Support For Enzyme Immobilization Research Articles

Advancements and potential of chitosan‐genipin complex in biotechnological applications: A comprehensive review

AbstractThe chitosan‐genipin complex has been extensively studied and applied in various fields, attracting increasing attention due to its unique characteristics and properties. This complex enables the development of novel materials for applications in biomedical engineering, biotechnology, and medicine. Chitosan is a natural biopolymer, and genipin is an extract obtained from some fruits, mainly from genipap and gardenia; both have demonstrated biodegradability, biocompatibility, and antimicrobial properties. Due to their versatile and reinforced structure, new materials have shown potential use in drug delivery, tissue engineering, scaffolds, curatives, food packaging, and enzyme immobilization. This review will discuss the chitosan and its modifications, the genipin and its reactivity, and finally, the complex chitosan‐genipin, showing some recent developments and applications and some prospectives for other applications in new fields, especially as support for enzyme immobilization.

Recombinant Esterase (BaCEm) Immobilized on Polyethyleneimine-Impregnated Mesoporous Silica SBA-15 Exhibits Outstanding Catalytic Performance.

A recombinant esterase, BaCEm, derived from Bacillus aryabhattai and heterologously expressed in Escherichia coli, was successfully immobilized on polyethyleneimine-impregnated mesoporous silica SBA-15. This immobilization utilized glutaraldehyde as a crosslinker. Optimal conditions were established with a PEI/SBA-15 ratio of 25% (w/w), a pH of 7.5, and a glutaraldehyde concentration of 0.5% (w/w), resulting in a loading capacity of 76.4mg/g, a recovery activity of 43.5%, and a specific activity of 7917 U/g for BaCEm. The immobilized BaCEm demonstrated high enantioselectivity, with an "E" value of 203.92, in the resolution assay of (R,S)-ethyl indoline-2-carboxylate. Notably, the immobilized enzyme, compared to its free counterpart, exhibited enhanced thermostability, maintaining 95.4% of its activity after 3h at 30°C. It also showed significant tolerance to organic solvents, retaining 48.4% and 28.7% residual activity in 10% v/v acetonitrile and acetone, respectively. Moreover, its storage stability was confirmed, with 68.5% residual activity preserved after 30days at 4°C. Remarkably, the immobilized BaCEm retained 58.1% of its activity after 10 reuse cycles, underscoring the potential of polyethyleneimine-impregnated mesoporous silica SBA-15 as an effective support for enzyme immobilization, promising for industrial applications.

A novel Affi-Cova magnetic nanoparticles for one-step covalent immobilization of His-tagged enzyme directly from crude cell lysate

Owing to the rapid advancement of in vitro synthetic biology, functional carriers capable of covalently binding target proteins from crude lysates under mild conditions have garnered escalating attention. Herein, a magnetic nanoparticle with affinity/covalent bifunction (MNP@Affi-Cova) was developed for the direct covalent immobilization of the recombinant enzyme of His-tagged birA (r-birA) from crude cell lysates in a single step. This innovative approach is attributed to the presence of chelated Ni2+ ions and epoxy groups on the surface of the beads. The fabricated magnetic nanoparticles were characterized by SEM, FT-IR spectrum, and zeta potential. The application conditions and stability of the MNP@Affi-Cova beads were systematically evaluated. Notably, the MNP@Affi-Cova beads exhibited a covalent capture efficiency of 91.25 μg r-birA/mg beads from a cell lysate supernatant containing 2.62 mg/mL crude protein. The immobilized r-birA exhibited significantly enhanced pH and thermal stability compared to the free counterpart. Additionally, the reusability of the immobilized r-birA on MNP@Affi-Cova demonstrated the retention of 76.1 % of its initial activity over ten cycles. These results suggest that the MNP@Affi-Cova presents considerable potential as a support for the covalent immobilization of recombinant His-tagged enzymes directly from crude lysates, thereby circumventing the labor-intensive purification process typically required before enzyme immobilization.

Activation of Inert Supports for Enzyme(s) Immobilization Harnessing Biocatalytic Sustainability for Perennial Utilization.

