Immobilization Of Α-amylase Research Articles

Covalent immobilization of α-amylase on hollow metal organic framework coated magnetic phase-change microcapsules for the improvement of its thermostability

Exploring efficient immobilized carrier for α-Amylase (α-Amy) to enhance its thermostability has significant influence in starch related industry. Here, hollow ZIF-8 (HZIF-8) with amino groups coated magnetic phase change microcapsules (PCM) was designed for covalent immobilization of α-Amy. Magnetic PCM consisting n-docosane core and SiO2/Fe3O4 hybrid shell were firstly synthesized. Then, HZIF-8 shell with amino groups was coated and α-Amy was subsequently immobilized through covalent cross-linking strategy. The morphology, chemical structure and magnetic property of PCM@HZIF-8@α-Amy (PCMHA) were comprehensively characterized. Moreover, heat control property of PCMHA was studied with encapsulation efficiency and thermal energy-storage efficiency of 50.55 % and 50.59 %, respectively. Catalytic activity of immobilized α-Amy was fully investigated with Km and Vmax of 2.773 mg/mL and 1.853 μmol/mL·min, respectively. From reusability and storage stability study, immobilized α-Amy not only maintained rather high activity after 9 cycles' reuse, but also exhibited good activity under high salt ion condition after 7 days.

Polydopamine-functionalized polyethersulfone membrane: A paradigm advancement in the field of α-amylase stability and immobilization

Biocatalytic membranes have great potential in various industrial sectors, with the immobilization of enzymes being a crucial stage. Immobilizing enzymes through covalent bonds is a complex and time-consuming process for large-scale applications. Polydopamine (PDA) offers a more sustainable and eco-friendly alternative for enzyme immobilization. Therefore, surface modification with polydopamine as mussel-inspired antifouling coatings has increased resistance to fouling. In this study, α-amylase enzyme was covalently bound to a bioactive PDA-coated polyethersulfone (PES) membrane surface using cyanuric chloride as a linker. The optimal activity of α-amylase enzyme immobilized on PES/PDA membrane was obtained at temperature and pH of 55°C and 6.5, respectively. The immobilized enzyme can be reused up to five reaction cycles with 55 % retention of initial activity. Besides, it maintained 60 % of its activity after being stored for five weeks at 4°C. Additionally, the immobilized enzyme demonstrated increased Michaelis constant and maximum velocity values during starch hydrolysis. The results of the biofouling experiment of various membranes in a dead-end cell demonstrated that the PES membrane’s water flux increased from 6722.7 Lmh to 7560.2 Lmh after PDA modification. Although α-amylase immobilization reduced the flux to 7458.5 Lmh due to enhanced hydrophilicity, compared to unmodified membrane. The findings of this study demonstrated that the membrane produced through co-deposition exhibited superior hydrophilicity, enhanced coating stability, and strong antifouling properties, positioning it as a promising candidate for industrial applications.

Artificial antibody-antigen-directed immobilization of α-amylase to hydrolyze starch for cascade reduction of 2-nitro-4-methylphenol to 2-amino-4-methylphenol

Nitrophenol is a hazardous substance that poses a threat to the environment and human health, and its treatment has attracted widespread attention. The purpose of this study is to establish an environmentally friendly α-amylase system for the hydrolysis of starch to reduce nitrophenol to aminophenol through cascade reactions. The α-amylase system was obtained through artificial antibody-antigen-directed immobilization, including the synthesis of artificial antibodies, synthesis of artificial antigens, and affinity assembly. In this process, catechol and protocatechuic aldehyde were used to prepare artificial antibodies and artificial antigens respectively through polymerization and Schiff base reactions. Then, artificial antibodies captured the catechol in the artificial antigen structure to form immobilized α-amylases. Compared with free α-amylase, the immobilized α-amylase showed a good reusability and excellent regenerative ability. Subsequently, the immobilized α-amylase were used in the reaction of catalyzing starch hydrolysis to synthesize 2-amino-4-methylphenol, and the yield of 2-amino-4-methylphenol was 58.88 ± 0.19 %. After 5 consecutive catalytic reactions, a yield of 47.61 ± 1.27 % can still be achieved.

