Immobilization Of Β-glucosidase Research Articles

Facile Construction of Stable Hierarchical Porous Metal–Organic Frameworks for In Situ Immobilization of β-Glucosidase Based on Competitive Coordination and Selective Etching Strategies

Facile Construction of Stable Hierarchical Porous Metal–Organic Frameworks for In Situ Immobilization of β-Glucosidase Based on Competitive Coordination and Selective Etching Strategies

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.

Hybrid Nano Supports of Nano Polyaniline, Magnetic Iron Oxide Nanoparticles and Nano Clay as Matrices for The Immobilization of β-Glucosidase, Fructosyltransferase and Carboxymethyl Cellulase

Hybrid Nano Supports of Nano Polyaniline, Magnetic Iron Oxide Nanoparticles and Nano Clay as Matrices for The Immobilization of β-Glucosidase, Fructosyltransferase and Carboxymethyl Cellulase

Immobilization of β-glucosidase on polyethersulfone membrane for cellobiose hydrolysis

Glucosidase is one of the enzyme cocktail that is used for converting lignocellulose to glucose. The significance of glucosidase is that it converts the cellobiose to glucose and subsequently avoid product inhibition caused by cellobiose. In this work, β-glucosidase was immobilized on polyethersulfone membrane via glutardehyde crosslinking for the hydrolysis of cellobiose. Based on the peaks of FTIR spectra of the membrane, enzyme was adsorbed based on the absorbance between 3100 and 3500 cm−1. The percentage of enzyme adsorption in the membrane was 6.8 % based on the initial protein solution. Kinetic parameters of β-glucosidase of immobilized on membrane have been studied and compared with free enzyme used. The Km values for the free and immobilized gained were 166.137 g/L and 36.176 g/L respectively. The immobilized enzyme showed lower Km indicating higher affinity of substrates than free enzyme. The Vmax value obtained was 60.606 g/L h for immobilized enzyme and 136.986 g/L h for free enzyme probably due to limited access of substrates towards the immobilized enzymes. The immobilized enzyme can be reused for at least seven cycles.

One-pot synthesis of rod-like lignin@zeolitic imidazolate framework-8 with enhanced immobilization of β-glucosidase

Developing value-added lignin-based biomaterials for enzyme immobilization has drawn great attention. This study developed a cost-effective and eco-friendly one-pot strategy to synthesize the rod-like lignin@zeolitic imidazolate framework-8 (ZIF-8) hybrid nanomaterial by using lignin derived from lignocellulosic biofuel production. The incorporation of lignin into the ZIF-8 network not only altered the morphology, but also modified the surface properties of the material, making it an ideal support for enzyme immobilization. The synthesized nanoscale hybrid materials were used to immobilize beta-glucosidase (BG) with high immobilization capacity and about 92–166 mg/g of BG was immobilized through physical adsorption. The immobilized BG exhibited good stability, catalytic activity and recycling properties, and was reused under acidic conditions for more than 8 cycles with more than 60% activity kept. The as-prepared hybrid material can serve as a great carrier for immobilizing various biomolecules.

Polyester fabric modification by chemical treatment to enhancing the β-glucosidase immobilization

This work reports the first approach to immobilizing the β-glucosidase enzyme on a modified polyester fabric support matrix. Herein, polyester fabric was successfully fabricated with hydrazide groups incorporated with graphene oxide, followed by glyoxal as the crosslinker. Various techniques, including Fourier transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, differential scanning calorimeter, and zeta potential analysis, were used to investigate their microstructural, dispersive, thermal, and physicochemical properties. β-glucosidase immobilization exhibited maximum activity at pH 6.0 with an immobilization yield (89.5%), immobilization efficiency (92%), and enzyme activity yields (82.3%). After fifteen reaction cycles, the remaining enzyme activity was 59%. Stored at 4 °C, immobilized β-glucosidase retained 74% of its activity, compared to a retain of 43% for soluble β-glucosidase, during the 6-weeks period. Soluble and immobilized enzyme exhibited similar optimal catalytic temperature at 60 °C, while the optimal catalytic pH was 5 and 6, respectively. Both soluble and immobilized β-glucosidase presented Michaelis–Menten kinetics with Vmax values of 1.82 and 2.94 U/mg, and Km values of 2.94 and 5.15 mM, respectively. This research provided a potential directed immobilization method for β-glucosidase, and robust biocatalyst for industrial applications.

