Enzyme Immobilization Efficiency Research Articles

Cellulase entrapment into alginate-PEG beads cross-linked with glutaraldehyde: Optimization of the immobilization conditions and kinetic characterization of the immobilized enzyme

Immobilization methods for cellulase entrapped into alginate beads with glutaraldehyde (GA) cross-linking were investigated. Cellulase entrapped into alginate-polyethylene glycol beads with glutaraldehyde cross-linking (SA-PEG-E) showed more superiority both in terms of catalytic performance and reusability than other methods. Several conditions during SA-PEG-E immobilization such as cellulase concentration, GA amount, pH of immobilization, cross-linking time and hardening temperature were evaluated to study their effect on immobilized enzyme activity, immobilization yield and immobilization efficiency. Based on the results, GA amount, pH of immobilization and hardening temperature were selected for process optimization of SA-PEG-E using the Box-Behnken design of response surface methodology. The results showed that the optimum immobilized conditions were obtained with a GA amount of 0.8 mL, pH of immobilization 4.7, and hardening temperature of 47.5 °C when the cellulase concentration was 7.5 mg/mL and cross-linking time was 2 h. Then, the characterization of SA-PEG-E was studied. The optimum pH of SA-PEG-E was observed to be 4, 1 unit lower than that of the free enzyme (pH 5). The optimum temperature was the same for both free and immobilized cellulase at 50 °C. In addition, the SA-PEG-E exhibited broad pH and temperature adaptability.

β-galactosidase immobilization on ceramic ultrafiltration membrane for simultaneous lactose hydrolysis and protein separation

Protein and lactose of cheese whey can be separated using ultrafiltration membranes and lactose can be hydrolyzed by β-galactosidase. This study investigated the integration of ultrafiltration and enzymatic hydrolysis for simultaneous protein concentration and lactose hydrolysis. β-galactosidase was immobilized on ceramic membrane via surface activation with gelatin and subsequent enzyme cross-linking with glutaraldehyde. Membrane geometries, buffer types, gelatin and enzyme concentrations were optimized. The optimal results yielded a 63 % enzyme immobilization efficiency and a 93 % lactose hydrolysis rate by using disc membrane, sodium acetate buffer (pH 4.8), sodium phosphate buffer (pH 7.5), 0.1 g/L gelatin concentration, and 5.0 g/L enzyme concentration. The optimized enzymatic membrane was evaluated with synthetic and real whey. In synthetic whey, 85 % lactose hydrolysis and 89 % protein rejection were achieved. While in real whey, 72 % lactose hydrolysis and 83 % protein recovery were obtained. This approach offers a single-step, efficient, and scalable method for whey valorization with potential for industrial applications.

Elucidation of enhanced cellulase immobilization onto synthetic magnetic nickel nanomaterials for lignocellulosic biomass hydrolysis

The immobilization of cellulase enzymes for the conversion of lignocellulosic biomass into sustainable biochemical products is essential for the stability and recovery of the enzymes. In this study, nickel nanoparticles (NiNPs) were synthesized and coated with 3-aminopropyl triethoxysilane (APTES) to serve as cellulase enzyme carriers. Cellulase enzyme immobilization on the prepared NiNPs was achieved using glutaraldehyde as a cross-linker. The physicochemical properties of the carrier were determined before and after enzyme immobilization using X-ray Powder Diffraction (XRD), Transmission Electron Microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), Vibrating-Sample Magnetometer (VSM), and Field Emission Scanning Electron Microscopy (FESEM). The impacts of different experimental factors on the performance of the cellulase enzymes immobilized on the synthesized NiNPs and commercial nickel nanoparticles were compared. The results showed comparable optimal cellulase enzyme immobilization conditions on both substrates in terms of the immobilization time, pH, and cellulase enzyme concentration. However, the recommended temperatures for cellulase enzyme immobilization on the synthesized and commercial NiNPs were 50 °C and 40 °C, respectively. Under these optimum conditions, the immobilized cellulase enzyme on the synthesized NiNPs had an activity of about 99.1% (in comparison to the activity of free cellulase enzyme), while the activity of the enzyme upon immobilization on commercial NiNPs was about 93%. The particle size of the NiNPs was found to be crucial for enzyme immobilization efficiency and its magnetic strength. Therefore, cellulase enzyme immobilization on tunable NiNPs could be a sustainable and eco-friendly approach towards high recovery of cellulose from lignocellulosic materials.