Fabrication of robust layered double-hydroxide nanoparticles for α-Amylase immobilization: Enhancing catalytic performance, stability, and prospective sustainable biocatalysis applications

Fabrication of robust layered double-hydroxide nanoparticles for α-Amylase immobilization: Enhancing catalytic performance, stability, and prospective sustainable biocatalysis applications

IMAC application of extracellular polymeric substances doped composite membranes for α-amylase immobilization and kinetic studies

Immobilized metal affinity chromatography (IMAC), extensively utilized technique in affinity chromatography, has proven to be highly effective in purifying α-amylase, a crucial enzyme in industrial applications. Here, Poly (2-hydroxyethyl methacrylate) (PHEMA) based composite membranes were fabricated using the radical copolymerization technique in a petri dish. Extracellular polymeric materials (EPS) as the metal binding agent. The attachment of Cu2+ ions to the membranes occurred through the utilization of EPSs (Cu2+-EPS). The membranes having Cu2+-EPS (Cu2+-EPS-PM) were subjected to various characterization techniques, including scanning electron microscopy (SEM), attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR), swelling tests, and surface area measurements. The investigation also encompassed the examination of various factors, including pH, initial α-amylase concentration, temperature, and ionic strength, that exert an influence on the adsorption of α-amylase from an aqueous medium. The Cu2+-EPS-PMs demonstrated maximum adsorption capacity with a value of 54.2 mg/g polymer (pH 5.0 sodium acetate buffer, temperature: 25°C, initial α-amylase concentration of 2 mg/mL). Additionally, it has been observed that the enzyme is able to be used in adsorption and desorption cycles on Cu2+-EPS-PM many times. The investigation also focused on examining the impact of pH, temperature, storage, and operational stability over the activities of both free and immobilized α-amylase. The outcomes showed that immobilization had positive effects on activity.

Copper-based metal-organic frameworks (BDC-Cu MOFs) as supporters for α-amylase: Stability, reusability, and antioxidant potential

Copper-based metal-organic frameworks (BDC-Cu MOFs) were synthesized via a casting approach using 1,4-benzene dicarboxylic (BDC) as organic ligand and their properties characterized. The obtained materials were then utilized to immobilize the α-amylase enzyme. The chemical composition and functional components of the synthesized support (BDC-Cu MOFs) were investigated with Fourier transform infrared spectroscopy (FTIR), the surface morphology was determined with scanning electron microscopy (SEM), and the elemental composition was established with energy dispersive X-ray (EDX) analyses. X-ray diffraction (XRD) was employed to analyze the crystallinity of the synthesized DBC-Cu MOFs. The zeta potentials of DBC-Cu MOFs and DBC-Cu MOFs@α-amylase were determined. The immobilized α-amylase demonstrated improved catalytic activity and reusability compared to the free form. Covalent attachment of the α-amylase to BDC-Cu provided an immobilization yield (IY%) of 81% and an activity yield (AY%) of 89%. The immobilized α-amylase showed high catalytic activity and 81% retention even after ten cycles. Storage at 4 °C for eight weeks resulted in a 78% activity retention rate for DBC-Cu MOFs@α-amylase and 49% retention for the free α-amylase. The optimum activity occurred at 60 °C for the immobilized form, whereas the free form showed optimal activity at 50 °C. The free and immobilized α-amylase demonstrated peak catalytic activities at pH 6.0. The maximum reaction velocities (Vmax) values were 0.61 U/mg of protein for free α-amylase and 0.37 U/mg of protein for BDC-Cu MOFs@α-amylase, while the Michaelis‒Menten affinity constants (Km) value was lower for the immobilized form (5.46 mM) than for the free form (11.67 mM). Treatments of maize flour and finger millet samples with free and immobilized α-amylase resulted in increased total phenolic contents. The enhanced antioxidant activities of the treated samples were demonstrated with decreased IC50 values in ABTS and DPPH assays. Overall, immobilization of α-amylase on BDC-Cu MOFs provided improved stability and catalytic activity and enhanced the antioxidant potentials of maize flour and finger millet.

