Free Enzyme Research Articles

Titanium carbide-based nanocomposite: A promising reinforcing material for enzymatic CO2 conversion

Enzymatic conversion of CO2 to formate is a highly significant pathway for CO2 utilization, but current level of conversion efficiency remains unsatisfactory due to low solubility of CO2 under mild conditions. CO2-philic materials can significantly augment the reaction by raising local concentration of substrate. Herein, the intercalated Ti3C2Tx, nano-silica (SiO2) and carbon nitride (CN) with abundant CO2 adsorption sites were introduced to fabricate Ti3C2Tx based nanocomposites, Ti3C2Tx/SiO2/CN (TSCN), for the first time. As the controls, binary composites including Ti3C2Tx/SiO2 (TS), Ti3C2Tx/CN (TCN) and SiO2/CN (SCN) were also studied. For each kind of material, the composition, structure, morphology, and application performance in the enzymatic CO2 conversion were characterized and influencing factors were investigated. Among these composites, TSCN exhibited the best application performance and the enzyme reaction was accelerated up to 23.9 times that of the blank system. The value could reach as high as 35.3 after polyethyleneimine (PEI) modification. Following that, PEI modified composites were used as carrier of formate dehydrogenase (FDH) and apparent specific activity was up to 2.8 times that of free enzyme, which could retain 88.2% of the initial value after 10 cycles associated with the structural integrity. This work provides new impetus to CO2 capture and conversion in a clean and mild way that shows bright application prospects.

Influence of Metal Ions and Organic Solutions on Activity and Stability of Beta‐Glucosidase Nanoflowers

Abstractβ‐Glucosidase, a pivotal enzyme in the synthesis of various glycosides with wide applications in industries such as food, cosmetics, beverages, bioenergy, nutraceuticals, pharmaceuticals, and detergents, undergoes immobilization to enhance its utility in industrial processes and improve enzymatic properties. In this study, the conventional hybrid nanoflower synthesis method was used for β‐glucosidase‐inorganic hybrid nanoflowers. Following a comprehensive pH and temperature scanning of the nanoflowers, the influence of metallic ions and various organic solvents on hybrid nanoflowers to enhance the activity and stability of the biocatalyst were investigated. The optimal morphology for β‐Glu/NFs was determined to be 0.01 mg/mL. To obtain the optimum activity conditions, we explored pH values ranging from 3 to 8 and temperatures between 30 and 80 °C. Both the free form and β‐Glu/NFs exhibited higher activity in acidic conditions, with a significant decrease in activity observed at pH 7 and 8. β‐Glu/NFs demonstrated superior activity compared to the free enzyme in the presence of metal ions, exhibiting a remarkable 3.6‐fold increase in activity in chloroform, and approximately 2.8 and 2.7 times increase in benzene and glycerol, respectively. Moreover, it displayed over a 2‐fold increase in activity in methanol, ethanol, isopropanol, n‐butanol, acetone, and DMSO.

Potential Application of Bacterial Lipase from Bacillus subtilis in the Production of Biodisel

This work aims to evaluate the potential of bacterial lipase of Bacillus subtilis in biodiesel production. Assays were performed to obtain, purify, and immobilize the enzyme and compare chemical and enzymatic transesterification tests. The Tryptic Soy Broth (TSB) culture medium without supplementation showed the highest enzymatic activity and purification yields when using 60% of (NH4)2SO4. Citral was the most efficient agent for lipase immobilization. Even though the free enzymes showed a higher activity rate, the immobilized enzymes are associated with a better-quality product. In conclusion, the lipase enzyme showed positive potential in biodiesel production.

Boosting Enzyme Activity in Enzyme Metal-Organic Framework Composites.

Enzymes, as highly efficient biocatalysts, excel in catalyzing diverse reactions with exceptional activity and selective properties under mild conditions. Nonetheless, their broad applications are hindered by their inherent fragility, including low thermal stability, limited pH tolerance, and sensitivity to organic solvents and denaturants. Encapsulating enzymes within metal-organic frameworks (MOFs) can protect them from denaturation in these harsh environments. However, this often leads to a compromised enzyme activity. In recent years, extensive research efforts have been dedicated to enhancing enzymatic activity within MOFs, leading to the development of new enzyme-MOF composites that not only preserve their catalytic potential but also outperform their free counterparts. This Review provides a comprehensive review on recent developments in enzyme-MOF composites with a specific emphasis on their enhanced enzymatic activity compared to free enzymes.