Sustainable 3D-printed β-galactosidase immobilization coupled with continuous-flow reactor for efficient lactose-free milk production

The production of low-lactose and lactose-free milk remains a critical challenge. Novel enzyme immobilization integrated with the flow reactor that could be applied to hydrolyze milk with cost-effectiveness, facilitation, and safety is highly expected. Here, we present a novel continuous-flow bioreactor loaded with β-galactosidase (β-Gal) covalently immobilized 3D-printed scaffolds for the production of lactose-free milk. 3D-printed polylactic acid (PLA) scaffolds modified by self-assembly of polyethyleneimine and sodium hyaluronate layers are rapidly branched with functional groups, followed by covalent immobilization of β-Gal using genipin as the cross-linker. The use of genipin allows efficient and stable immobilization of the enzyme (92.87 % immobilization efficiency), and the immobilized enzyme retains 90.53 % activity after 6th repeated use. Moreover, the 3D-printed scaffolds integrated into the flow reactor perturbed the fluids in the different reactors through directional channels, which improved the internal mass transfer to achieve high catalytic activity (94.20 % lactose hydrolysis efficiency). The lactose residues of milk reacted in the β-Gal flow reactor for 6 and 12 h were 1.87 g dL-1 and 0.38 g dL-1, which fully met the requirements of the national food safety standards for low-lactose and lactose-free milk. Due to the high efficiency of enzyme immobilization and the stable catalytic activity of the reactor, it has a broad application prospect for the production of low-lactose and lactose-free milk and other biocatalytic fields.

Enhanced Laccase Activity and Stability as Crosslinked Enzyme Aggregates on Magnetic Copper Ferrite Nanoparticles for Biotechnological Processes

AbstractHighly stable and reusable magnetic crosslinked enzyme aggregates (m‐CLEAS) of laccase are synthesized with simultaneous improved enzymatic activity. Magnetic copper ferrite nanoparticles (CFNPs) were synthesized by solvothermal procedure with an average size of ~8 nm. The nanometric m‐CLEAS were formed by co‐aggregation of enzyme with CFNPs and crosslinked using glutaraldehyde. Different mass ratios of CFNPs:Laccase were assayed (1 : 2, 1 : 3, and 1 : 6), where 1 : 6 resulted in the highest activity recovery (97 %). The m‐CLEAS showed an average size of ~239 nm, ~24 % enzyme immobilization efficiency, and loading as high as 1.75 g of protein per g of support. As expected, m‐CLEAS oxidized the substrate with a higher transformation rate (kcat) and catalytic efficiency (kcat/Km) than the free enzyme. m‐CLEAS showed superior storage and thermostability compared to free enzyme and non‐magnetic CLEAS. In particular, the m‐CLEAS showed ~150 % and ~100 % residual activity after 30 days of storage at 4 °C and room temperature, respectively. Furthermore, m‐CLEAS showed good recyclability, retaining ~78 % and ~54 % laccase activity after 5 and 10 cycles of reuse, respectively. This work highlights the facile and cost‐effective synthesis of nanometric m‐CLEAS with exceptional storage stability and simultaneously improved laccase activity, making them suitable for a range of green industrial processes.

Design and Optimization of Laccase Immobilization in Cellulose Acetate Microfiltration Membrane for Micropollutant Remediation

The industrial and environmental applications of laccase, especially in wastewater treatment, have gained focus in recent years. Therefore, developing the proper laccase immobilization techniques, which could improve the stability of the enzymes and simplify the required downstream processes, is needed. A novel two-step immobilization process was developed, resulting in cross-linked enzyme aggregates (CLEA) in the pores of the membrane. Laccase adsorption on a biodegradable cellulose acetate microfiltration membrane along with cross-linking was investigated to maximize the enzyme load and immobilization efficiency. The optimization was done regarding the: pH, temperature, enzyme concentration, adsorption time, cross-linker concentration, and temperature. It was concluded that the highest immobilization efficiency (76%) could be achieved in acidic buffers at 29 °C with high surface activity (1174 U·m−2) at the cost of partial denaturation and membrane fouling. The membrane was successfully utilized for the enzymatic treatment of diclofenac, and 58% removal efficiency was achieved. The results indicated that cellulose acetate is a suitable carrier for adsorption-based immobilization of laccase for the potential for environmental utilisation.

