Development of a biocomposite based on alginate/gelatin crosslinked with genipin for β-galactosidase immobilization: Performance and characteristics
Graphical abstract
Introduction
Enzyme technology is becoming gradually more important for many applications in several industrial areas. Not only for their versatility and efficiency but also environmental issues, since its use in industrial processes has been replacing conventional procedures that use chemical catalysts, generating environmentally friendly methods. Thereby, enzymatic immobilization is an excellent tool, due to the possibility to improve enzyme properties, including increased activity under adverse conditions, better specificity, and possible recovery and reuse, thus reducing costs (Bilal & Iqbal, 2019; Miletić et al., 2012). However, it is necessary to recognize the limitations in enzymatic immobilization such as decreased catalytic efficiency, enzyme inactivation in the presence of support due to multiple interactions (Basso & Serban, 2019), or even mass transfer limitations promoted by immobilization (Mateo et al., 2007).
Among different solid supports described in the literature for enzymatic immobilization (Eskandarloo & Abbaspourrad, 2018; Ricardi et al., 2018), biopolymers have attracted researchers due to their characteristics of biocompatibility, hydrophilicity, biodegradability, and adhesion properties (Bilal & Iqbal, 2019), being also compatible to immobilization with genipin (Amaro-Reyes et al., 2019; Díaz-Hernández et al., 2018; Gracida et al., 2019).
Alginate is an anionic polysaccharide extracted mainly from brown seaweed, and it is composed of repeated units of α-1,4-L-guluronate (G) and β-1,4-D-mannuronate (M). Its composition depends on the extraction source (Wang et al., 2019), as well as its physical-chemical characteristics, which are strongly dependent on the monomer composition (M/G ratio) and distribution, and molecular weight. One of the excellent properties of sodium alginate is its ionotropic gelation capacity in the presence of divalent ions, which leads to the formation of an “egg-box” structure (Bennacef et al., 2021), leading to a rod-like interconnected network (Traffano-Schiffo et al., 2018).
Gelatin is a polymer derived from collagen by hydrolysis, is cheap and abundant as it is present in animal skin and bones. Hydrolytic depolymerization can be performed through acidic or basic pre-treatment, producing gelatin type A and B, respectively (Duconseille et al., 2015). These different treatments impact its isoelectric point, where gelatin type A has pI ~8.5 and gelatin type B has pI ~4.9, which can drastically affect its functionality (Smith et al., 2016). Also, gelatin has limited use due to high hygroscopicity and poor mechanical properties (Wang et al., 2019). To overcome these drawbacks, chemical crosslinking reagents, such as genipin, could be added into gelatin formulations to improve mechanical properties.
Moreover, mechanical properties of supports for enzyme immobilization can be improved by the crosslinking with agents as genipin. This substance is an iridoid, obtained from the fruit of Genipa americana L. and flowers of Gardenia jasminoides Ellis by direct extraction or after extraction and hydrolysis of the geniposide (Paik et al., 2001). Genipin can react spontaneously with primary amine groups of amino acids, proteins, or peptides through crosslinking (Ramos-de-la-Peña et al., 2016), and can be used as a natural crosslinking agent in some polymers, such as chitosan and gelatin. According Sung et al. (1999) the cytotoxicity of genipin in a studied in vitro with 3 T3 fibroblasts resulted 10,000 times lower than that of glutaraldehyde, a conventional crosslinker.
β-galactosidase from Aspergillus oryzae is an extracellular monomeric enzyme and hydrolyze the (1 → 4) linkage of lactose [galactosyl (1 → 4) glucose] to glucose and galactose in several products such as milk and whey, giving rise to products with low lactose content, which benefit people intolerant to this sugar. Besides, in the presence of concentrated lactose, this enzyme synthesizes galactooligosaccharides, a prebiotic ingredient (Klein et al., 2016).
In this context, this study aimed to develop a biocomposite formulated with alginate and gelatin using genipin as a crosslinker for the immobilization of β-galactosidase as a model enzyme. Besides a detailed analysis of the hybrid material microstructure using Small-Angle X-ray Scattering (SAXS), other techniques such as TGA, thermal stability, storage, and operational stabilities were made revealing details about molecular associations and structural conformations. Therefore, this research provides new insights and valuable information for future research, especially for food applications.
Section snippets
Materials
β-Galactosidase from Aspergillus oryzae and o-nitrophenyl-β-D-galactopyranoside (ONPG) were purchased from Sigma-Aldrich (São Paulo, Brazil). Sodium alginate, molecular weight 198.11 g/mol, was purchased from Cromato Produtos Químicos Ltda (São Paulo, Brazil), and gelatin type A from porcine skin (GECOLL) was provided by the company Vêneto Mercantil Importadora LTDA (Flores da Cunha, RS, Brazil). A d-glucose determination kit was purchased from Labtest Diagnóstica SA (São Paulo, Brazil).
Influence of sodium alginate, gelatin, genipin, and enzyme concentration in the immobilization parameters
Preliminarily, the effect of sodium alginate, gelatin, genipin, and enzyme concentration was investigated through immobilization parameters. According to Tables S1, S2, S3, and S4 (Supporting Information), in general, there is a decrease in yield and activity recovered parameters with an excess in the concentration of sodium alginate, gelatin, genipin, and enzyme. This decrease in the parameters is associated with the smaller pore sizes of calcium alginate that may have caused substrate
Conclusions
A successful method for the immobilization of β-galactosidase from Aspergillus oryzae in biocomposites composed of alginate-gelatin and crosslinked with genipin was developed and showed good immobilization parameters. Although immobilization did not change the optimal pH, the AGG-β-gal was more resistant to alkaline conditions and more active at lower temperatures than the free enzyme. The structural characterization of biocomposites showed that the addition of genipin and β-galactosidase
CRediT authorship contribution statement
Camila Regina Hackenhaar: Methodology, Validation, Formal analysis, Investigation, Writing – original draft, Visualization. Carolina Flores Rosa: Investigation. Elí Emanuel Esparza Flores: Formal analysis, Investigation. Patricio Román Santagapita: Formal analysis, Resources, Writing – review & editing, Supervision. Manuela Poletto Klein: Methodology, Formal analysis, Resources, Writing – review & editing, Supervision. Plinho Francisco Hertz: Conceptualization, Methodology, Formal analysis,
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
The authors would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), from Brazilian government. This work was supported by the Brazilian Synchrotron Light Laboratory (LNLS, Brazil, proposal SAXS1-20190143), Agência Nacional de Promoción Científica y Tecnológica (ANPCyT PICT-2017-0569).
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