Immobilization Yield Research Articles

Sustainable pretreatment method of lignocellulosic depolymerization for enhanced ruminant productivity using laccase protein immobilized agarose beads

Laccase, the selectively lignin degrader, vital to the initiation of lignocellulosic deconstruction was immobilized onto activated agarose beads to increase its reuse potential. Laccase cross-linked beads (~ 3.42 mm) recorded a specific activity of 23 Umg− 1, retaining about 80.43% enzyme activity after 45 days of storage. The immobilization yield and efficiency were 89% and 97% respectively. The equilibrium data fitted the Freundlich equation (R2 = 0.9987) demonstrating multilayer adsorption and the presence of Cu, Fe, and S in the elemental analysis of immobilized beads established effective binding between activated agarose beads and the laccase protein. Characterization studies of the immobilized laccase-treated crop residues revealed significant differences in the lignin polymer after each treatment cycle. An increase in digestibility of 26.21% and 7.62% was observed in paddy and finger millet-treated straws respectively, over the controls corroborating efficient lignin depolymerization. The propitious performance of laccase beads authenticated in the batch enzymatic reactor to treat crop residues paves headway as a sustainable green technology in the deconstruction of crop residues for use as ruminant feed, augmenting productivity.

Covalent Lysozyme Immobilization on Enzymatic Cellulose Nanocrystals.

Nanostructured materials represent promising substrates for biocatalyst immobilization and activation. Cellulose nanocrystals (CNCs), accessible from waste and/or renewable sources, are sustainable and biodegradable, show high specific surface area for anchoring a high number of enzymatic units, and high thermal and mechanical stability. In this work, we present a holistic enzyme-based approach to functional antibacterial materials by bioconjugation between the lysozyme from chicken egg white and enzymatic cellulose nanocrystals. The neutral CNCs were prepared by endoglucanase hydrolysis from Avicel. We explore the covalent immobilization of lysozyme on enzymatic CNCs and on their TEMPO oxidized derivatives (TO-CNCs), comparing immobilization yields, material properties, and enzymatic activities. The materials were characterized by X-ray diffractometry (XRD), attenuated total reflectance Fourier Transform infrared spectroscopy (ATR-FTIR), bicinchoninic acid (BCA) assay, field-emission scanning electron microscopy (FE-SEM) and dynamic light scattering (DLS). We demonstrate the higher overall efficiency of the immobilization process carried out on TO-CNCs, based on the success of covalent bonding and on the stability of the isolated bioconjugates.

Biocatalytic Production of Solketal Esters from Used Oil Utilizing Treated Macauba Epicarp Particles as Lipase Immobilization Support: A Dual Valorization of Wastes for Sustainable Chemistry

This study describes the production of solketal esters from used soybean cooking oil (USCO) via enzymatic hydroesterification. This process consists of the complete hydrolysis of USCO into free fatty acids (FFAs) catalyzed by crude lipase extract from Candida rugosa (CRL). The resulting FFAs were recovered and utilized as the raw material for an esterification reaction with solketal, which was achieved via an open reaction. For this purpose, lipase Eversa® Transform 2.0 (ET2.0) was immobilized via physical adsorption on treated epicarp particles from Acrocomia aculeata (macauba), a lignocellulosic residue. A protein loading of 25.2 ± 1.3 mg g−1 with a support and immobilization yield of 64.8 ± 2.5% was achieved using an initial protein loading of 40 mg g−1 of support. The influence of certain parameters on the esterification reaction was evaluated using a central composite rotatable design (CCRD). Under optimal conditions, a FFAs conversion of 72.5 ± 0.8% was obtained after 150 min of reaction at 46 °C using a biocatalyst concentration of 20% wt. and a FFAs–solketal molar ratio of 1:1.6. The biocatalyst retained 70% of its original activity after ten esterification batches. This paper shows the conversion of two agro-industrial waste into valuable materials (enzyme immobilization support and solketal esters).

