an electron acceptor, while the oxidized substrate

Future perspectives

Chitosan is a ubiquitous biopolymer with great potential for enzyme immobilization due to its low cost, biocompatibility, good adsorption properties, and the possibility of binding with different enzymes without the need for a cross-linking agent [14,147,794]. Nevertheless, Chitosan fibers and beads suffer from low mechanical stability, hindering their use for industrial purposes [829,830]. Cross-linking agents can be used to enhance these properties, enabling the obtainment of biocatalysts

Conclusions

Chitosan is an excellent candidate for acting as a support for enzyme immobilization, since the existence of multiple functional groups in its structure makes it possible to easily carry out modifications that improve its physical-chemical properties, in addition to allowing the material to behave as a support, even without the use of a crosslinker. However, the use of such compounds can optimize the final properties of these biocatalysts. To perform these chemical modifications, a choice of

Acknowledgments

We gratefully acknowledge the financial support of the following Brazilian Agencies for Scientific and Technological Development: Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, 408790/2016-4, 422942/2016-2, 311062/2019-9), and Coordenação de Aperfeiçoamento de Ensino Superior.

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