Immobilization of α-amylase onto functionalized molybdenum diselenide nanoflowers (MoSe2-NFs) as scaffolds: Characterization, kinetics, and potential applications in starch-based industries

The current study presents the application of molybdenum diselenide nanoflowers (MoSe2-NFs) as an innovative substrate for immobilizing α-amylase by glutaraldehyde activation. This approach results in the development of a nanobiocatalyst that exhibits remarkable advantages compared to a standalone enzyme. Several physical methods, such as fluorescence microscopy, FT-IR, SEM, TEM, XRD, AFM, and Raman spectroscopy, were used to confirm that α-amylase was successfully attached to MoSe2-NFs. By employing the Box-Behnken design of the RSM, the parameters were optimized, resulting in an immobilization efficiency of roughly 87.33%. The immobilized variant of α-amylase demonstrated superior thermostability, pH stability, reusability, and storage stability in comparison to the soluble enzyme. The catalytic activity of α-amylase was highest when immobilized on MoSe2-NFs at the same pH and temperature as the soluble enzyme. However, there was an expansion in the range of parameters in which this activity was observed. Furthermore, the immobilized enzyme exhibited a retention of nearly 80% residual activity following 12 successive reuses. The immobilized enzyme exhibited around 82% residual activity after being stored for 120 days. It is possible that the immobilization process changed the Michaelis-Menten constant, which means that the substrate could no longer reach certain active sites on the enzyme because it had become longer. The study's findings suggest that the α-amylase-MoSe2-NFs system could be useful in industry because it can work in a wider range of temperature and pH conditions. Furthermore, the intrinsic non-toxic characteristics of the matrix, along with its ability to be kept for prolonged periods and recycled, render nano biocatalysts very well-suited for the effective synthesis of maltose in the food and pharmaceutical industries.

A newly synthesized magnetic nanoparticle coated with glycidyl methacrylate monomer and 1,2,4-Triazole: Immobilization of α-Amylase from Bacillus licheniformis for more reuse, stability, and activity in the presence of H2O2

α-Amylase is a secretory enzyme commonly found in nature. The α-Amylase enzyme catalyzes the hydrolysis of α-D-(1,4)-glucosidic bonds in starch, glycogen, and polysaccharides. The chemical characterization of the composite carrier and the immobilized enzyme was performed, and the accuracy of the immobilization was confirmed by FTIR, SEM, and EDS analyses. The X-ray diffraction (XRD) analysis indicates that the magnetic nanoparticle retained its magnetic properties following the modification process. Based on the Thermogravimetric Analysis (TGA) outcomes, it was evident that the structural integrity of the FPT nanocomposite remained unchanged at 200°C. The optimal pH was determined to be 5.5, and no alteration was observed following the immobilization process. Purified α-amylases usually lose their activity rapidly above 50°C. In this study, Bacillus licheniformis α-Amylase enzyme was covalently immobilized on the newly synthesized magnetic composite carrier having more azole functional group. For novelty-designed immobilized enzymes, while there is no change in the pH and optimum operating temperature of the enzyme with immobilization, two essential advantages are provided to reduce enzyme costs: the storage stability and reusability are increased. Furthermore, our immobilization technique enhanced enzyme stability when comparing our immobilized enzyme with the reference enzyme in industrial applications. The activity of the immobilized enzyme was higher in presence of 1–3% H2O2.

Immobilization of α-amylase in calcium alginate-gum odina (CA-GO) beads: An easily recoverable and reusable support

A natural polysacharide, gum odina was collected from Odina wodier tree and purified. Purified gum odina was used with sodium alginate for immobilization of α-amylase. Calcium alginate-gum odina (CA-GO) beads were prepared by ionotropic gelation method to find the improvement of immobilization efficiency and reusability of α-amylase over calcium alginate (CA) beads. XRD, SEM, FTIR, beads diameter, enzyme leaching from beads, moisture content, total soluble matter and swelling study have been carried out to understand the physical morphology and mechanism of immobilization of enzyme in beads matrix. It has been observed that if the polymer ratio changes (keeping enzyme conc. & calcium Chloride conc. constant) then the size and shape of the beads will vary and at a particular range of polymer ratio, the optimal beads forms. At a certain conc.(4%w/v of SA and 1%w/v GO), the immobilization efficiency of CA-GO and CA beads were 92.71 ± 0.85 % (w/w) and 89.19 ± 0.35 %(w/w) respectively. After 8th time use, the CA-GO beads remain (~4 fold) more active than that of CA beads. The FTIR confirms that GO does not interfere with α-Amylase and alginate. Here, it can be concluded that CA-GO beads show better efficiency in respect to immobilization, reusability than CA beads only.