Evaluation on potential application of lipase immobilized on rice husks in enzymatic glycerolysis reaction

Owing to high production cost and low reaction yield, immobilized lipase is rarely used in industrial glycerolysis. This research characterizes the performance of lipase immobilized on rice husk in glycerolysis reaction. By utilizing hexamethylenediamine (HMDA) and glutaraldehyde (GA) as coupling agents, lipase from Thermomyces lanuginosus was immobilized on oxidized rice husk (ORH). For comparison, another sample was prepared where the lipase was directly immobilized on ORH without the use of HMDA and GA. Then, monoglyceride production was performed via glycerolysis using the immobilized lipase. The FTIR analysis verify interactions on rice husk including rice husk oxidation, HMDA coupling, GA activation and lipase immobilization on rice husk. The study found that within the examined range of added lipase for immobilization (10–40 mg-protein/g-support), ORH–HMDA–GA–Lipase possessed superior outcomes in terms of protein loading, immobilization yield, and recovered glycerolysis activity compared to ORH–Lipase. Besides, ORH–HMDA–GA–Lipase exhibits better storage stability (60°C, 44.9 %) and higher reusability (90.0 % monoglyceride yield at the 8th cycle) against ORH–Lipase. The results confirm satisfying performance of the prepared immobilized lipase in glycerolysis and highlight its enhancements facilitated by coupling agents.

Single stage bi-substrate transglycosylation reaction for the synthesis of ascorbic acid 2 glucoside using immobilized α-glucosidase

Alpha glucosidases are multifunctional glycoside hydrolases with hydrolysis and transglycosylation ability. It can be utilized for the glycosidic bond synthesis or glycosylation of Ascorbic acid/Vitamin C to its stable analogue, Ascorbic acid 2 glucoside (AA2G), a compound with wide applications in cosmetics and pharma. The application of α-glucosidases for industrial scale transglycosylation is limited due to the low transglycosylation yield of free enzymes. Enzyme immobilization techniques could enable the development of efficient, reusable catalysts. Only a few glycoside hydrolases have been studied in immobilized form for transglycosylation reactions, and α-glucosidases are probably the least explored in this form. Transglycosylation activity of immobilized α-glucosidase from Aspergillus carbonarius BTCF 5 was studied for AA2G synthesis, where different immobilization techniques like calcium alginate encapsulation, adsorption on chitosan beads, covalent cross-linking on magnetic nanoparticles, and cross-linked enzyme aggregates (CLEA) were employed for the immobilization. The immobilization yield of calcium alginate encapsulated enzyme, enzyme immobilized on Fe-MNP support, enzyme immobilized on chitosan beads and as CLEA were 107%, 99%, 46% and 486%, respectively. CLEA was identified as the best immobilization technique for this bi-substrate reaction due to the high immobilization yield and activity retention (30% activity retained after 5 consecutive cycles). Enzyme immobilization increased the transglycosylation activity by 38%, yielding 118 mM AA2G against 72 mM by the free enzyme. This indicates the potential of immobilized α-glucosidase as a catalyst for synthesizing AA2G at an industrial scale.

Investigating a siderophore-based approach for the recovery of critical metals from waste streams and leach solutions using microorganisms

As technological advancements progress and unsustainable consumerism of electrical devices rapidly increases, the demand for critical metals continues to rise. The limited supply of these metals, driven by the depletion of non-renewable natural resources, calls for the recycling of waste materials and the development of sustainable and cost-effective extraction methods. Conventional methods of leaching such as pyrometallurgy pose threats to the environment by creating slags and releasing toxic secondary materials. Biohydrometallurgy emerges as an environmentally friendly method to extract metals from low-grade ores and waste material. Siderophores are secondary metabolites secreted by microorganisms that have the ability to selectively chelate certain metals. By immobilizing these siderophores for subsequent bioleaching, target metals can be extracted from multielement solutions. This research focuses on optimizing the immobilization efficiency of the siderophore desferrioxamine B (DFOB) in sodium alginate using physical entrapment for the removal of gallium. Four parameters were varied to find optimal conditions: sodium alginate concentration (1 – 4 % w/v), calcium chloride concentration (1 – 10% w/v), agitation time (0.25 – 10 hours), and DFOB concentration (1 – 4 mM). Using UV-Vis spectroscopy, free siderophore concentration could be determined. After 30 runs, the highest immobilization yield of 80.05% was determined at 2.5% sodium alginate, 10% calcium chloride—the highest concentration tested—after 5.13 hours with a concentration of 2.25 mM DFOB. Design Expert-13 software was used to analyze all experimental results. 2D graphs support that DFOB immobilization depends on responsive parameters, CaCl2 demonstrating the largest impact.