Precursor concentration effects on crystallite size and enzyme immobilization efficiency of Enzyme@ZIF-8 composite

Composites of zeolitic imidazolate framework-8 (ZIF-8) and biomolecules have garnered much attention because the environmental tolerance of biomolecules is enhanced in the composites with ZIF-8. Those composites are formed easily by dissolving biomolecules with precursors of ZIF-8 in an aqueous solution. However, correlation between the composites’ properties and forming conditions remains unclear. In this study, we specifically examined composites of ZIF-8 and an enzyme (glucose oxidase): a much-researched composite for application purposes. For such composites, the crystallite size dependence of ZIF-8 on prepared molar ratio of precursors (2-methyimidazole / Zn ion, i.e., Hmim/Zn) was assessed and compared to that found in an earlier study for pure ZIF-8 without seeding. Results show that the ZIF-8 crystallite size decreased along with the decrease of the molar ratio of precursors. This correlation is opposite to that in cases of pure ZIF-8. Thus, the formation process of ZIF-8 in presence of enzyme is supposed to be much different from that of pure ZIF-8, and such process was discussed focusing on the pH-dependent coordination state of Zn ion in preformed amorphous phase of composite. Furthermore, the molar ratio of precursors affected the enzyme immobilization efficiency. Along with molar ratio of precursors, variations of counter anion of Zn were also investigated and discussed.

A novel construct of an electrochemical acetylcholinesterase biosensor for the investigation of malathion sensitivity to three different insect species using a NiCr2O4/g-C3N4 composite integrated pencil graphite electrode.

Herein, an electrochemical biosensor has been prepared to assess the sensitivity of an organophosphate insecticide, malathion, to acetylcholinesterase (AChE) enzyme of three insects including Apis mellifera (honeybee), Tribolium castaneum (red flour beetle), and Zootermopsis nevadensis (dampwood termite). A composite of nickel chromite (NiCr2O4) and graphitic carbon nitride (g-C3N4) was prepared and characterized for its morphological, chemical and electrical properties. The NiCr2O4/g-C3N4 composite integrated pencil graphite electrodes were used to covalently immobilize insect AChE enzymes and amperometric response of bioelectrodes was determined through cyclic voltammetry. The prepared bioelectrodes exhibited high enzyme immobilization efficiency and electro-catalytic performance. The integrated bioelectrodes could efficiently detect malathion induced inhibition of insects' AChEs. The linear ranges for malathion were found to be 0.1–1.6 μM, 1–40 nM and 2–100 nM, and LODs were 2 nM, 0.86 nM and 2.3 nM for A. mellifera, T. castaneum, and Z. nevadensis, respectively. Additionally, the biosensing platform developed using A. mellifera AChE was found highly sensitive and effective for malathion recoveries from spiked wheat flour samples with high recovery rates. Moreover, the proposed method was adequately reproducible and selective. The results revealed that A. mellifera AChE is less sensitive to inhibition by malathion as compared to T. castaneum, and Z. nevadensis AChE. The experimental results were validated through computational docking of malathion with insect AChEs and the results were in correspondence to experimental outcomes. The proposed method can be a plausible alternate to conventional analytical methods to assess the pesticide sensitivity and toxicity of various compounds against insect enzymes.

Convenient Immobilization of α‐L‐Rhamnosidase on Cerium‐based Metal‐Organic Frameworks Nanoparticles for Enhanced Enzymatic Activity and Recyclability

AbstractThe α‐L‐Rhamnosidase (Rha) is a useful glycoside hydrolase for selectively hydrolyzing the terminal L‐rhamnose residues in flavonoids, being vital to food and pharmaceutical industries. However, Rha suffers from low recyclability and poor stability in harsh environments. Herein, we explored five typical metal‐organic frameworks (MOFs) as porous carriers to immobilize Rha, and the activities of the resultant Rha@MOF composites were compared with the free enzyme. The locations of the enzyme in MOFs were proved by a series of characterization techniques. It was found that Rha@Ce‐BTC (with enzyme immobilization efficiency of 23 % and enzyme loading content of 8.8 %) showed the highest enzymatic activity. The immobilized Rha@Ce‐BTC showed 80 % residual activity after five consecutive cycles, suggesting a limited leaching effect. Also, Rha@Ce‐BTC manifested markedly enhanced enzyme‐substrate affinity and catalytic efficiency compared to free Rha, as supported by Michaelis‐Menten kinetic studies. Accordingly, the Ce‐BTC would be an appealing carrier for enzyme immobilization, and the as‐designed enzyme/Ce‐BTC composites are promising candidates for industrial use with remarkably high activity, recyclability, and storage stability.