Mesocellular Silica Foam as Immobilization Carrier for Production of Statin Precursors.

The employment of 2-deoxyribose-5-phosphate aldolase (DERA) stands as a prevalent biocatalytic route for synthesizing statin side chains. The main problem with this pathway is the low stability of the enzyme. In this study, mesocellular silica foam (MCF) with different pore sizes was used as a carrier for the covalent immobilization of DERA. Different functionalizing and activating agents were tested and kinetic modeling was subsequently performed. The use of succinic anhydride as an activating agent resulted in an enzyme hyperactivation of approx. 140%, and the stability almost doubled compared to that of the free enzyme. It was also shown that the pore size of MCF has a decisive influence on the stability of the DERA enzyme.

Support Materials of Organic and Inorganic Origin as Platforms for Horseradish Peroxidase Immobilization: Comparison Study for High Stability and Activity Recovery.

In the presented study, a variety of hybrid and single nanomaterials of various origins were tested as novel platforms for horseradish peroxidase immobilization. A thorough characterization was performed to establish the suitability of the support materials for immobilization, as well as the activity and stability retention of the biocatalysts, which were analyzed and discussed. The physicochemical characterization of the obtained systems proved successful enzyme deposition on all the presented materials. The immobilization of horseradish peroxidase on all the tested supports occurred with an efficiency above 70%. However, for multi-walled carbon nanotubes and hybrids made of chitosan, magnetic nanoparticles, and selenium ions, it reached up to 90%. For these materials, the immobilization yield exceeded 80%, resulting in high amounts of immobilized enzymes. The produced system showed the same optimal pH and temperature conditions as free enzymes; however, over a wider range of conditions, the immobilized enzymes showed activity of over 50%. Finally, a reusability study and storage stability tests showed that horseradish peroxidase immobilized on a hybrid made of chitosan, magnetic nanoparticles, and selenium ions retained around 80% of its initial activity after 10 repeated catalytic cycles and after 20 days of storage. Of all the tested materials, the most favorable for immobilization was the above-mentioned chitosan-based hybrid material. The selenium additive present in the discussed material gives it supplementary properties that increase the immobilization yield of the enzyme and improve enzyme stability. The obtained results confirm the applicability of these nanomaterials as useful platforms for enzyme immobilization in the contemplation of the structural stability of an enzyme and the high catalytic activity of fabricated biocatalysts.

Bioactivity of Myrtus communis from the Montenegro coastline

Although numerous studies have demonstrated the biomedical potential of Myrtus communisL., (Myrtaceae) data on myrtle from Montenegro are scarce. To evaluate antioxidant, antimutagenic and antibacterial activity of myrtle methanolic extracts. Antioxidant activity was evaluated by measuring free radicals scavenging activity, reducing power and enzyme inhibition. The strongest scavenging activity was towards DPPH radical (2,2-diphenyl-1-picrylhydrazyl) (IC501.69-2.25 mg/mL) and superoxide anion (IC500.56 to 0.88 mg/mL), followed by high reducing power (428-472 mgAA/g.DE) and inhibition of XOD (IC500.308-0.6261mg/mL). Antimutagenic activity was evaluated in reverse mutation assays with Escherichia coliWP2 oxyR mutant IC202 and deficient in the induction of antioxidant enzymes. The myrtle extracts strongly inhibited mutagenesis induced by t-BOOH, reaching 70% at the highest concentration applied. Antimicrobial activity was examined on eight different bacterial strains. Gram-positive bacteria, S. epidermis, S. aureusand M. flavus demonstrated the highest sensitivity towards extracts (MICs 4.5-9 mg/mL), but significantly lower towards essential oil (MIC 0.42-3.32 mg/mL).