Agro-industrial Waste Utilization, Medium Optimization, and Immobilization of Economically Feasible Halo-Alkaline Protease Produced by Nocardiopsis dassonvillei Strain VCS-4.

The oceanic actinobacteria have strong potential to secrete novel enzymes with unique properties useful for biotechnological applications. The Nocardiopsis dassonvillei strain VCS-4, associated with seaweed Caulerpa scalpeliformis, was a halo-alkaline protease producer. Further investigation focuses on medium optimization and the use of agro-industrial waste for economically feasible, high-yield protease production. A total of 12 experimental runs were designed using Minitab-20 software and Placket-Burman design. Among the 7 physicochemical parameters analyzed, incubation time and gelatin were detected as significant factors responsible for higher protease production. Incubation time and gelatin were further analyzed using OVATs. Optimal protease production was achieved with 2% gelatin, 0.1% yeast extract, 0.1% bacteriological peptone, 7% NaCl, pH 8, 5% inoculum, and a 7-day incubation period, resulting in a maximum protease activity (Pmax) of 363.97 U/mL, generation time of 11.9h, specific growth rate of 0.161g/mL/h, and protease productivity (Qp) of 61.65 U/mL/h. Moreover, utilizing groundnut cake as an agro-industrial waste led to enhanced production parameters: Pmax of 408.42 U/mL, generation time of 9.74h, specific growth rate of 0.361g/mL/h, and Qp of 68.07 U/mL/h. The immobilization of crude protease was achieved using Seralite SRC 120 as a support matrix resulting in 470.38 U/g immobilization, 88.20% immobilization yield, and 28.90% recovery activity. Characterization of both crude and immobilized proteases revealed optimal activity at pH 10 and 70°C. Immobilization enhanced the shelf-life, reusability, and stability of VCS-4 protease under extreme conditions.

Laccase Immobilized on Arginine-Functionalized Boron Nitride Nanosheets for Enhanced Atrazine Degradation.

Enzyme-mediated systems have been widely employed for the biotransformation of environmental contaminants. However, the catalytic performance of free enzymes is restricted by the rapid loss of their catalytic activity, stability, and reusability. In this work, we developed an enzyme immobilization platform by elaborately anchoring fungal laccase onto arginine-functionalized boron nitride nanosheets (BNNS-Arg@Lac). BNNS-Arg@Lac showcased ∼75% immobilization yield and enhanced stability against fluctuating pH values and temperatures, along with remarkable reusability across six consecutive cycles, outperforming free natural laccase (nlaccase). A model pollutant, atrazine, was selected for a proof-of-concept demonstration, given the substantial environmental and public health concerns in agriculture runoff. BNNS-Arg@Lac-catalyzed atrazine degradation rate was nearly twice that of nlaccase. Moreover, BNNS-Arg@Lac consistently demonstrated superior atrazine degradation in synthetic agricultural wastewater and various mediator systems compared to nlaccase. Comprehensive product analysis unraveled distinct degradation pathways for BNNS-Arg@Lac and nlaccase. Overall, this research provides a foundation for the future development of enzyme-nanomaterial hybrids for degrading environmental chemicals and may unlock new potential for green and efficient resource recovery and waste management strategies.