Reusability of immobilized β-glucosidase on sodium alginate-coated magnetic nanoparticles and high productivity applications

The purpose of this study is to detect the impact of the covalent immobilization of β-glucosidase (β-Glu) from Thermatoga maritima on sodium alginate-based magnetically recyclable iron nanoparticles (SA-Fe3O4 MNPs) for boosting robustness, facile retrieval, and recycling of enzyme. We developed a novel green method to amend the transformation of lactose to galacto-oligosaccharides (GOS). The interaction of the Immobilized β-Glu and SA-Fe3O4 MNPs were characterized by SDS-PAGE, Transmission electron microscope (TEM) and FT-IR spectroscopy. After immobilization, the ideal pH maintained 7.0, the optimum temperature (temp.) increased to 85 °C and deceptive Km and Vmax raised by 65 and 41%, respectively. Immobilization provided low activation energy and enhanced pH and heat stability. The SA-Fe3O4 MNPs maintained the effectiveness of enzyme by 39.52% up to the 6th cycle of reuse and the hydrolysis power exceed 28.13% of lactose to GOS. Conclusively, thermal stability, improved pH, and excellent reusability over β-Glu make SA-Fe3O4 MNPs a viable option for long-term GOS production as well as other industrial uses.

One-step immobilization of β-glucosidase in crude enzyme solution by recyclable UCST-responsive polymer with enhanced uniformly biocatalytic performance

In this study, the upper critical solution temperature (UCST)-responsive polymers poly (ethylene oxide) monomethyl ether-block-poly(acrylamide-co-acrylonitrile) (PEG-b-p(AAM-co-AN) were synthesized and successfully utilized to immobilize β-glucosidase in crude enzyme solution. These UCST-responsive β-glucosidase biocatalysts (PEG-b-p(AAM-co-AN@LytA-Glu) have specific UCST with tunable transition temperature, which could be tuned the separation temperature to the desired temperature range. The P2 @ LytA-Glu with an UCST of about 42.9 ℃ was exploited by one-step covalent immobilization of β-glucosidase in crude enzyme solution. The prepared P2 @ LytA-Glu exhibited significantly improved temperature, pH, storage, and operation stabilities compared with that of free enzyme. The catalytic rate of P2 @ Glu-LytA was 14.5% higher than that of P2-Glu (immobilized pure β-glucosidase), which indicated that one-step immobilization of crude enzyme directly from crude enzyme solution was feasible, and it can greatly save the purification step and reduce the experimental cost. The engineered UCST-responsive immobilized enzymes are potentially useful for the practical green biocatalysis.

Adsorption and immobilization of β-glucosidase from Thermoascus aurantiacus on macroporous cryogel by hydrophobic interaction

Enzyme immobilization has been reported as a promising approach to improving parameters such as thermal stability, pH and reusability. In this study, a polyacrylamide cryogel functionalized with L-phenylalanine was prepared to be used in the adsorption of β-glucosidase from Thermoascus aurantiacus, aiming at its separation and also its immobilization on the cryogel matrix. The enzyme was produced by solid state fermentation. First, the adsorption was studied as a function of the pH and the resulting yield (Y, %) and purification factor (PF , dimensionless) were determined (1.57–5.13 and 64.19–91.20, respectively). The PF and yield from eluate samples obtained at pH 3.0 were the highest (5.13 and 91.20, respectively). Then, β-glucosidase was immobilized on the hydrophobic cryogel and the recovery activities (%) were determined as a function of temperature and in the presence of different saline solutions. The values ranged from 14.45 to 45.97. As expected, salt type and ionic strength affected the activity remained in the immobilized β-glucosidase. The average bioreactor activity was 39.9 U/g of dry cryogel and its operational stability was measured, with no decrease in activity being observed during seven cycles. Kinetic parameters of free and immobilized enzyme were determined according to different models.