Cross-linked enzyme aggregates of xylanase, XynR8(N58D), for effective degradation of untreated lignocellulosic biomass

Enzymatic degradation of biomass is preferred over chemical methods for environmental protection. However, in most cases, a chemical pretreatment is still required to help improve enzyme accessibility of the biomass. Therefore, it is necessary to develop highly efficient enzymes for those reasons and to save costs accordingly. Costs can be further reduced if the enzymes are recycled. XynR8(N58D) is an engineered and highly active xylanase originating from the rumen fungus Neocallimastix patriciarum. In this study, XynR8(N58D) was extracellularly over-expressed by Pichia pastoris. The resulting crude enzyme solution, with a purity of XynR8(N58D) of over 80%, was used to prepare the cross-linked enzyme aggregates (CLEA) of XynR8(N58D). Specific activity of the crude enzyme solution was 11,599.39 IU/mg, and that of CLEA was 6129.42 IU/mg. The immobilization yield and the immobilization efficiency were 62.68 ± 16.20% and 84.31 ± 3.11%, respectively. The thermostability, stress tolerance, and shelf life of CLEA were significantly better than those of the free enzyme. The free enzyme and CLEA both effectively decomposed insoluble xylan and autoclaved corncob powders. Therefore, XynR8(N58D) made into CLEA not only maintained high activity, but also improved its stability and permitted its reuse or recyclability. Both forms of the enzyme can effectively degrade lignocellulosic biomass without relying on chemical pretreatment to reduce recalcitrance of the biomass structure. The application of XynR8(N58D) and its CLEA to biomass degradation can help establish an eco-friendly and more cost-effective process.

Comparative study on different immobilization sites of immobilized β-agarase based on the biotin/streptavidin system

β-Agarase was biotinylated and immobilized onto streptavidin-conjugated magnetic nanoparticles to provide insights into the effect of immobilization sites on β-agarase immobilization. Results showed that, compared with free enzyme, the stability of prepared immobilized β-agarases through amino or carboxyl activation were both significantly improved. However, the amino-activated immobilized β-agarase showed higher thermostability and catalytic efficiency than the carboxyl-activated immobilized β-agarase. The relative activity of the former was 65.00 % after incubation at 50 °C for 1 h, which was 1.77-fold higher than that of the latter. Additionally, amino-activated immobilization increased the affinity of the enzyme to the substrate, and its maximum reaction rate (0.68 μmol/min) was superior to that of carboxyl-activated immobilization (0.53 μmol/min). The visualization results showed that the catalytic site of β-agarase after carboxyl-activated immobilization was more susceptible to the immobilization process, and the orientation of the enzyme may also hinder substrate binding and product release. These results suggest that by pre-selecting appropriate activation sites and enzyme orientation, immobilized enzymes with higher catalytic activity and stability can be obtained, making them more suitable for the application of continuous production.

From PET bottle waste to enzyme support for removal of estrogens from wastewaters

Recycling poly(ethylene terephthalate) disposables into electrospun functional membranes creates new opportunities for pollution reduction. In this study, two types of electrospun membranes made of poly(ethylene terephthalate) (PET) recycled from bottles and PET recycled from recycled bottles (rPET) were utilized in order to produce four types of biosystems with immobilized laccase for the removal of synthetic hormones micropollutants from wastewater. The enzyme was adsorbed onto the fibers directly or after a prior 24 h treatment with 10% glutaraldehyde. Obtained removal efficiency of 17α-ethynylestradiol from aqueous systems was in a range of 71% to 85%, depending on the type of PET utilized for the fabrication of the membranes and the way of laccase immobilization. Moreover, fabricated biosystems showed highly improved storage stability of 72% to 85% compared to only 55% activity exhibited by free laccase after 20 days of storage. Immobilization of the laccase on the PET-based fibrous substrates also increased their thermal stability after 2 h at 50 °C with preserved activity in a range of 27% to 55%, compared to only 5% registered for the free enzyme. Produced biosystems exhibited high reusability and the activity of the enzyme remains over 60% after seven catalytic cycles for all tested samples, with 79% activity after eight cycles obtained for the enzyme immobilized on PET membranes modified with glutaraldehyde prior to immobilization of laccase. The presented approach has been proven to have great potential as a sustainable tool for the enzymatic removal of synthetic hormones from wastewaters showing superior properties compared to the utilization of the enzyme in its basic form.