Co-administration of xylo-oligosaccharides produced by immobilized Aspergillus terreus xylanase with carbimazole to mitigate its adverse effects on the adrenal gland

Carbimazole has disadvantages on different body organs, especially the thyroid gland and, rarely, the adrenal glands. Most studies have not suggested any solution or medication for ameliorating the noxious effects of drugs on the glands. Our study focused on the production of xylooligosaccharide (XOS), which, when coadministered with carbimazole, relieves the toxic effects of the drug on the adrenal glands. In addition to accelerating the regeneration of adrenal gland cells, XOS significantly decreases the oxidative stress caused by obesity. This XOS produced by Aspergillus terreus xylanase was covalently immobilized using microbial Scleroglucan gel beads, which improved the immobilization yield, efficiency, and operational stability. Over a wide pH range (6–7.5), the covalent immobilization of xylanase on scleroglucan increased xylanase activity compared to that of its free form. Additionally, the reaction temperature was increased to 65 °C. However, the immobilized enzyme demonstrated superior thermal stability, sustaining 80.22% of its original activity at 60 °C for 120 min. Additionally, the full activity of the immobilized enzyme was sustained after 12 consecutive cycles, and the activity reached 78.33% after 18 cycles. After 41 days of storage at 4 °C, the immobilized enzyme was still active at approximately 98%. The immobilized enzyme has the capability to produce xylo-oligosaccharides (XOSs). Subsequently, these XOSs can be coadministered alongside carbimazole to mitigate the adverse effects of the drug on the adrenal glands. In addition to accelerating the regeneration of adrenal gland cells, XOS significantly decreases the oxidative stress caused by obesity.

Two‐Step Biocatalytic Synthesis of (1S)‐Nor(pseudo)ephedrine Catalyzed by Immobilized Enzymes

AbstractNor(pseudo)ephedrines [N(P)Es] are naturally occurring compounds showing sympathomimetic activity that have many applications especially in the chemical industry and in the pharmaceutical field. Recently a two‐step biocatalytic cascade for the preparation of (1S)‐N(P)E was designed, consisting of a benzoin‐type condensation catalyzed by the (S)‐selective acetoin:dichlorophenolindophenol oxidoreductase (Ao : DCPIP OR) followed by a transamination mediated by either a (S)‐ or (R)‐selective amine transaminase (ATA). In this study, we successfully immobilized both Ao : DCPIP OR and two ATAs of opposite enantioselectivity and used them in the biosynthetic cascade to N(P)Es. Immobilization yield, activity recovery, and stability of the immobilized enzymes, both after use (enzyme recycling) and storage (shelf‐life), were assessed. Ao : DCPIP OR immobilized on glyoxyl‐agarose exhibited remarkable stability under storage conditions throughout the 30‐days monitoring period. Moreover, it was successfully reused in the synthesis of (S)‐phenylacetylcarbinol [(S)‐PAC] yielding high conversion (>94 %) and enantiomeric excess (>99 %). The (R)‐selective At‐ATA from Aspergillus terreus, immobilized on Eupergit® C, demonstrated a slightly higher storage stability when compared to the (S)‐selective Sbv333‐ATA from Streptomyces sp. Bv333 immobilized on the same carrier. Remarkably, both enzymes were effectively reused for ten reaction cycles in the synthesis of N(P)Es, starting from the enzymatically synthesized (S)‐PAC, achieving complete conversions and excellent diastereoselectivity (>99 %).

Chitinase-functionalized UiO-66 framework nanoparticles active against multidrug-resistant Candida Auris

Candida auris (C. auris) is a yeast that has caused several outbreaks in the last decade. Cell wall chitin plays a primary role in the antifungal resistance of C. auris. Herein, we investigated the potential of chitinase immobilized with UiO-66 to act as a potent antifungal agent against C. auris. Chitinase was produced from Talaromyces varians SSW3 in a yield of 8.97 U/g dry substrate (ds). The yield was statistically enhanced to 120.41 U/g ds by using Plackett–Burman and Box–Behnken design. We synthesized a UiO-66 framework that was characterized by SEM, TEM, XRD, FTIR, a particle size analyzer, and a zeta sizer. The produced framework had a size of 70.42 ± 8.43 nm with a uniform cubic shape and smooth surface. The produced chitinase was immobilized on UiO-66 with an immobilization yield of 65% achieved after a 6 h loading period. The immobilization of UiO-66 increased the enzyme activity and stability, as indicated by the obtained Kd and T1/2 values. Furthermore, the hydrolytic activity of chitinase was enhanced after immobilization on UiO-66, with an increase in the Vmax and a decrease in the Km of 2- and 38-fold, respectively. Interestingly, the antifungal activity of the produced chitinase was boosted against C. auris by loading the enzyme on UiO-66, with an MIC50 of 0.89 ± 0.056 U/mL, compared to 5.582 ± 0.57 U/mL for the free enzyme. This study offers a novel promising alternative approach to combat the new emerging pathogen C. auris.