The effect of nano-calcium carbonate on β-glucosidase immobilized by alginate and chitosan

Nanotechnology has become the most promising domain to boost the efficiency of enzymes. Enzymes are vital as a green catalyst in many industries, food, pharmaceutical and biomedical, etc. The immobilization process of the enzyme increases its catalytic properties. In this research, a novel method is presented to describe the effect of nano-calcium carbonate on the characteristics of immobilized β-glucosidase, which was extracted from the Agrocybe aegirit. The nano-CaCO3 was produced using the eco-friendly natural deep eutectic solvent. The pure nano -CaCO3 was observed as vaterite, with a size of about 300 nm. The nano-calcium carbonate was coated by a natural polymer sodium alginate compound and then adsorbed chitosan. Further, this obtained composite is cross-linked by the bioactive genipin to immobilize the β-glucosidase. The enzyme/protein loading ratio to the supports was 1:4, respectively. The recovery efficiency of immobilized β-glucosidase was 89.3%, and immobilization yield was 96.452%. Chitosan-coated nano-CaCO3 was used as a carrier for immobilization of β-glucosidase to improve its stability and reusability. In addition to stability and reusability, pH tolerance, temperature tolerance, and enzyme kinetics are the significant parameters that illustrate the proficiency of an immobilized enzyme. The measured optimal enzymatic reaction conditions for the immobilized β-glucosidase were 50 °C and pH 6. Furthermore, it has shown noticeable improvements in thermo-stability and pH tolerance. Temperature tolerance was observed 50% to the initial activity of the immobilized enzyme even after the 3 h of incubation at 50 °C, while pH tolerance was noticed more than 50% and 40% at pH 7 and 8, respectively. The Km and Vmax values of free and immobilized β-glucosidase to 4-nitrophenyl β-D-glucopyranoside were 1.549 μmol/L/min, 0.346 mmol/L and 0.532 μmol/L/min, 0.080 mmol/L, respectively. The immobilized β-glucosidase retains its storability 80% even after 30 days of storage at 4 °C and maintains 93.1% of its residual activity by reusing up to 10 cycles.

Magnetically recyclable catalytic nanoparticles grafted with Bacillus subtilis β-glucosidase for efficient cellobiose hydrolysis

This study reports covalent immobilization of β-glucosidase (BGL) from Bacillus subtilis PS on magnetically recyclable iron nanoparticles for enhancing robustness, facile recovery and reuse of enzyme. Immobilized BGL iron nanoparticles (BGL-INPs) were characterized by various biophysical techniques viz. TEM, DLS, FTIR and CD spectroscopy. The efficiency and yield of immobilization were 89.78 and 84.80%, respectively. After immobilization, optimum pH remained 6.0 whereas optimum temperature upraised to 70 °C whereas apparent Km and Vmax shifted from 0.819 mM to 0.941 mM and 54.46 to 57.67 μmole/min/mg, respectively. Immobilization conferred lower activation energy and improved pH and thermal stabilities. The BGL-INPs retained 85% activity up to 10th cycle of reuse and hydrolyzed more than 90% of cellobiose to glucose within 30 min. Conclusively, improved pH, thermal stability and excellent reusability over free enzyme make BGL-INPs a promising candidate for sustainable bioethanol production and other industrial applications.