Immobilization of α-amylase on mesoporous silica nanoparticles as a target fishing tool for rapid affinity recognition of an active component from Schisandra chinensis (Turcz.) Baill

α-Amylase inhibitors constitute a category of compounds capable of diminishing α-amylase activity and hold significant promise for the development of novel drugs. This study focused on assessing the potential of Schisandra chinensis Baill extract to inhibit α-amylase. However, sieving and segregating inhibitors from intricate mixtures presents an obstacle. Consequently, this study devised and refined a methodology centered on immobilized α-amylase to identify and isolate natural inhibitors from S. chinensis. The immobilized α-amylase, featuring a load capacity of 254.18 ± 1.20 μg/mg and a specific activity of 2.69 ± 0.01 U/mg, was achieved through meticulous optimization of preparation conditions. Subsequently, this immobilized enzyme was used to isolate bioactive compounds from S. chinensis extract. The results revealed the presence of an active compound with an IC50 of 324.12 ± 11.23 μg/mL. This compound was isolated via magnetic immobilized enzyme-based ligand fishing, employing high-speed countercurrent chromatography, and was conclusively identified as 5-hydroxymethylfurfural. The molecular docking analyses revealed its ability to interact with the active pocket of α-amylase through multiple hydrogen bonds.

Immobilization of Aspergillus fumigatus α-Amylase via Adsorption onto Bentonite/Chitosan for Stability Enhancement

Stability enhancement attempted in this study demonstrated that significant improvement in the stability of the α-amylase isolated from Aspergillus fumigatus was achieved by immobilizing the enzyme on a bentonite/chitosan hybrid matrix using the adsorption method. Centrifugation was used to isolate the α-amylase, which was then refined using (NH4)2SO4 salt precipitation and dialysis. The purity of the α-amylase improved 19.40 times when compared to that of the crude extract. The optimal temperature for free α-amylase is 50˚C, while the optimum temperature for α-amylase/bentonite/chitosan is 60˚C. The KM value of α-amylase/bentonite/chitosan was 1.69 ± 0.08 mg mL-1 substrate and the Vmax value was 52.32 ± 3.29 µmol mL-1 min-1, whereas for free α-amylase, the KM value of 2.56 ± 0.09 mg mL-1 substrate and the Vmax value of 3.78 ± 0.09 µmol mL-1 min-1 were obtained. The ΔGi value of free α-amylase is 102.68 ± 0.30 kJ mol-1 and the t½ is 21.23 ± 0.23 min, whereas the ΔGi value of α-amylase/bentonite/chitosan is 104.43 ± 0.00 kJ mol-1 and the t½ is 94.29 ± 0.91 min. The higher values of ΔGi and t½demonstrated that α-amylase/bentonite/chitosan has better stability than that of free α-amylase. Another important finding is that α-amylase /bentonite/chitosan was able to retain their activity as high as 47.61 ± 0.53% after six recycles, indicating that the enzyme has the potential to be used in industry. Doi: 10.28991/ESJ-2023-07-05-023 Full Text: PDF

Preparation of water-insoluble lignin nanoparticles by deep eutectic solvent and its application as a versatile and biocompatible support for the immobilization of α-amylase

