Immobilization Yield Research Articles

Development of a rapid, efficient, and reusable magnetic bead-based immunocapture system for recombinant human procollagen type II isolation from yeast fermentation broth

Recombinant collagen production, especially using yeasts as expression systems, could represent a promising alternative over traditional extractive methods from animal sources, offering controllable, scalable, and high-quality products. Monitoring the efficiency and efficacy of procollagen/collagen expression, especially in the initial fermentation phases, can be difficult and time consuming, as biological matrices necessitate purification and commonly used analytical methods are only partially informative. We propose a straightforward, efficient, and reusable immunocapture system able to specifically isolate human procollagen type II from fermentation broths and to release it in few experimental steps. A recovered sample allows for a detailed characterization providing information on structural identity and integrity, which can strongly support the monitoring of fermentation processes. The immunocapture system relies on the use of protein A–coated magnetic beads which have been functionalized and cross-linked with a human anti-procollagen II antibody (average immobilization yield of 97.7%) to create a stable and reusable support for the specific procollagen fishing. We set up the binding and release conditions ensuring specific and reproducible binding with a synthetic procollagen antigen. The absence of non-specific interaction with the support and binding specificity was demonstrated, and the latter was also confirmed by a peptide mapping epitope study in reversed-phase liquid chromatography high-resolution mass spectrometry (RP-LC-HRMS). The bio-activated support proved to be reusable and stable over 21 days from the initial use. Finally, the system was successfully tested on a raw yeast fermentation sample to provide a proof of concept of the applicability within recombinant collagen production.Graphical

Wax esters from waste fish oil catalysed by immobilized Candida rugosa lipase

Herein, the evaluation of waste fish (WF) oils as feedstock for the production of wax esters through transesterification reactions was reported. In particular, the synthesis of an emollient ester in the presence of oleyl alcohol and using an immobilized lipase catalyst was proposed. Lipase from Candida rugosa (CRL) was immobilized (0.5 enzyme/support wt. ratio) on a magnetic amino-functionalized hypercross-linked resin (M-HCLR-NH2) by ion exchange, interfacial activation, and covalent anchoring, mediated by 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC). The experimental results demonstrated the successful immobilization of lipase on the as-produced M-HCLR-NH2 with a high immobilization yield of 90%. The immobilized CRL showed very high activity and an activity recovery of ∼ 94%. The immobilized enzyme on the magnetic resin results in more stability and less sensitivity to temperature change than the free counterpart. In addition, during emollient ester synthesis in a solvent system, the immobilized enzyme exhibited very high activity, stability, and excellent reusability, with a yield of 94% after 12 h at 45 °C at an oil:alcohol molar ratio of 1:4 and an immobilized enzyme concentration of 15 wt%./wt of oil. Esters synthesized showed excellent physicochemical properties.

Investigation of adsorption and biosorption features of bio-functionalized poly(GMA-Co-EGDMA) polymer beads in the treatment of nicotine from tobacco industry

The investigation of multifunctional materials for modern enzyme immobilization is an attractive subject in advanced adsorption and biosorption applications. In the present study, the feasibility of immobilization of Lipozyme TL 100L (LPZM) on 3-aminopropyltriethoxysilane (APTES) modified poly-(GMA-co-EGDMA) (PEGDMA) was investigated for adsorption and biosorption of nicotine from aqueous solution. Characterization tests confirmed successful immobilization of lipozyme which significantly altered thermal behavior, surface characteristics, and surface morphology of PEGDMA and PEGDMA/APTES. In addition, the immobilization yields were calculated as 85.0% and 72.0% onto PEGDMA/APTES using physical adsorption and covalent immobilization methods, respectively. The nicotine removal efficiencies were calculated to be 66.4%, 79.0%, 98.9%, and 85.7%, using raw PEGDMA, PEGDMA/APTES, PEGDMA/APTES@LPZM, and PEGDMA/APTES/GU@LPZM, respectively. For the raw PEGDMA, the Langmuir isotherm was best fitted to the adsorption data, while Langmuir-Freundich model described well the adsorption process on PEGDMA/APTES and PEGDMA/APTES@LPZM. The maximum adsorption capacities of Langmuir-Freundlich model increased from 8.118 to 17.32mg/g after enzyme immobilization. The negative enthalpy value, ΔH° (- 10.37kJ/mol), revealed that the nicotine adsorption on PEGDMA/APTES@LPZM was exothermic in nature, which was corroborated by the decrease observed in the number of adsorbed molecules with increasing temperature. In the kinetic experiments, the adsorption on PEGDMA and PEGDMA/APTES@LPZM reached equilibrium with the removal percentages as 66.4% and 98.9% at the end of 3h, respectively. The nicotine adsorption performances in real water matrices were also investigated, and PEGDMA/APTES@LPZM showed satisfactory reusability with removal percentage decreased from 98.9% (1st cycle) to 83.0% (6th cycle).

Functionalized polyacrylonitrile particles as a promising support for the immobilization of laccase fromTrametes versicolor

AbstractThis work evaluates the potential of polyacrylonitrile particles (PAN) as support for laccase fromTrametes versicolorimmobilization. The slurry polymerization method forms mesoporous particles with low surface area and low maximum pore volume in desorption. The particles were chemically modified by consecutive alkaline and acid hydrolysis, followed by amination and activation with glutaraldehyde to enzyme immobilization. The laccase immobilization yield was 99.48% and 14.29% using the functionalized and non‐functionalized particles, respectively. The enzyme activity was measured by the oxidation of 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid) at different pHs and temperatures. The PAN/laccase derivative was hyperactivated at pH 3, up to 3 times higher than the free enzyme, and performed better at 50°C after 6 h of incubation, with relative activity up to 33% higher than the free enzyme. However, both enzymes denatured when the conditions reached pH 8 and 70°C. The PAN/laccase retained 89% of the initial activity after 30 days of storage at 5°C. It was possible to reuse the enzymatic derivative for 5 cycles, with up to 50% residual activity, under 50°C and pH 3. These results show the potential of this new support for laccase immobilization and further applications of industrial interest.