Immobilization of β-Glucosidase over Structured Cordierite Monoliths Washcoated with Wrinkled Silica Nanoparticles

The enzymatic conversion of biomass-derived compounds represents a key step in the biorefinery flowsheet, allowing low-temperature high-efficiency reactions. β-Glucosidases are able to hydrolyze cellobiose into glucose. Wrinkled silica nanoparticles (WSNs) were demonstrated to be a good support for the immobilization of β-glucosidases, showing better performance than free enzymes in batch reaction; on the other hand, immobilized enzyme microreactors (IEMs) are receiving significant attention, because small quantities of reagents can be used, and favorable heat and mass transfer can be achieved with respect to conventional batch systems. In this work, we prepared, characterized, and tested structured enzymatic reactor compounds by a honeycomb monolith, a WSN washcoat, and β-glucosidases as the active phase. Powder and structured materials were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), N2 physisorption, thermogravimetric analysis (TGA), and Fourier-transform infrared spectroscopy (FT-IR). Structured catalysts were tested under both batch and continuous flow reaction conditions and compared to powder catalysts (batch reaction). The WSN washcoat was attached well onto the monolith walls, as suggested by the negligible weight loss after ultrasound treatment; the WSNs preserved their shape, porosity, and individual nature when deposited onto the monolith walls. The immobilized enzyme microreactors proved to be very efficient in hydrolysis of cellobiose to glucose, showing a complete conversion under continuous flow reaction at a batch-equivalent contact time equal to 120 min vs. 24 h obtained in the batch experiments. The apparent KM value showed a 20-fold decrease with respect to the batch process, due to the absence of external diffusive transport limitations.

Stabilization of Glycosylated β-Glucosidase by Intramolecular Crosslinking Between Oxidized Glycosidic Chains and Lysine Residues.

Many industrial enzymes can be highly glycosylated, including the β-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized β-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of β-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of β-glucosidase from Aspergillus niger and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme.

Mesoporous silica nanoparticles for β-glucosidase immobilization by templating with a green material: Tannic acid

Bioconversion of lignocellulosic biomass to biofuels through enzymatic hydrolysis has become a promising strategy to reduce our dependence on fossil fuels. β-Glucosidase (BG) is a key enzyme in the enzymatic degradation of cellulose. However, free β-glucosidase is relatively unstable, costly and difficult to recover. Immobilization of β-glucosidase may help to improve its reusability and stability, allowing developing a sustainable low-carbon and resource-efficient circular economy. BG was immobilized by adsorption on synthesised tannic acid templated mesoporous silica nanoparticles (TA-MSNPs). The synthesis procedure adopts a green templating agent cheap and biocompatible. Textural and morphological characterization was performed. We studied the effect induced by the morphology, pore size and surface features of supports, by using different amounts of templating agent, on the catalytic performance of the biocatalyst. The immobilization procedure used preserved most of the secondary structure of the enzyme and consequently its catalytic activity. The biocatalyst exhibiting the best activity and operational stability is obtained with the sorbent prepared with the largest quantity of tannic acid. In this case, the mesopores are too small for the enzyme to enter them, and the large amount of adsorbed enzyme is due to its peculiar surface features, favouring the formation of a multilayer enzyme corona.

Immobilization of \u03b2-Glucosidase from Thermatoga maritima on Chitin-functionalized Magnetic Nanoparticle via a Novel Thermostable Chitin-binding Domain