Partial purification, characterization and immobilization of a novel lipase from a native isolate of Lactobacillus fermentum.

Background and Objectives:Due to the widespread use of lipase enzymes in various industries, finding native lipase producing microorganisms is of great value and importance. In this study, screening of lipase-producing lactobacilli from native dairy products was performed.Materials and Methods:Qualitative evaluation of lipolytic activity of lipase-producing lactobacilli was performed in different media containing olive oil. A clear zone observation around the colonies indicated the lipolytic activity. The strain with the highest enzymatic activity was identified. Determination of optimal pH and temperature of lipase activity was measured by spectrophotometry using p-nitrophenyl acetate (ρ-NPA) substrate. Partial purification of lipase enzyme was performed using 20–90% saturation ammonium sulfate. Eventually, lipase was immobilized by physical adsorption on chitosan beads.Results:Among screened lipolytic bacterial strains, one sample (5c isolate) which showed the highest enzymatic activity (5329.18 U/ml) was close to Lactobacillus fermentum. During characterization, the enzyme showed maximum activity in Tris-HCl buffer with pH 7, while remaining active over a temperature range of 5°C to 40°C. The results of the quantitative assay demonstrated that the fraction precipitated in ammonium sulfate at 20% saturation has the highest amount of lipolytic activity, with a specific activity of 22.0425 ± 3.6 U/mg. Purification folds and yields were calculated as 8.73 and 44%, respectively. Eventually, the enzyme was immobilized by physical adsorption on chitosan beads with a yield of 56.21%.Conclusion:The high efficiency of enzyme immobilization on chitosan beads indicates the suitability of this method for long-term storage of new lipase from native 5c isolate.

Oxytetracycline removal by biological/chemical activated mesoporous carbon

The presence of emerging pollutants in water matrices is an increasing concern; therefore, the development of sustainable technologies that enable the removal of these pollutants is required. The goal of the present study was to propose a methodology to prepare a sustainable catalyst using functionalized mesoporous activated carbon and an enzyme for the removal of oxytetracycline. Four strategies of laccase immobilization were evaluated using a 32 experimental design to evaluate the enzyme immobilization efficiency. The activated carbon was functionalized with hydrochloric acid, glutaraldehyde, or carbodiimide, resulting in four different enzyme immobilization carriers. The response variables evaluated were the protein-specific load capacity and specific activity of immobilized laccase. All developed carriers successfully immobilized the enzyme; among these, the carriers treated with hydrochloric acid and glutaraldehyde had significant protein-specific capacities with yields of 90%, while the carbodiimide carrier had a yield below 40%. In addition, the hydrochloric acid-treated carrier achieved the highest specific activity (168 ± 52 U/g) and specific protein load (13.48 ± 0.90 mg/g). The antibiotic removal assays that employed this catalyst showed better performance (100%) than the free enzyme, which was used as a reference. However, the apparent antibiotic removal mechanism was found to be pollutant adsorption instead of degradation, which was ascribed to the high affinity of the hydrochloric acid-treated carrier for the studied pollutant.

Recent Advances in Feedstock and Lipase Research and Development towards Commercialization of Enzymatic Biodiesel

Biodiesel is a biodegradable, renewable, and carbon-neutral alternative to petroleum diesel that can contribute to the global effort of minimizing the use of fossil fuels and meeting the ever-growing energy demands and stringent environmental constraints. The aim of this work was to (1) review the recent progress in feedstock development, including first, second, third, and fourth-generation feedstocks for biodiesel production; (2) discuss recent progress in lipase research and development as one of the key factors for establishing a cost-competitive biodiesel process in terms of enzyme sources, properties, immobilization, and transesterification efficiency; and (3) provide an update of the current challenges and opportunities for biodiesel commercialization from techno-economic and social perspectives. Related biodiesel producers, markets, challenges, and opportunities for biodiesel commercialization, including environmental considerations, are critically discussed.