Comparative study for enzymatic hydrolysis of sugarcane bagasse using free and nanoparticle immobilized holocellulolytic enzyme cocktail

This study aimed at a comparative analysis of enzymatic hydrolysis of sugarcane bagasse biomass by using a free holocellulase cocktail of an enzyme (Cellic CTec2) and immobilized enzyme system on IOMNP@SiO2-NH2 support to make the process cost-effective. The immobilization was substantiated by examining the nano biocatalysts using FTIR, TEM, and DLS analysis. IOMNP@SiO2 –NH2 and immobilized enzyme system have average particle sizes of 81.1, and 455.9 nm confirmed by DLS. Response surface methodology (RSM) based on the Box Behnken design was employed to optimize the saccharification process conditions. The free cellulase cocktail showed a maximum hydrolysis yield of 70.71 % at 12.5FPU/gm enzyme dose, while the immobilized enzyme at a dose of 20 FPU/gm achieved a conversion rate of 74.19 % under optimized parameters likes hydrolysis time, surfactant and substrate concentration. Pretreated sugarcane bagasse substrate was used to evaluate the reusability potential of magnetically immobilized cellulase enzyme compared to recovered free enzyme up to 5 cycles. In the second and third cycles, the conversion rates of cellulose to glucose were determined to be 68.21 %, and 52.6 %, respectively. The findings of this study demonstrate that the immobilized Cellic CTec2 enzyme cocktail on functionalized IOMNP is effective for hydrolyzing sugarcane bagasse which also provides the advantages of easy recovery and reusability of enzyme for multiple hydrolysis cycles.

Exploring the impact of taurine on the biochemical properties of urate oxidase: response surface methodology and molecular dynamics simulation

This paper investigates the impact of taurine as an additive on the structural and functional stability of urate oxidase. First, the effect of the processing parameters for the stabilization of Urate Oxidase (UOX) using taurine was examined using the response surface methodology (RSM) and the central composite design (CCD) model. Also, the study examines thermodynamic and kinetic parameters as well as structural changes of urate oxidase with and without taurine. Fluorescence intensity changes indicated static quenching during taurine binding. The obtained result indicates that taurine has the ability to preserve the native structural conformation of UOX. Furthermore, molecular dynamics simulation is conducted in order to get insights into the alterations in the structure of urate oxidase in the absence and presence of taurine under optimal conditions. The molecular dynamics simulation section investigated the formation of hydrogen bonds (H-bonds) between different components as well as analysis of root mean square deviation (RMSD), root mean square fluctuations (RMSF) and secondary structure. Lower Cα-RMSD and RMSF values indicate greater stabilization of the taurine-treated UOX structure compared to the free enzyme. The results of molecular docking indicate that the binding of taurine to the UOX enzyme through hydrophobic interactions is associated with a negative value for the Gibbs free energy.

Determination of chloropropanol esters and glycidyl esters in instant noodles based on solid-phase microextraction with chitosan-β-cyclodextrin coated fiber

We developed a method for the determination of chloropropanol esters and glycidyl esters (GE) in instant noodles using solid-phase microextraction with chitosan-β-cyclodextrin (CS-β-CD) coated fiber coupled with gas chromatography-tandem mass spectrometry. The developed low-cost fiber coating can improve the sensitivity of the method. Immobilized enzymes can improve operational stability and reusability compared to free enzymes, thereby reducing costs. The adsorption isotherm was modeled using the Langmuir model, while the adsorption kinetics followed the pseudo second-order model. The limit of detection was 0.3 ng/L. The method exhibited satisfactory recoveries for the analytes, ranging from 80.2 % to 105.3 %, with relative standard deviations < 9.9 %. Furthermore, the results of the exposure assessment showed that chloropropanol esters do not pose unacceptable risks to different age groups. However, the margin of exposure for GE suggested a potential health risk for populations between the ages of 3 and 12 years old.

Enhancement of photocatalytic performance of cu-decorated bifunctional g-C3N4-laccase ICPB system

Printing and dyeing wastewater contains high concentrations of dyes which have been released into the environment and lead serious threat to the ecological environment and human health. This article uses a covalent grafting method to immobilize laccase onto mesoporous g-C3N4 (CN) and Cu is doped to improve the degradation performance of intimate coupling photocatalysis and biodegradation (ICPB) system. The crystal structure of the CN is no changed after the immobilization of laccase. In addition, more oxygen-containing functional groups of the immobilized carrier is increased that improve the separation efficiency of photogenerated electron hole pairs of the original carrier. Compared to free enzymes, mesoporous g-C3N4 immobilized laccase (CN-LC) and copper doped mesoporous g-C3N4 immobilized laccase (Cu-CN-LC) both have a wider adaptation range and better stability. Because the addition of Cu, the PL of Cu-CN-LC becomes significantly weaker, and the bandgap is decreased. The removal efficiency of Cu-CN-LC for methyl orange and active blue is 1.34 times and 1.66 times higher than of CN and possessed good reusability. A novel technical contribution of ICPB proposed in this work is a feasible solution for printing and dyeing wastewater.