Although Nature's evolution and intelligence have gifted humankind with noteworthy enzyme candidates to simplify complex reactions with ultrafast, overselective, effortless, mild biological reactions for millions of years, their availability at minute-scale, short-range time-temperature stability, and purification costs hardly justify recycling/or reuse. Covalent immobilization, particularly via multipoint bonds, prevents denaturing, maintains activities for long-range time, pH, and temperature, and makes catalysts available for repetitive usages; which attracts researchers and industries to bring more immobilized enzyme contenders in science and commercial progressions. Inert-support activation, the most crucial step, needs appropriate activators; under mild conditions, the activator's functional group(s) still present on the activated support rapidly couples the enzyme, preventing unfolding and keeping the active site alive. This review summarizes exciting experimental advances, from the 1950s until today, in the activation strategies of various inert supports with five different surface activators, the cyanogen bromide, the isocyanate/isothiocyanate, the glutaraldehyde, the carbodiimide (with or without N-hydroxysuccinimide (NHS)), and the diazo group, for the immobilization of diverse enzymes for broader applications. These activators under mild pH (7.5 ± 0.5) and temperature (27 ± 3 °C) and ordinary stirring witnessed support activation and enzyme coupling and put off unfolding, harnessing addressable activities (CNBr: 40 ± 10%; -N═C═O/-N═C═S: 32 ± 7%; GA: 70 ± 15%; CDI: 60 ± 10%; -N+≡N: 80 ± 15%), while underprivileged stability, longevity, and reusabilities keep future investigations alive.

Obtaining a biodegradable biocatalyst – study on lipase immobilization on spent coffee grounds as potential carriers

Coffee waste can be reused as matrices for enzyme immobilization, as it contains various organic compounds able to adsorb catalytic proteins. In this study, spent coffee grounds were used as a support in the immobilization process in their native form and after being pretreated with hexane, ethanol, and sulphuric acid solution, respectively. Microbial lipases from Aspergillus oryzae, Thermomyces lanuginosus, and Rhizomucor miehei were adsorbed on the carrier, and as a reference, the abovementioned lipases were also immobilized on a synthetic matrix - Lewatit VP OC 1600. The research investigated the impact of the purification step on the immobilization process and the full characteristics of the obtained biocatalysts. The hydrolytic and synthetic activities of the immobilized enzyme preparations were tested, as well as substrate specificity, recovery, and temperature and pH activity profiles. SEM and FTIR analyses were also performed. The results showed that the chemical composition of coffee waste influenced the activity of the obtained biocatalysts, and the lack of hemicellulose caused a reduction in lipolytic activity. The study reveals that spent coffee grounds can be a potential support for enzyme immobilization, and lipases adsorbed on them have improved properties such as hydrolytic or synthetic activity, and stability in pH.

Hydroxyapatite: An Eco-Friendly Material for Enzyme Immobilization.

Biocatalysis has emerged in the last decade as a valuable and eco-friendly tool in chemical synthesis, allowing in several instances to reduce or eliminate the use of hazardous reagents, environmentally dangerous solvents and harsh reaction conditions. Enzymes are indeed able to catalyse chemical transformations on non-natural substrates under mild reaction conditions, still maintaining their high chemo-, regio-, and stereoselectivity. Enzyme immobilization, i. e. the grafting of enzymes on solid supports, can be viewed as an enabling technology, as it allows a better control of the reaction and the recycling of the biocatalyst, thus rendering economically viable the use of expensive enzymes also on a large scale. To pursue a sustainable approach, the supports for enzyme immobilization should be eco-friendly and possibly renewable. This review highlights the use of hydroxyapatite (HAP), an inorganic biomaterial able to confer strength and stiffness to the bone tissue in animals, as carrier for enzyme immobilization. HAP is a cheap, non-toxic and biocompatible material, with high surface area and protein affinity. Different enzyme classes, immobilization strategies, and the use of diverse HAP-based supports will be discussed, underlining the immobilization conditions and the properties of the obtained biocatalysts.

Synthesis and characterization of mesoporous silicas with dendritic and spongy-like structures: Potential supports for human lactate dehydrogenase-based microreactors aimed at anticancer inhibitor screening

Mesoporous silica are versatile materials with wide-ranging potential. Notably, they excel as enzyme supports. This work examines the influence of three distinct siliceous mesoporous materials used as supports for the enzyme human lactate dehydrogenase (hLDH-A). Drugs with inhibition effects have recently shown favorable effects on diminishing the proliferation of cancerous cells. The ultimate goal of this research is to produce a stable and effective biocatalyst suitable for being employed in a microreactor for the screening of hLDH-A inhibitors. The synthesized mesoporous silica exhibited distinctive structural features, including a quasi-mesocellular network, bent-channels structure, and a dendritic geometry with radial symmetry, as evidenced by FESEM and HR-TEM. These materials were functionalized with amino and aldehyde groups to covalently immobilize hLDH-A. Characterization of both pristine and functionalized materials involved a comprehensive examination of their physico-chemical properties. The CO dosing revealed Brønsted acidity characteristic of mesoporous silica, while FT-IR spectroscopy and N2 physisorption at 77 K confirmed their successful functionalization. Enzyme immobilization on the functionalized supports, performed with stabilizing agents such as PEG (0.05 mg mL−1) or trehalose (300 mM), produced promising results. The immobilization yield consistently exceeded 80 %, with retained activity reaching values as high as 15 %. The immobilization of the enzyme on mesoporous silica increased the stability of hLDH-A against alkaline and organic solutions. These findings hold significance for those exploring siliceous porous supports for enzyme immobilization, paving the way for the development of stable and active biocatalysts.