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.

Bromelain Immobilized onto Clay-Carboxymethylcellulose Composites for Improving Nutritive Value of Soybean Meal.

Improvement of nutritional value and reduction of antinutritional factors (ANFs) of soybean meal (SBM) for animal feed applications could be achieved by using bromelain immobilized onto bentonite (Bt)-carboxymethylcellulose (CMC) composites. The composite with mass ratio between CMC to calcium ion (Ca2+) at 1:20 provided the highest enzyme activity, immobilization yield higher than 95%, with superior thermal and storage stabilities. Performance of the immobilized bromelain for soybean protein hydrolysis was further studied. The results showed that at 60 °C, the immobilized bromelain exhibited the highest efficiency in enzymatic hydrolysis to release free alpha amino nitrogen (FAN) as a product with high selectivity and to effectively reduce SBM allergenic proteins within 30 min. In conclusion, immobilization of bromelain onto Bt-CMC composites leads to stability enhancement of the enzyme, enabling effective improvement in SBM quality in a short treatment time and showing great potential for application in animal feed industries.

Phenol Removal through Horseradish Peroxidase Immobilization on Ultrafiltration Membranes: Comparative Analysis of Immobilization Methods and Fouling Patterns

This research investigates the removal of phenol using pure peroxidase from horseradish grade I in conjunction with a dead-end ultrafiltration membrane. Various horseradish peroxidase (HRP) immobilization techniques— physical adsorption, covalent bonding, and cross-linking with glutaraldehyde—were applied to a regenerated cellulose (RC) membrane with a surface area of 44 m2 and a molecular weight cut-off of 30 kDa. The investigation examined factors influencing phenol removal, including phenol concentration, membrane fouling, and the reusability of immobilized enzymes. Results indicated that covalent bonding was the most suitable enzyme immobilization technique, achieving a remarkable 90.1% immobilization yield. Phenol removal efficiency reached 100% at 30 min under specific conditions: phenol concentration of 1 mg/L, pH 6.0, hydrogen peroxide concentration of 0.5 mM, and operating pressure set at 3 psig, with temperature maintained at 28 ± 3 °C. Membrane fouling resulted in a decrease in flux. The performance of fouling models was found to be influenced by phenol concentration, with the Cake Formation Model (CFM) proving most effective at low concentrations, while the Complete Pore Blocking Model (CBM) emerged as more suitable at higher concentrations. The immobilized enzyme exhibited reusability for five cycles, maintaining a phenol removal efficiency exceeding 50%. These findings contribute to understanding the enzymatic phenol removal process and the use of appropriate enzyme immobilization techniques for the effective and sustainable treatment of phenol-contaminated water.

Magnetically separable spent coffee grounds as a potential novel support for the covalent immobilization of β‐glucosidase for cellobiose hydrolysis

AbstractBACKGROUNDThe industrial‐scale application of enzymes faces obstacles due to elevated costs and difficulties in stability and reuse. In this study, magnetic spent coffee grounds, an ecotoxic waste, have been utilized successfully for the first time to immobilize β‐glucosidase to overcome these challenges.RESULTSThe spent coffee grounds were magnetized and amine‐functionalized, followed by characterization using various techniques. Under optimized conditions, forming an imine bond between the functionalized support and β‐glucosidase resulted in a 62% immobilization yield (92.81 mg g−1 enzyme loading) and 12.5 U mg−1 activity after immobilization. A relatively small kinetic change was observed in the Km value (902 to 946 μmol L−1) after immobilization, suggesting minimal hindrance by AMSCG3 on substrate access or product release. Moreover, Glu@AMSCG3 showed exceptional stability (>90% residual activity) within a pH range of 3 to 6 after 2 h of incubation at 25 °C. A residual activity of 87.94% was maintained even at 80 °C and pH 5 after 2 h of incubation compared to the free β‐glucosidase, which showed only 6.5% residual activity at the same temperature. When cellobiose was hydrolyzed using Glu@AMSCG3 under optimum conditions, 91.33% cellobiose conversion was achieved initially, and over 79% conversion was maintained for 10 reusability cycles.CONCLUSIONThe improved stability of β‐glucosidase after covalent immobilization on amine‐modified magnetically separable spent coffee grounds indicates their potential as a support matrix for application in enzyme immobilization. © 2024 Society of Chemical Industry (SCI).