Preparation of amino-functionalized polyethylene-silica composite membrane and FDH immobilization

Enzymatic CO2 conversion represents a highly efficient, energy-conserving, and environmentally sustainable approach to mitigating the increasing pressure caused by huge carbon emissions and generating valuable products. However, several challenges associate with the process including the instability and recovery difficulty of free enzyme, as well as low solubility of CO2 in mild conditions. Immobilizing enzyme on the amino-functionalized supports presents a viable solution to these issues. Considering the wide application of polymer hollow fiber membranes in the field of CO2 gas–liquid membrane contactor and the characteristics of hydrophilic, biocompatible, and high specific surface area of silica nanoparticles, the amino-functionalized polyethylene (PE)-silica (APS) composite membranes were fabricated via different routes. The morphologies, the loading rates of modified SiO2 and reusability of APS membranes were characterized for a crude screening. Following that, seven kinds of APS membranes were used as the carriers of formate dehydrogenase (FDH) that could catalyze the reduction of CO2 to formate, and their immobilization efficiency and catalytic performance were compared. The results show that 1# and 2# membranes based on carboxyl-modified PE with a loading rate of 5.5 %∼5.6 % and the reusability of above 99 % were more ideal carriers than others. The enzyme activity recovery rate of APS-immobilized FDH (FDH-APS) was separately 367 % and 256 %, proving the intensification action of carriers on the enzymatic reaction. After 10 cycles of use, the relative activity of two kinds of FDH-APS was around 70 % and 78 %, much higher than corresponding particles or membrane attached FDH (25.3 % and 20.4 %). After storage at 4 °C for 20 days, the relative activity of FDH-APS was 82.7 % and 81.0 %, while it was only 61.4 % for free enzyme.

Affinity-assisted covalent self-assembly of PduQ-SpyTag and Nox-SpyCatcher to construct multi-enzyme complexes on the surface of magnetic microsphere modified with chelated Ni2+

It is of great significance to study the effect of multi-enzyme aggregation behavior at the interface on the formation of multi-enzyme complexes and their co-catalytic characteristics, which is helpful for us to design and construct immobilized multi-enzyme complex systems for in vitro synthetic biology. Here, a magnetic microsphere with chelated Ni2+, was prepared to explore the self-assembly characteristics of PduQ-SpyTag (P-T) and Nox-SpyCatcher (NC) on its surface, based on the mixed interaction mode consisting the affinity of His-tag/Ni2+ and covalent binding of SpyTag/SpyCatcher. After studying the effect of saturated or unsaturated adsorption of P-T on the covalent binding between P-T and NC at the interface, a possible multienzyme interaction mechanism for the affinity-assisted covalent self-assembly on the Ni2+ chelating surface was proposed. The time evolution of NADH showed that the immobilized P-T/N-C complex formed by this method and the free P-T/N-C complex exhibited similar synergistic catalytic properties, and presented higher catalytic efficiency than the simple mixing of P-T and NC. The optimal catalytic conditions, stability and reusability of the immobilized multi-enzyme complexes prepared in this study were also discussed by comparing them with free enzymes. In this study, we demonstrate a simple and effective strategy for self-assembling SpyTag/SpyCatcher fusion proteins on the surface of magnetic beads, which is inspiring for the construction of more cascade enzyme systems at the interface. It provides a new method for facilitating the rapid construction of immobilized multi-enzyme complexes in vitro from the crude cell lysis.

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.