Activated carbon from sugarcane bagasse as support for lipase immobilization by physical adsorption technique

Lipases are recognized as the most important group of catalysts in biotechnology. However, utilization of free enzymes is often hampered by the need for more operational stability, high cost, and non-reusability. Most of these obstacles can be solved by lipase immobilization. This work's objective was to evaluate the performance of the activated carbon obtained from sugarcane straw (SAC) as a support for lipase immobilization. Two lipases were immobilized by physical adsorption on SAC: Aspergillus niger 11T53A14 lipase and CalB (lipase B from Candida antarctica, Novozymes). Results revealed that the lipase had been anchored on the activated carbon with the lipase binding efficiency of 89 % (A. niger lipase) and 100 % (CalB) at the optimum experimental conditions (initial protein concentration 0.1 mg mL-1, 0.15 g of SAC, 25 °C, and 120 min). Langmuir isotherm fitted the adsorption equilibrium data of the lipases on SAC. SAC presents a high surface area and protein adsorption capacity. These results show that activated carbon synthesized from the sugarcane straw is a promising support for enzyme immobilization.

DIMENSIONING OF VINYLSULFONIC SUPPORTS FROM CASHEW APPLE BAGASSE BIOMASS IN THE IMMOBILIZATION OF LIPASES

In this work, the support, cashew apple bagasse (CAB), was chemically modified with divinyl sulfone (DVS), and it was evaluated to immobilize Candida antarctica lipase A (CAL-A). The best activation conditions of CAB support were defined by an advanced experimental design using the Taguchi method, assessing five factors at four levels (concentration of DVS, ionic strength, pH, temperature, and time). The support and biocatalyst (CAB-DVS-CAL-A) were characterized by Fourier transform infrared spectroscopy (FTIR), elemental analysis, thermogravimetry (TGA), scanning microscopy (SEM), fluorescence spectroscopy (XRF) and electrophoresis. The optimal conditions to activate the support were DVS concentration of 1.4 mol L-1 (3 mL of DVS in 20 mL of reaction volume), a concentration of sodium bicarbonate buffer at 5 mmol L-1, pH 3.0 at 30 °C for 12 h. The immobilization on CAB-DVS promoted increased thermal stability at 70 °C and different pHs of CAL-A. Therefore, the selected conditions allowed for a catalyst with a catalytic activity of 6.8 U g-1 and more stable than the free enzyme (CAL-A). This demonstrates that pretreated and DVS-activated CAB is a promising support for enzyme immobilization.

Enhancing Enzyme Stability and Functionality: Covalent Immobilization of Trypsin on Magnetic Gum Arabic Modified Fe3O4 Nanoparticles.

This study aimed to fabricate gum Arabic (GA)-coated Fe3O4 nanoparticles bearing numerous active aldehyde groups on their surface, followed by an assessment of their capability as a magnetic support for the covalent immobilization of the trypsin enzyme for the first time. FT-IR, XRD, TGA, and SEM results demonstrated the successful synthesis of GA-coated Fe3O4 nanoparticles, along with the covalent immobilization of the enzyme onto the support. Immobilization enhanced the relative enzymatic activity across a range of aqueous solution pH levels (ranging from 4 to 11) and temperatures (ranging from 20 to 80 °C) without altering the optimum pH and temperature for trypsin activity. Kinetic studies using Michaelis-Menten plots revealed changes in kinetic parameters, including a lower Vmax and higher Km for immobilized trypsin compared to the free enzyme. The immobilization onto magnetic gum Arabic nanoparticles resulted in an improved stability of trypsin in the presence of various solvents, maintaining a stability order comparable to that of the free enzyme due to the stabilizing effect of the support. The reusability results showed that the immobilized enzyme can retain over 93% of its activity for up to 15 cycles.