Immobilizing Enzymes on Noble Metal Hydrogel Nanozymes with Synergistically Enhanced Peroxidase Activity for Ultrasensitive Immunoassays by Cascade Signal Amplification.

Enzyme immobilization plays an essential role in solving the problems of the inherently fragile nature of enzymes. Although prominent stability and reuse of enzymes can be achieved by enzyme immobilization, their bioactivity and catalytic efficiency will be adversely affected. Herein, PdCu hydrogel nanozymes with a hierarchically porous structure were used to immobilize horseradish peroxidase (HRP) to obtain PdCu@HRP. In addition to the improvement of stability and reusability, PdCu@HRP displayed synergistically enhanced activities than native HRP and PdCu hydrogels. Not only the specific interactions between PdCu hydrogel nanozymes and enzymes but also the enrichment of substrates around enzymes by electrostatic adsorption of hydrogels was proposed to expound the enhanced catalytic activity. Accordingly, by taking advantage of the excellent catalytic performance of the PdCu@HRP and the glucose oxidase encapsulated in zeolitic imidazolate framework-8, colorimetric biosensing of the carcinoembryonic antigen via catalytic cascade reactions for achieving signal amplification was performed. The obtained biosensor enhanced the detection sensitivity by approximately 6.1-fold as compared to the conventional HRP-based enzyme-linked immunosorbent assay, demonstrating the promising potential in clinical diagnosis.

Preparation of AChE immobilized microspheres containing thiophene and furan for the determination of pesticides by the HPLC-DAD method

In this study, acetylcholinesterase (AChE) enzyme immobilized polymeric microspheres were prepared to detect the dichlorvos (DDVP: 2,2-dichlorovinyl dimethyl phosphate) and the chlorpyrifos (O, O-Diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate) pesticides. To produce polymeric supports, tris(2-aminoethyl)amine polystyrene (2AEPS) was reacted with thiophene carboxaldehyde and its derivatives; and 2AEPS was also reacted with furaldehyde and its derivatives. A total of six different ligands were synthesized (2AEPS-Tio, 2AEPS-5-CH3Tio, 2AEPS-5-NO2Tio, 2AEPS-Fur, 2AEPS-5-CH3Fur, 2AEPS-5-NO2Fur). Polystyrene's spherical porous structure having large surface area and aldehyde groups were modified to enhance the efficiency of enzyme immobilization. In terms of the number of times it can be reused, the highest relative activity was found to be with 2AEPS-Tio-AChE at 71.28% after 10 cycles. HPLC-DAD system was used for detection of pesticides. For this purpose, the parameters which were thought to affect the response in HPLC-DAD analysis were optimized. Method validation was performed for pesticides. The decomposition reaction of the pesticide was followed by free and immobilized AChE enzyme using validated conditions. Using free AChE, the detection of limit (LOD) for DDVP and chlorpyrifos was found to be 2.57 µg L−1 and 0.45 µg L−1, respectively. When using immobilized AChE, the LOD values for DDVP and chlorpyrifos was obtained in the range of 0.42-0.75 µg L−1 and 0.34-4.23 µg L−1, respectively.

IMMOBILIZATION OF Bacillus subtilis E6-5 PROTEASE AND COMMERCIAL PROTEASE IN NANOFIBRILS CONTAINING DIFFERENT AMINO ACIDS