Enzyme immobilization is a powerful tool not only as a protective agent against harsh reaction conditions but also for the enhancement of enzyme activity, stability, reusability, and for the improvement of enzyme properties as well. Herein, immobilization of β-glucosidase from Thermotoga maritima (Tm-β-Glu) on magnetic nanoparticles (MNPs) functionalized with chitin (Ch) was investigated. This technology showed a novel thermostable chitin-binding domain (Tt-ChBD), which is more desirable in a wide range of large-scale applications. This exclusive approach was fabricated to improve the Galacto-oligosaccharide (GOS) production from a cheap and abundant by-product such as lactose through a novel green synthesis route. Additionally, SDS-PAGE, enzyme activity kinetics, transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) revealed that among the immobilization strategies for Thermotoga maritime-β-Glucosidase thermostable chitin-binding domain (Tm-β-Glu-Tt-ChBD) on the attractive substrate; Ch-MNPs had the highest enzyme binding capacity and GOS production ratio when compared to the native enzyme. More interestingly, a magnetic separation technique was successfully employed in recycling the immobilized Tm-β-Glu for repetitive batch-wise GOS without significant loss or reduction of enzyme activity. This immobilization system displayed an operative stability status under various parameters, for instance, temperature, pH, thermal conditions, storage stabilities, and enzyme kinetics when compared with the native enzyme. Conclusively, the GOS yield and residual activity of the immobilized enzyme after the 10th cycles were 31.23% and 66%, respectively. Whereas the GOS yield from native enzyme synthesis was just 25% after 12 h in the first batch. This study recommends applying Tt-ChBD in the immobilization process of Tm-β-Glu on Ch-MNPs to produce a low-cost GOS as a new eco-friendly process besides increasing the biostability and efficiency of the immobilized enzyme.

Organosolv lignin/Fe3O4 nanoparticles applied as a β-glucosidase immobilization support and adsorbent for textile dye removal

In this paper, the liquor obtained from organosolv pretreatment of green coconut fiber (GCF) was used for the synthesis of lignin/Fe3O4 nanoparticles. The coating of Fe3O4 nanoparticles was evaluated by X-ray and dynamic light scattering analysis. β-glucosidase immobilized on lignin/Fe3O4 nanoparticles by covalent bonding exhibited better stability than free enzyme. Compared to the free enzyme, β-glucosidase immobilized on lignin/Fe3O4 nanoparticles achieved good digestibility performance of crystalline cellulose (∼ 21.9 g/L reducing sugars) and delignified GCF (∼ 6.4 g/L reducing sugars). In adsorption experiments, lignin/Fe3O4 nanoparticles exhibited low equilibration time and high adsorption capacity of Methylene blue (203.66 mg/g), Cibacron blue (112.36 mg/g) and Remazol red (96.46 mg/g) dyes. After four cycles, the adsorption of the Methylene blue dye on lignin/Fe3O4 nanoparticles reduced 43.68 % using 0.1 M HCl as eluent.

Covalent Immobilization of β-Glucosidase into Mesoporous Silica Nanoparticles from Anhydrous Acetone Enhances Its Catalytic Performance.

An immobilization protocol of a model enzyme into silica nanoparticles was applied. This protocol exploited the use of the bifunctional molecule triethoxysilylpropylisocyanate (TEPI) for covalent binding through a linker of suitable length. The enzyme β-glucosidase (BG) was anchored onto wrinkled silica nanoparticles (WSNs). BG represents a bottleneck in the conversion of lignocellulosic biomass into biofuels through cellulose hydrolysis and fermentation. The key aspect of the procedure was the use of an organic solvent (anhydrous acetone) in which the enzyme was not soluble. This aimed to restrict its conformational changes and thus preserve its native structure. This approach led to a biocatalyst with improved thermal stability, characterized by high immobilization efficiency and yield. It was found that the apparent KM value was about half of that of the free enzyme. The Vmax was about the same than that of the free enzyme. The biocatalyst showed a high operational stability, losing only 30% of its activity after seven reuses.

Fabrication of three-dimensional porous cellulose microsphere bioreactor for biotransformation of polydatin to resveratrol from Polygonum cuspidatum Siebold & Zucc