Optimization and characterization of immobilized thermostable α-amylase from germinating Sword bean (Canavalia gladiata (Jacq.) DC.) seeds on DEAE-cellulose and chitosan bead for operational stability.

Thermostable α-amylase from germinating Sword bean (Canavalia gladiata (Jacq.) DC.) seeds has been successfully immobilized on DEAE-cellulose (ICgAmy1) and chitosan bead (ICgAmy2) support materials. Optimum conditions of immobilization for DEAE-cellulose and chitosan bead revealed 97% and 96% immobilization yield, respectively. The optimum pH and temperature of both DEAE-cellulose and chitosan bead immobilized α-amylases were pH 7 and 70°C. Both ICgAmy1 and ICgAmy2 were high stability over a wide pH range of pH 5-9 and a temperature range of 70-90°C. In addition, ICgAmy1 and ICgAmy2 led to an operationally stable biocatalyst with above 74% and 76% residual activity after 10 reuses, respectively. Immobilized α-amylases showed high storage stability with 81% (ICgAmy1) and 85% (ICgAmy2) residual activity after 120 days of storage. The easy immobilization process on low-cost, biodegradable, and renewable support materials exhibited an increase in the enzyme operation range and storage stability which reduces production costs. This makes immobilized amylases an effective biocatalyst in various industrial applications especially a potential candidate for bioethanol production, a key renewable energy source.

Effect of Process Parameters on Immobilization of Recombinant Escherichia coli on Coconut Fiber

Escherichia coli (E. coli) is the earliest and most widely used host to produce recombinant proteins, one such example being cyclodextrin glucanotransferase (CGTase) which has found application in numerous industries such as food, environmental engineering and pharmaceutical. The recombinant protein in E. coli was applied to overcome the low output of CGTase from Bacillus species such as Bacillus lehensis and Bacillus licheniformis. However, cell lysis and plasmid instability have been some of the stumbling blocks during the production of recombinant protein in E. coli. Therefore, cell immobilization has been proposed as a way to increase CGTase production while maintaining high cell stability. The objective of this study is to immobilize recombinant E. coli on coconut fiber. The effect of the process parameters, namely pH level (5, 6, 7, 8, and 9), contact time (12, 15, 18, 21, 24, and 27 hours), and temperature (20, 25, 30, 35, and 40 °C), towards the immobilization of recombinant E. coli onto coconut fiber was studied. The optimal pH resulted in the highest immobilization yield was pH 8 (58.90%). The best contact time for the highest immobilization yield (57.59%) was 24 hours. The optimum temperature was identified at 25 °C with immobilization yield of 58.78%. Hence, the optimum conditions could improve the immobilization of recombinant E. coli on the carrier and the coconut fibre was an appropriate carrier material for cell immobilization process for the high CGTase production with high cell stability.