In this study, polyamide 6 polymer surfaces that have a high surface area were produced by electrospinning method with the participation of Glycine, Tyrosine and Glutamic acid amino acids, and lyophilized Bacillus subtilis E6-5 protease and commercial protease enzymes were immobilized on nanofibrils. Enzyme reusability were investigated. The immobilization efficiencies of the enzymes were approximately between 50-55 %. In studies with lyophilized Bacillus protease, glutaraldehyde activated PA6 nanofibrils and glutaraldehyde unactivated PA6 nanofibrils were found to be more immobilized in the presence of Glutamic acid. Although the lyophilized protease enzyme immobilized on non-glutaraldehyde activated and activated surfaces has been used 4 times, the best functional stability has been achieved with 2 times use. In pure PA6/Glutamic acid nanofibrils, the immobilization yield of the two times used enzymes was found to be 38 %. In glutaraldehyde-activated PA6 nanofibrils, the PA6/Glutamic acid nanofibril surfaces were found to have 65 % immobilization yield of the two repetitive used enzymes. The enzyme immobilization efficiency has been doubled by glutaraldehyde activation of the nanofibrils. In studies with commercial protease, the most functional stability was obtained for 3 repeated uses, although the enzyme was used 6 times on the non-glutaraldehyde activated nanofibril surfaces. The most successful immobilization was found in 58 % of PA6 nanofibrils. In glutaraldehyde-activated PA6 nanofibrils, the enzyme was found to be used 6 times, but the functional stability was maintained as much as 4 times of repeated use.

Starch hydrolysis using maltogenase immobilized via different techniques

A series of modified poly(urethane-urea) carriers for immobilization of maltogenase from Bacillus stearothermophilus were presented. Poly(urethane-urea) microparticles (PUU) from poly(vinyl alcohol) and isophorone diisocyanate were modified with ethylenediamine, glutaraldehyde, trichlorotriazine and gold nanoparticles (AuNPs). Covalent attachment of enzyme to carrier via active isocyanate, aldehyde or aromatic chlorine group or electrostatic adsorption to AuNPs was performed. Maximal surface area (218.3 m2/g) was estimated for PUU microparticles modified with ethylenediamine and decorated with AuNPs. The high efficiency and yield of enzyme immobilization onto PUU-EDA/AuNPs were determined. Properties of maltogenase immobilized onto PUU carriers were compared to crosslinked enzyme aggregates from maltogenase (CLEA-maltogenase). The determined values of KM were 21.29 mM, 22.46 mM and 61.72 mM for soluble, CLEA-maltogenase and immobilized onto PUU-EDA/AuNPs, respectively. Hydrolysis of wheat starch catalyzed by immobilized maltogenase in continuous mini-reactor was more effective in comparison to CLEA-maltogenase; obtained dextrose equivalent was 28.9% and amount of maltose was 26.6%.

EFFECT OF IMMOBILIZATION PARAMETERS ON THE IMMOBILIZATION OF CYCLODEXTRIN GLUCANOTRANFERASE ON HOLLOW FIBER MEMBRANE

Cyclodextrin (CD) is a non-reducing maltooligosaccharides which able to form inclusion complexes with many hydrophobic molecules, changing their physical and chemical properties. With these properties, CD has been discovered to have numerous applications in food industries, pharmaceutical, agricultural and environmental engineering. CD is produced by the enzymatic reaction between cyclodextrin glucanotransferase (CGTase) and starch. Various enzyme immobilization techniques such as adsorption, entrapment, encapsulation and cross-linking have been applied to improve the production of CD. Some of the immobilization parameters such as contact time, agitation rate and pH of the immobilization solution play a vital role in enzyme immobilization process, in order to achieve high production of CD. In the present study, the CGTase from Bacillus licheniformis was immobilized on polyvinylidene difluoride (PVDF) hollow fiber membrane via adsorption technique. The efficiency of enzyme immobilization appears to be affected by various factors (immobilization parameters) such as contact time, agitation rate and pH. Therefore, the effect of contact time (6-72 h), agitation rate (50-250 rpm) and pH (3-10) on the immobilization of CGTase on PVDF hollow fiber membrane were investigated in this study. The immobilized CGTase exhibited the highest immobilization yield of 69.37% under the conditions of 24 h contact time, 100 rpm and pH 7.0. Therefore, the influence of the immobilization parameters during the enzyme immobilization process is vital in order to achieve the high production of CD. Hence, high immobilization yield contributed to the high production of CD which in turn may be beneficial for the industrial purposes.