Macro porous ZIF-8 beads: Promising supports for enzyme immobilization

In recent years, metal–organic frameworks (MOFs) have emerged as promising supports for enzyme immobilization because they are highly designable and structurally diverse. However, the high porosities of conventional MOFs cannot be fully utilized for reactions involving large molecules, such as triglyceride transesterification reactions that produce biodiesel. Macroporous MOFs have been used to enhance diffusivity and, consequently, the reaction rate. Modifying MOFs with alginate to form MOF–polymer beads has also been proposed as a method for simplifying separation and reusability. Macroporous ZIF-8 (M-ZIF-8), sodium alginate ZIF-8 beads (SA@ZIF-8), and M-ZIF-8 combined with SA@ZIF-8 (SA@M-ZIF-8) were prepared for immobilizing lipase. M-ZIF-8 showed the highest adsorption capacity of 113.7 mg/g at 40 °C and was found to be more thermodynamically stable than ZIF-8. The experimental data for all three supports are best fitted to the Freundlich isotherm model. The adsorption kinetic data for M-ZIF-8 and SA@M-ZIF-8 are best described by a pseudo-first-order model, whereas a pseudo-second-order model provided a better fit for SA@M-ZIF-8. Lipase immobilized on M-ZIF-8 showed the highest catalytic activity, whereas that immobilized on SA@M-ZIF-8 showed the best reusability.

Investigation of Graphene Oxide/Mesoporous Silica Supports for Enhanced Electrochemical Stability of Enzymatic Electrodes

Mesoporous silica materials (MSMs) are widely used materials in many applications due to their diverse pore structures. However, the electrical conductivity of MSMs is poor which limits their use in electrochemical applications. In this study, widely used MSMs of different structural properties such as MCM-41, MCM-48, SBA-15, and SBA-16 were synthesized and reinforced with graphene oxide (GO) to obtain conductive composite supports for enzyme immobilization. MSMs were first synthesized using a hydrothermal method and characterized by Fourier-transform infrared spectroscopy, X-ray crystallography, scanning electron microscopy/energy dispersive X-ray, and MAPPING techniques. Aqueous dispersion of GO:MSM composites were prepared with as-synthesized materials and coated on screen-printed electrodes (SPE). The best composites were chosen based on their electroanalytical performance. Glucose oxidase (GOx) was then immobilized on modified SPEs using a simple drop-casting method to produce enzymatic electrodes. The electroanalytical performance of the enzymatic electrodes was investigated using different glucose concentrations to demonstrate biocatalytic activity. Stability tests were performed using intraday and interday measurements which revealed that SPE/GO:MCM-41/GOx electrode showed a more stable performance (3-folds) than SPE/GO/GOx electrode. This study presents an investigation of MSM mixed with GO in enzymatic electrochemical systems providing insight into the use of such materials to preserve enzyme activity.Graphical

Fabrication of lignin-based sub-micro hybrid particle as a novel support for adenylate cyclase immobilization

This study developed a surface functionalized lignin-based sub-microsphere as an innovative support for enzyme immobilization. Lignin was first modified with a silane reagent leading to lignin/SiO2 (LS) organic/inorganic hybrid particles, displayed as regular sub-micro spheres with a SiO2 shell as demonstrated in SEM images. The LS particles were further modified to introduce nickel ions, as evidenced in XPS spectra, facilitating affinity adsorption with a his-tagged enzyme. The immobilization of adenylate cyclase from Haloactinopolyspora alba (HaAC), expressed in Escherichia coli, was conducted on the surface functionalized LS (LS-G-NTA-Ni). The immobilization conditions were optimized to achieve the highest relative activity, which were determined to be using a Ni2+ concentration of 62.5 mM, at pH=9.5 and 25 °C, with an enzyme-to-support ratio of 4.0 for a duration of 2 h. The immobilized HaAC shows maximum relative activity at pH=9.5 and 40 °C, and exhibits significantly improved thermal stability compared to the free enzyme. After undergoing five reusing cycles, the immobilized HaAC maintains a satisfactory activity (54.15%), which is due to the surface chemistry and the structural stability of the functionalized LS. This work provides a valuable exploration for high-value application of industrial lignin.