Xylooligosaccharides as emerging prebiotics and their sustainable generation from xylan catalysed by endoxylanase immobilized ordered mesoporous silica

The enzyme endo-1,4-β-xylanase has been a prominent candidate for research in recent years due to its ability to catalyse prebiotic production in an environmentally friendly manner. However, the loss of stability and activity when employed in industrial processes has prompted enzyme immobilization. The present study aims to compare the efficiencies of amorphous (such as fumed silica and silicic acid) and ordered mesoporous silica materials (including SBA-15, IITM-41, and MCM-41) as carriers for endoxylanase immobilization, particularly focussing on the novel silicate IITM-41. Recombinant endoxylanase from Bacillus subtilis KCX006 purified by Ni-NTA columns has been used in this study. XRD, TEM, and BET analyses of these nanostructured materials confirmed the mesoporous structure and the nature of the pores. FT-IR spectroscopy confirmed protein binding through characteristic functional group signatures of silica materials and proteins. Zeta potential values of the nanostructure materials became more negative after enzyme binding, and the confirmation of enzyme immobilization was achieved through fluorescent tagging. Although amorphous silica showed a higher yield of immobilization, it resulted in lower recovered activity due to the formation of protein multilayers. Among the ordered mesoporous silica materials, IITM-41 exhibited the highest recovered activity at 74 %, with a maximum yield of xylooligosaccharides reaching 797.61 mg/g. The optimum pH of the immobilized enzymes remained unchanged, but there was a shift in the optimum temperature from 50 to 60 °C, along with a broader optimal range, attributed to increased enzyme rigidity and substrate diffusion limitations. Kinetic parameters Km and Vmax were higher for immobilized enzymes compared to the free enzyme, particularly with higher Vmax values observed for enzymes immobilized on mesoporous materials compared to those on amorphous silica. Generally, mesoporous materials showed superior performance in terms of xylooligosaccharides production, storage stability, and recycling efficiency. Over 10 cycles of reuse, mesoporous materials exhibited a higher yield of total xylooligosaccharides (ranging from X2 to X6) compared to amorphous silica.

Enhanced Enzymatic Performance of Immobilized Pseudomonas fluorescens Lipase on ZIF-8@ZIF-67 and Its Application to the Synthesis of Neryl Acetate with Transesterification Reaction.

In this study, hybrid skeleton material ZIF-8@ZIF-67 was synthesized by the epitaxial growth method and then was utilized as a carrier for encapsulating Pseudomonas fluorescens lipase (PFL) through the co-precipitation method, resulting in the preparation of immobilized lipase (PFL@ZIF-8@ZIF-67). Subsequently, it was further treated with glutaraldehyde to improve protein immobilization yield. Under optimal immobilization conditions, the specific hydrolytic activity of PFL@ZIF-8@ZIF-67 was 20.4 times higher than that of the free PFL. The prepared biocatalyst was characterized and analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FT-IR). Additionally, the thermal stability of PFL@ZIF-8@ZIF-67 at 50 °C was significantly improved compared to the free PFL. After 7 weeks at room temperature, PFL@ZIF-8@ZIF-67 retained 78% of the transesterification activity, while the free enzyme was only 29%. Finally, PFL@ZIF-8@ZIF-67 was applied to the neryl acetate preparation in a solvent-free system, and the yield of neryl acetate reached 99% after 3 h of reaction. After 10 repetitions, the yields of neryl acetate catalyzed by PFL@ZIF-8@ZIF-67 and the free PFL were 80% and 43%, respectively.

Immobilized chitinase as effective biocatalytic platform for producing bioactive di-N-acetyl chitobiose from recycled chitin food waste

Described is chitinase immobilization on magnetic nanoparticles (MNPs) as biocompatible support for enzymatic production of di-N-acetyl chitobiose from chitin waste. Chitinase immobilization was feasible with an immobilization yield of 88.9 ± 1.6 % with 97.8 ± 1.0 % retention of activity and compared to free enzyme work, immobilization conferred better thermal and storage stability. As practical benefit the attachment to magnetic nanocarriers enabled easy enzyme recovery after repeated application runs and thus sustainable reuse. In fixed state chitinase retained a remarkable 39.7 ± 2.6 % of the starting activity after 16 reaction cycles. Furthermore, immobilized chitinase showed higher catalytic activity than free chitinase in converting shrimp shells and squid-pens chitins into di-N-acetyl chitobiose in a single-step reaction. The final yield of purified compound was 37.0 ± 1.2 % from shrimp shells and 61.1 ± 0.5 % from squid-pens chitin. In conclusion, an efficient MNP-based chitinase immobilization system with the potential for large-scale production was developed.