Synthesis of a polydopamaine nanoparticle/bacterial cellulose composite for use as a biocompatible matrix for laccase immobilization

Bacterial cellulose has attracted attention as a scaffolding material for enzyme immobilization because of its excellent mechanical properties, nanoporous structure, high purity, and super hydrophilicity. However, the lack of reactive group and the relatively low binding capacity toward biomolecules limit its application. In this study, we have synthesized a polydopamine nanoparticle/bacterial cellulose hybrid material with hierarchical multiscale architecture for enzyme immobilization, via a simple in situ self-assembly of polydopamine nanoparticles on cellulose surface. Commercial enzyme laccases were employed to evaluate the enzyme immobilizability of this hybrid material, and various key parameters of enzyme immobilization such as reaction time, enzyme concentration, temperature and pH were also systematically studied. This hybrid composite displayed excellent enzyme immobilization efficiency (~ 165 mg laccase per g composite), and the immobilized laccase also exhibited better thermostability and operational stability over a broad temperature range as compared with free laccase. By using the immobilized laccase, fast and efficient dye removal ability and good recyclability were achieved during the dye decolorization test, which demonstrated its potential application in various industrial relevant wastewater treatment processes.

Application of carboxymethyl cellulose-g-poly(acrylic acid-co-acrylamide) hydrogel sponges for improvement of efficiency, reusability and thermal stability of a recombinant xylanase

Enzyme immobilization onto supports could improve its specificity, storage stability, reusability, pH tolerance and thermal stability. Aiming to provide a proficient immobilization carrier for a recombinant xylanase (PersiXyn1), the high-performance 3D copolymeric network of carboxymethyl cellulose (CMC)-based hydrogel sponges were synthesized using in situ graft radical polymerization. While typical purified gel drying in a vacuum oven made CMC/Acrylate-Hydrogel, the freeze-drying of the resultant swelled Hydrogel containing different amounts of water, resulted in two samples of sponges (i.e., CMC/Acrylate-Sponge1 and CMC/Acrylate-Sponge2). The structural characteristics and water absorbency properties of all the as-prepared samples were evaluated. CMC/Acrylate-Sponge2 was selected for PersiXyn1 immobilization due to its homogeneous porous polymeric skeleton and maximum water absorbency (166.6 g/g) offering superb enzyme immobilization efficiency (100%). Moreover, presence of numerous oxygenated functionalities and cage-like characteristics in the CMC/Acrylate-Sponge2, led to effective electrostatic attractions between support and enzyme providing physically stabilized and reusable bio-conjugates which exhibited up to 60% recovered activity even after eight continuous reuse cycles. The results of kinetic studies indicated that the specific activities of free and immobilized PersiXyn1 were 4157 and 5508 µmol min−1 mg−1, respectively. Interestingly, CMC/Acrylate-Sponge2 support could noticeably broaden the temperature endurance ranges of immobilized PersiXyn1. While free enzyme could preserve only 10% of its maximum activity, the immobilized enzyme maintained 35% of its maximum activity after 60 min incubation at 60 °C. Lastly, CMC/Acrylate-Sponge2 support provided remarkable enzyme protection from denaturing, which caused shifting enzyme unfolding temperature from 71.5 to 78.0 °C, indicating the effectual shielding effects of polymeric support on PersiXyn1.

Immobilization of Peroxidase on Functionalized MWCNTs-Buckypaper/Polyvinyl alcohol Nanocomposite Membrane

Surface modified Multi-walled carbon nanotubes (MWCNTs) Buckypaper/Polyvinyl Alcohol (BP/PVA) composite membrane was synthesized and utilized as support material for immobilization of Jicama peroxidase (JP). JP was successfully immobilized on the BP/PVA membrane via covalent bonding by using glutaraldehyde. The immobilization efficiency was optimized using response surface methodology (RSM) with the face-centered central composite design (FCCCD) model. The optimum enzyme immobilization efficiency was achieved at pH 6, with initial enzyme loading of 0.13 U/mL and immobilization time of 130 min. The results of BP/PVA membrane showed excellent performance in immobilization of JP with high enzyme loading of 217 mg/g and immobilization efficiency of 81.74%. The immobilized system exhibited significantly improved operational stability under various parameters, such as pH, temperature, thermal and storage stabilities when compared with free enzyme. The effective binding of peroxidase on the surface of the BP/PVA membrane was evaluated and confirmed by Field emission scanning electron microscopy (FESEM) coupled with Energy Dispersive X-Ray Spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR) and Thermogravimetric Analysis (TGA). This work reports the characterization results and performances of the surface modified BP/PVA membrane for peroxidase immobilization. The superior properties of JP-immobilized BP/PVA membrane make it promising new-generation nanomaterials for industrial applications.