In this study, in order to bio-transform polydatin to resveratrol from Polygonum cuspidatum extract, a 3D-porous structured cellulose microsphere (CMs) immobilized with β-glucosidase acted as a micro-bioreactor. To successfully immobilize β-glucosidase, CMs was treated with epoxidation to attach three different functional amion groups, and finally combined with β-glucosidase. CMs provided large surface area (32.0 m²/g) and many sites for immobilization of β-glucosidase and chemical modification. The porosity of CMs was 90 % and its density was about 1.00 g/m3. According to SEM images, the 3D-porous structure was obviously found and the result of FTIR proved the successful modification of amino groups. The crystal structure of CMs was changed from crystalline cellulose I-β to crystalline cellulose II during the sol-gel method. In the biotransformation tests of the Polygonum cuspidatum extracts, the conversion rate of polydatin reached 93.06 %, and after conversion, the concentration of resveratrol was two times higher than the original Polygonum cuspidatum extract. In addition, the cellulose microsphere micro-bioreactor also possessed good reusability, which could maintain 89.2 % activity after being repeatedly operated 8 times. Therefore, this cellulose microsphere micro-bioreactor can be successfully used to produce resveratrol from polydatin, and this micro-bioreactor may also have the potential to convert other plant glycosides into aglycones. Moreover, because of their good biocompatibility, the 3D porous cellulose microspheres can also be used for immobilization of other enzymes to prepare efficient bioreactors.

Immobilization of β-glucosidase by self-catalysis and compared to crosslinking with glutaraldehyde

Nanoparticles have been successfully used for immobilization of different enzymes. The enzyme immobilized by nanomaterials has promising application in the biotechnological industry. The β-glucosidase is very important in industrial field. The immobilization of β-glucosidase by nanomaterials increases its activity and reusability. In this work, the β-glucosidase was extracted from fruiting bodies of Agrocybe aegirit and was purified by anion exchange. The β-glucosidase was immobilized by SiO2 nanoparticles (nano-SiO2) crosslink with glutaraldehyde (GA). On the other hand, the β-glucosidase firstly immobilized by process adsorption and then crosslink with genipin (GP) which was produced by the hydrolysis of geniposide by β-glucosidase. Then we compared the properties of both immobilized GA-crosslinked β-glucosidase and GP-crosslinked β-glucosidase, their immobilization yields were 83.34% and 96.29% respectively. The GA-crosslinked β-glucosidase and free β-glucosidase revealed optimal pH at 6.0. The optimal pH of GP-crosslinked β-glucosidase was ranging between 4.5 and 7.5. The GA-crosslinked β-glucosidase and GP-crosslinked β-glucosidase revealed the optimal temperature at 50 °C, 70 °C respectively. For the free β-glucosidase, the optimal temperature was 55 °C. Furthermore, the GA-crosslinked β-glucosidase and GP-crosslinked β-glucosidase were characterized by scanning electron microscope (SEM). The geniposide consumption in the reaction system of GA-crosslinked β-glucosidase was analyzed by high performance liquid chromatography (HPLC).

Green construction of recyclable amino-tannic acid modified magnetic nanoparticles: Application for β-glucosidase immobilization

The β-glucosidase (BGL) enzyme in food industry is great interest due to its role in food conversion to produce functional food products. In this study, the BGL was covalently immobilized onto amino-tannic acid modified Fe3O4 magnetic nanoparticles (ATA-Fe3O4 MNPs) as biocompatible nanoplatform by modified poly-aldehyde pullulan (PAP) as a cross-linker to enhance the ability and strength of the nanoparticle connection to the enzyme. The properties of support were subsequently characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier transformed infrared (FTIR), X-ray diffraction (XRD), and vibrating sample magnetometer (VSM). The highest percentage of loading and immobilization yield was obtained with 0.1 mg enzyme/mL citrate buffer (pH 6, 1 M) enzyme solution, carrier solution of 10 mg ATA-Fe3O4/3 mL citrate buffer (pH 6, 1 M), and PAP solution of 20% total reaction system volume. Optimum pH and temperature were found for free (pH 5.0 and temperature 30 °C) and immobilized (pH 6.0 and temperature 40 °C) enzyme. The immobilized BGL maintains its activity to 83% after 10 cycles. Therefore, immobilization of BGL by this method is an efficient procedure to improve the properties of enzyme.