As one of the most abundant biopolymers, lignin is a widely available resource. However, its potential largely remains untapped, with most of it ending up as waste from industries like paper production, pulp processing, and bio-refining. The research undertaken in this study focused on the extraction of lignin from agroforestry waste using a deep eutectic solvent (DES) as a carrier for α-amylase immobilization, resulting in high stability and reusability. Several techniques, including Nuclear Magnetic Resonance (NMR), Scanning Electron Microscopy (SEM), Energy-Dispersive X-ray Spectroscopy (EDS), and the Brunauer-Emmett-Teller (BET) method were employed to examine the structure and morphology of both the extracted lignin and the immobilized enzyme. The temperature used to recover lignin by DES would affect immobilization efficiency and enzyme loading by influencing its specific surface area, pore size, and volume distribution. Investigations using Nuclear Overhauser Effect Spectroscopy (NOESY) uncovered that the hydroxyl groups in G, H, and S units and the β-O-4 structure of lignin primarily serve as binding sites for enzyme molecules. Immobilized α-amylase demonstrated a higher pH and thermal stability level, with an optimal pH of 7.0 and temperature of 100 °C, compared to the free enzyme, which exhibited optimal activity at a pH of 6.5 and temperature of 90 °C. Importantly, immobilized α-amylase retained >80 % of its initial activity even after 28 days at room temperature, and it maintained 70 % of its activity after being reused 12 times. These findings strongly suggest that lignin derived from agroforestry residues holds promising potential as a future versatile immobilization material, a prospect integral to society's sustainable development.

Functionalized maghemite superparamagnetic iron oxide nanoparticles (γ-Fe2O3-SPIONs)-amylase enzyme hybrid in biofuel production

The current study describes a straightforward, biologically and environmentally friendly method for creating magnetic iron oxide (γ-Fe2O3) nanoparticles. We report here that the Bacillus subtilis SE05 strain, isolated from offshore formation water near Zaafarana, the Red Sea, Hurghada, Egypt, can produce highly magnetic iron oxide nanoparticles of the maghemite type (γ-Fe2O3). To the best of our knowledge, the ability of this bacterium to reduce Fe2O3 has yet to be demonstrated. As a result, this study reports on the fabrication of enzyme-NPs and the biological immobilization of α-amylase on a solid support. The identified strain was deposited in GenBank with accession number MT422787. The bacterial cells used for the synthesis of magnetic nanoparticles produced about 15.2 g of dry weight, which is considered a high quantity compared to the previous studies. The XRD pattern revealed the crystalline cubic spinel structure of γ-Fe2O3. TEM micrographs showed the spherically shaped IONPs had an average size of 7.68 nm. Further, the importance of protein-SPION interaction and the successful synthesis of stabilized SPIONs in the amylase enzyme hybrid system are also mentioned. The system showed the applicability of these nanomaterials in biofuel production, which demonstrated significant production (54%) compared to the free amylase enzyme (22%). Thus, it is predicted that these nanoparticles can be used in energy fields.

An Accessible Method to Improve the Stability and Reusability of Porcine Pancreatic α-Amylase via Immobilization in Gellan-Based Hydrogel Particles Obtained by Ionic Cross-Linking with Mg2+ Ions.

Amylase is an enzyme used to hydrolyze starch in order to obtain different products that are mainly used in the food industry. The results reported in this article refer to the immobilization of α-amylase in gellan hydrogel particles ionically cross-linked with Mg2+ ions. The obtained hydrogel particles were characterized physicochemically and morphologically. Their enzymatic activity was tested using starch as a substrate in several hydrolytic cycles. The results showed that the properties of the particles are influenced by the degree of cross-linking and the amount of immobilized α-amylase enzyme. The temperature and pH at which the immobilized enzyme activity is maximum were T = 60 °C and pH = 5.6. The enzymatic activity and affinity of the enzyme to the substrate depend on the particle type, and this decreases for particles with a higher cross-linking degree owing to the slow diffusion of the enzyme molecules inside the polymer's network. By immobilization, α-amylase is protected from environmental factors, and the obtained particles can be quickly recovered from the hydrolysis medium, thus being able to be reused in repeated hydrolytic cycles (at least 11 cycles) without a substantial decrease in enzymatic activity. Moreover, α-amylase immobilized in gellan particles can be reactivated via treatment with a more acidic medium.