Multi-point immobilization of GH 11 endo-β-1,4-xylanase on magnetic MOF composites for higher yield of xylo-oligosaccharides

GH 11 endo-β-1,4-xylanase (Xy) was a crucial enzyme for xylooligosaccharides (XOS) production. The lower reusability and higher cost of purification has limited the industrial application of Xy. Addressing these challenges, our study utilized various immobilization techniques, different supports and forces for Xy immobilization. This study presents a new method in the development of Fe3O4@PDA@MOF-Xy which is immobilized via multi-point interaction forces, demonstrating a significant advancement in protein loading capacity (80.67 mg/g), and exhibiting remarkable tolerance to acidic and alkaline conditions. This method significantly improved Xy reusability and efficiency for industrial applications, maintaining 60 % activity over 10 cycles. Approximately 23 % XOS production was achieved by Fe3O4@PDA@MOF-Xy. Moreover, the yield of XOS from cobcorn xylan using this system was 1.15 times higher than that of the free enzyme system. These results provide a theoretical and applicative basis for enzyme immobilization and XOS industrial production.

Circadian-based approach for improving physiological, phytochemical and chloroplast proteome in Spinacia oleracea under salinity stress and light emitting diodes

Salt stress is a recognized annihilating abiotic stress that has a significant impact on agricultural and horticulture crop productivity. Plant development faces three distinct dangers as a result of salt stress: oxidative stress, osmotic stress, and ionic toxicity. It has been shown that plants can forecast diurnal patterns using the circadian clock; moreover, they can manage their defensive mechanism for the detoxification of reactive oxygen species (ROS). Circadian rhythmicity in gene expression assembles transcription and translation feedback networks to govern plant shape, physiology, cellular and molecular activities. Both external and internal variables influence the systemic rhythm via input routes. The Malav Jyoti (MJ) and Delhi Green (DG) genotypes of spinach (Spinacia oleracea) were grown in the plant growth chamber. The chamber had an optimized temperature of 25 °C and humidity of 65% containing light emitting diode (LED) having Red: Blue: white (one side) and White fluorescent (other side) under salinity stress. The samples were collected on the basis of 4 h intervals of circadian hours (0 h, 4 h, 8 h and 12 h) during Day-10 and Day-20 of salt treatments. Under salt stress, the circadian and light-emitting diode-based strategy had a substantial influence on spinach's anti-oxidative responses, stomatal movement, CO2 assimilation, PS-I and II efficiency, phytochrome pigment efficiency, and photosynthesis. Based on the findings of the free radical scavenging enzyme tests, the photoperiodic hours for the proteome analysis were set to 11 am and 3 pm on Day-20. When compared to white fluorescent, this study found that LED has the capacity to influence the entrainment cues of the circadian clock in the cultivation of salt-sensitive spinach genotypes. According to our findings, changing the cellular scavenging mechanism and chloroplast proteome has increased the survival rate of spinach genotypes under LED when compared to white fluorescent.

Xylanase immobilization using activated carrier of gellan gum-agar beads: Improved stability and catalytic activity for the production of antioxidant and anti-proliferative xylooligosaccharides

Immobilization of xylanase produced by the fungus, Aspergillus flavus, was achieved utilizing the activated gellan gum-agar as a carrier. The immobilization conditions were optimized based on Box-Behnken design which comprised the addition of agar (1.23%) to gellan-gum (GL) and processing the obtained beads at pH 7.7 with 1.62% polyethyleneimine (PEI) as the optimum conditions. Alteration of the surface morphologies and elemental constituents of the carrier during the synthesis and immobilization process were inspected using energy-dispersive X-ray analysis (EDX) and scan electron microscope (SEM). The yield and efficiency of the immobilization process were 45.85% and 97.41%, respectively. The immobilized enzyme possessed the highest activity at pH 5–8 and temperature up to 70 °C with activation energy lower than half that of the free form. Higher half-lives and Gibbs free energy were achieved for the immobilized enzyme, sustaining the suitability of the used carrier for promoting enzyme stability. Moreover, the negative entropy values for both free enzyme and the immobilized forms estimated the high stability of the enzyme during the thermal deactivation process. The Km value of the immobilized form was lower than that of the free enzyme, reflecting its enhanced affinity toward xylan substrate. Furthermore, the immobilized enzyme retained 99.3 and 85.7% of its initial xylan hydrolyzing activity after 7, and 11 cycles, respectively. Finally, the applicability of the immobilized form in the hydrolysis of xylan and production of antioxidant and breast cancer anti-proliferative xylooligosaccharide mixture was also estimated.