Magnetic macroporous chitin microsphere as a support for covalent enzyme immobilization

In this study, a novel magnetic macroporous chitin microsphere (MMCM) was developed for enzyme immobilization. Chitin nanofibers were prepared and subsequently subjected to self-assembly with magnetic nanoparticles and PMMA (polymethyl methacrylate). Following this, microspheres were formed through spray drying, achieving a porous structure through etching. The MMCM serves as an effective support for immobilizing enzymes, allowing for their covalent immobilization both on the microsphere's surface and within its pores. The substantial surface area resulting from the porous structure leads to a 2.1-fold increase in enzyme loading capacity compared to non-porous microspheres. The MMCM enhances stability of the immobilized enzymes under various pH and temperature conditions. Furthermore, after 20 days of storage at 4 °C, the residual activity of the immobilized enzyme was 2.93 times that of the free enzyme. Even after being recycled 10 times, the immobilized enzyme retained 56.7 % of its initial activity. It's noteworthy that the active sites of the enzymes remained unchanged after immobilization using the MMCM, and kinetic analysis revealed that the affinity of the immobilized enzymes rivals that of the free enzymes.

Sustainable Immobilization of β-Glucosidase onto Silver Ions and AgNPs-Loaded Acrylic Fabric with Enhanced Stability and Reusability.

Modified polymer design has attracted significant attention for enzyme immobilization, offering promising applications. In this study, amine-terminated polymers were synthesized by incorporating functional groups into polyacrylonitrile using hexamethylenediamine. This work highlights the successful enzyme immobilization strategy using modified polymers, offering improved stability and expanded operational conditions for potential biotechnological applications. The resulting amino groups were utilized to capture silver ions, which were subsequently converted to silver nanoparticles (AgNPs). The obtained materials, AgNPs@TA-HMDA (acrylic textiles coated silver nanoparticles AgNPs) and Ag(I)@TA-HMDA (acrylic textiles coated with Ag ion) were employed as supports for β-glucosidase enzyme immobilization. The highest immobilization yields (IY%) were achieved with AgNPs@TA-HMDA at 92%, followed by Ag(I)@TA-HMDA at 79.8%, resulting in activity yields (AY%) of 81% and 73%, respectively. Characterization techniques such as FTIR, FE-SEM, EDX, TG/DTG, DSC, and zeta potential were employed to investigate the structural composition, surface morphologies, elemental composition, thermal properties, and surface charge of the support materials. After 15 reuses, the preservation percentages decreased to 76% for AgNPs@TA-HMDA/β-Glu and 65% for Ag(I)@TA-HMDA/β-Glu. Storage stability revealed that the decrease in activity for the immobilized enzymes was smaller than the free enzyme. The optimal pH for the immobilized enzymes was broader (pH 5.5 to 6.5) compared to the free enzyme (pH 5.0), and the optimal temperature for the immobilized enzymes was 60 °C, slightly higher than the free enzyme's optimal temperature of 50 °C. The kinetic analysis showed a slight increase in Michaelis constant (Km) values for the immobilized enzymes and a decrease in maximum velocity (Vmax), turnover number (Kcat), and specificity constant (Kcat/Km) values compared to the free enzyme. Through extensive characterization, we gained valuable insights into the structural composition and properties of the modified polymer supports. This research significantly contributes to the development of efficient biotechnological processes by advancing the field of enzyme immobilization and offering valuable knowledge for its potential applications.

Preparation and Characterization of a Novel Morphosis of Dextran and Its Derivatization with Polyethyleneimine.

Dextran, a variant of α-glucan with a significant proportion of α-(1,6) bonds, exhibits remarkable solubility in water. Nonetheless, the precipitation of dextran has been observed in injection vials during storage. The present study aimed to establish a technique for generating insoluble dextran and analyze its structural properties. Additionally, the potential for positively ionizing IS-dextran with polyethyleneimine was explored, with the ultimate objective of utilizing IS-dextran-PEI as a promising support for enzyme immobilization. As a result, IS-dextran was obtained by the process of slow evaporation with an average molecular weight of 6555 Da and a yield exceeding 60%. The calculated crystallinity of IS-dextran, which reaches 93.62%, is indicative of its irregular and dense structure, thereby accounting for its water insolubility. Furthermore, positive charge modification of IS-dextran, coupled with the incorporation of epichlorohydrin, resulted in all zeta potentials of IS-dextran-PEIs exceeding 30 mV, making it a promising supporting factor for enzyme immobilization.