Magnetic Amine-Functionalized ZIF-8 with Hierarchical Pores for Efficient Covalent Immobilization of α-Amylase

Magnetic Amine-Functionalized ZIF-8 with Hierarchical Pores for Efficient Covalent Immobilization of α-Amylase

Understanding the versatile roles of cellulose nanocrystals in α-amylase immobilization, starch hydrolysis, colloidal stability and printability towards advanced starch based bio-latex performance

Recently starch based bio-latexes are facing serious bottlenecks in their preparation and applications especially at the aspects of high concentration, high colloidal stability and high rheological performance. Starch paste for bio-latex production at high concentration would be prone to retrogradation and inevitably deteriorate its performance. The excessive and uncontrollable α-amylase hydrolysis behavior of starch granules at low concentration below 30 wt% could severely affect the strengthening or binding ability of bio-latex. Herein, this study proposes a practical and effective strategy by using cationic cellulose nanocrystals (CCNC) as versatile carriers of α-amylase for the preparation of starch based bio-latex (SBL) to improve starch hydrolysis efficiency even at 50 wt% concentration. CCNC immobilized α-amylase (CCNC/E) can effectively regulate starch hydrolysis behavior with controllable amylose/ amylopectin molecular weight/ distribution for enhanced colloidal stability and viscoelasticity. Furthermore, the multifunctional roles of CCNC as excellent dispersants, nano-fillers and enhancers within starch colloids could impart SBL excellent rheological property and printability in pigmented coating and printing performance, by improving the coating surface, higher printing density/ picking velocity, and lower dot gain phenomenon, compared with the control latex samples. This study proposes a promising and practical approach for efficient production of 50 wt% starch based bio-latex with advanced performance and further valorizes the cellulose nanocrystals in the starch based biomaterials applications.

Immobilization of α-Amylase by Pseudomonas sp isolated from Indian Chicken

Immobilization of α-Amylase by Pseudomonas sp isolated from Indian Chicken

Immobilization of the Bacillus licheniformis α-Amylase on Azole Functionalized Nanoparticle: More Active, Stable, and Usability.

Enzymes are a powerful tool employed in industrial applications due to their high specificity and efficiency. Amylase enzymes play an important role in detergent, textile, analytical chemistry, and paper industries. Here we present the design, synthesis, and characterization of azole functionalized nanoparticles for the immobilization of α-amylase from Bacillus licheniformis (BlA). A modest binding efficiency (47%) was determined by the BCA assay. Enzymatic activity was measured using DNS method and illustrated the immobilization of amylase with the designed nanoparticles enhanced the thermal stability and long-term storage of amylases at a wide range of temperatures and pHs. With the required scale-up study, these implications amplify novel ways to implement this Fe3O4-PGMA-5A immobilized BlA enzyme in particular industrial applications.

Cellulose tosylate as support for α-amylase immobilization

Tremendous potential exists to use cellulose as a support for the immobilization of enzymes. In the current study, cellulose was transformed into cellulose tosylate, and α-Amylase was immobilized using the derivatized polymer. Techniques like Fourier transform infrared, scanning electron microscopy, thermogravimetric analysis, and X-ray diffraction methods were used to characterize the support. The support is a perfect illustration of how both covalent and hydrophobic/ionic types of immobilizations can be experimentally used on support. The support was found to show max degradation at 320.2 °C with 3.2 % residual substance and crystallinity of about 56.6 %. The support presented maximum enzyme loading at the support: enzyme ratio of 1:4. The immobilized enzyme displayed two ideal pH values (about 4.6 and 7) and two ideal temperatures (approximately 60 °C & 40 °C). It was discovered that the immobilized α-amylase could be used easily eight times with 9.9 % residual activity. The findings of this study show that the immobilized cellulose tosylate enzyme has the potential for application in both acidic and neutral pH environments with the best activity for commercial use.

Immobilization of α-Amylase onto Quantum Dots Prepared from Hypericum perforatum L. Flowers and Hypericum capitatum Seeds: Its Physicochemical and Biochemical Characterization

Immobilization of α-Amylase onto Quantum Dots Prepared from Hypericum perforatum L. Flowers and Hypericum capitatum Seeds: Its Physicochemical and Biochemical Characterization