Rational post-synthesis of lipase-magnetic MOF conjugates with boosted enzymatic performance

Metal-organic frameworks (MOFs) have shown great potential as supports for enzyme immobilization, but their application is limited by separation difficulties due to their nanoscale size. Herein, magnetic MOFs (Fe3O4@MIL-53-NH2(Al)) were prepared under mild conditions, and an ionic liquid (IL) was used as linker to covalently immobilize lipase in Fe3O4@MIL-53-NH2-IL/PPL. The IL was composed of imidazolium cations and bis(trifluoromethylsulfonyl)imide anions, which enhanced the hydrophobicity of the support. The synthesized biocatalyst Fe3O4@MIL-53-NH2-IL/PPL showed excellent activity, 2.4 times higher than free lipase, and its denaturation, thermal, and storage stability were all satisfactory. The activity of the biocatalyst remained at up to 88.4% after ten recycles. The excellent magnetic responsiveness facilitated the collection of the biocatalyst, which may be beneficial to its reusability, and the multiple interactions between lipase and support may enhance the structural stability of lipase, which was investigated by circular dichroism spectroscopy. Compared to free lipase, both the substrate affinity and catalytic efficiency of Fe3O4@MIL-53-NH2-IL/PPL were improved. The magnetic MOF composite materials prepared through the post-synthesis method exhibited good potential in the field of lipase immobilization. The preparation method of immobilized lipase in this work provides a meaningful reference for the application of other MOF composites in the field of enzyme immobilization.

Enzyme technology in bioethanol production from lignocellulosic biomass: Recent trends with a focus on immobilized enzymes

Enzyme immobilization is a useful tool to produce biocatalysts with improved performance, such as high activity, high stability at operational conditions, and easy recovery and reuse. In this context, the production of second-generation ethanol using immobilized enzymes was reviewed. Emphasis was placed on the pre-treatment and/or hydrolysis steps to increase efficiency and reduce the cost of the process. In addition, the process design of bioethanol using immobilized enzymes was critically reviewed. In the enzymatic pre-treatment, laccases, manganese peroxidases, lignin peroxidases, and lytic polysaccharide monooxygenases are the main enzymes involved. When considering processes with heterogeneous biocatalysts, laccases are the most explored enzyme. In the hydrolysis step, cellulases and hemicellulases or a mixture of them are the main enzymes explored in the literature frequently using magnetic nanoparticles as support for enzyme immobilization. Although enzyme immobilization is a mature technology, the use of these biocatalysts is frequently limited to only one step of the bioethanol production process, such as pre-treatment or hydrolysis performed using Benchmark Technology. However, some recent papers have explored innovative design processes using a mixture of immobilized enzymes or co-immobilized enzymes to join some process steps, thereby decreasing the cost of enzymes and equipment.

A critical review of enzymes immobilized on chitosan composites: characterization and applications.

Enzymes with industrial significance are typically used in biological processes. However, instability, high sensitivity, and impractical recovery are the major drawbacks of enzymes in practical applications. In recent years, the immobilization technology has attracted wide attention to overcoming these restrictions and improving the efficiency of enzyme applications. Chitosan (CS) is a unique functional substance with biocompatibility, biodegradability, non-toxicity, and antibacterial properties. Chitosan composites are anticipated to be widely used in the near future for a variety of purposes, including as supports for enzyme immobilization, because of their advantages. Therefor this review explores the effects of the chitosan's structure, molecular weight, degree of deacetylation on the enzyme immobilized, effect of key factors, and the enzymes immobilized on chitosan based composites for numerous applications, including the fields of biosensor, biomedical science, food industry, environmental protection, and industrial production. Moreover, this study carefully investigates the advantages and disadvantages of using these composites as well as their potential in the future.

Chitosan with modified porosity and crosslinked with genipin: A dynamic system structurally characterized

In this work, a deep structural characterization of the chitosan-genipin complex was performed by techniques like small-angle X-ray scattering, dye absorption, atomic force microscopy, compression strength, nitrogen adsorption/desorption, scanning electronic microscopy, and thermogravimetric analysis to understand the changes in the structure through all steps of the process. For this, chitosan beads were modified with Na2CO3 to change porosity and then crosslinked with genipin. The prepared particles were used as support for enzyme immobilization. Our results demonstrated modification in the porosity of the chitosan beads, changing the number of pores and pore volume. After crosslinking process (pH 9), especially the microstructure of chitosan-genipin beads was modified, enhancing the fractality and, after enzyme immobilization (pH 4.5) the fractality increased at the higher scale (∼100 nm) and reduced at the lower scale (∼1–10 nm). β-galactosidase immobilized on the modified particles presented high stability on the continuous production of galactooligosaccharides for 30 days. Our results indicate that the chitosan-genipin complex can be characterized as a plastic and dynamic system due to its structural modifications, particularly in response to changes in the pH environment, even after the crosslinking process.