β-Sitosterol Oleate Synthesis by Candida rugosa Lipase in a Solvent-Free Mini Reactor System: Free and Immobilized on Chitosan-Alginate Beads

Candida rugosa lipase (CRL) was immobilized by the ionic gelling technique using alginate and chitosan as encapsulating agents. An immobilization yield of 99% and an immobilization efficiency of 51% were obtained. Maximum hydrolytic activity for free and immobilized CRL was detected at 40 °C and for synthesis activity at 35 °C. The optimum pH for immobilized and free CRL hydrolysis activity was 8.0. The Vmax obtained for the hydrolysis reaction was higher for free CRL (4121.4 μmol/min/g) compared to immobilized CRL (2359.13 μmol/min/g). A Vmax of 2.24 μmol/min/g was detected for the synthetic activity of free CRL. The Km obtained for the hydrolysis reaction was higher (660.02 μmol/L) for immobilized CRL than for free CRL (403.06 μmol/L). For the synthetic activity, a Km of 234.44 μmol/L was calculated. The conversion of β-sitosterol oleate ranged from 80.85 to 96.84% for free CRL, higher than the maximum found for immobilized CRL (32%). The scale-up (scale factor: 50) with the free CRL was successfully performed, achieving a high conversion value (92%) in a 500 mL bioreactor. This conversion value was within the range predicted by the mathematical model obtained using mini reactors. These mini reactors are good models to test several conditions of enzyme reactions that are intended for large scales.

Removal of emerging pollutants from water using enzyme-immobilized activated carbon from coconut shell

This work reports the removal of diclofenac, amoxicillin, carbamazepine, and ciprofloxacin by utilizing three commercially available granular activated carbons (GACs) (Activated carbon, Silcarbon, and Donau) loaded with laccase. Adsorption was used to successfully immobilize laccase on the GACs, as revealed by scanning electron microscopy and energy dispersive X-ray analysis (SEM-EDX) and Fourier transform infrared spectroscopy (FTIR). In the three types of GACs tested, pH 5, 30 °C, and 2 mg mL−1 laccase content were found to be the optimum immobilization parameters. Laccase immobilization yields of 65.2%, 63.1%, and 62.9% were achieved with activated carbon, Silcarbon, and Donau respectively. The adsorption behaviors of the pharmaceuticals onto the tested activated carbons are best described as a spontaneous endothermic process that follows Langmuir isotherm and first-order kinetics. The reusability of the immobilized enzyme was evaluated using 2, 2′-azino-bis 3-ethylbenzothiazole-6-sulphonic acid (ABTS) as a substrate within six cycles for all adsorbents. In 120 mins, nearly a complete removal of the pharmaceutical compounds (50 mg L−1) was obtained in the case of activated carbon type and more than 90% for other adsorbent types when synergistic adsorption and enzymatic degradation were applied. With adsorption alone, 74% removal was obtained with activated carbon and < 56% for other adsorbents. The finding of this study suggests that biochar produced from coconut shell (same as the one used in this study) can effectively be used as a substrate and adsorbent for pharmaceutical removal. This enzymatic physical removal system has the potential to be applied on a large-scale.

β-Xylosidase mutant immobilization on UiO-66-NH2 for continuous production of ginsenoside Rg1 and selective production of furfural from notoginsenoside R1

In order to improve the activity and reusability of β-xylosidase, the β-xylosidase mutant S160N-R333H (R333H) which could convert notoginsenoside R1 to ginsenoside Rg1 and xylose was immobilized on the metal-organic framework (MOF) UiO-66-NH2. The physical and biochemical properties of immobilized β-xylosidase (R333H@UiO-66-NH2) were studied in detail. The results showed improved performances, i.e. the high immobilization yield of 93% and recovery activity of 192.8%, better stability under acidic or trypsin-containing conditions, the higher activity in the reaction containing ions, and the improved catalytic efficiency (was 1.37-fold than that of free R333H). Importantly, the ginsenoside Rg1 yields remained more than 80% after six recycles in McIlvaine buffer and pure water. The UiO-66-NH2 could catalyze xylose dehydration to produce furfural and the yield reached 69%. Thus this study provides a promising method for production of bioactive or platform chemicals via continuous conversion of xyloylated substrates using nanocatalyst synthesized by β-xylosidase and MOF.

Preparation and characterization of silica nanoparticles as an efficient carrier for two bio‐detergents based enzymes

AbstractBio‐detergents are new bio‐friendly formulas that contain biobased ingredients, including enzymes. In the present study, alkaline protease and α‐amylase were immobilized via physisorption onto silica nanoparticles (SNPs). The derivatized SNPs served as major components of a prepared bio‐detergent. Alkaline protease was produced by the recombinant Bacillus subtilis cells that carry the protease genes on a multiple‐copy plasmid, while α‐amylase was commercially purchased. SNPs were prepared by the sol–gel method and well‐characterized through the Brunauer–Emmett–Teller (BET) method, scanning electron microscopy (SEM), transmission electron microscopy (TEM), zeta potential, X‐ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. The adsorption capacity of the SNPs was determined via colorimetry through the adsorption of methylene blue dye (MB), with approximately 97% adsorption achieved under the conditions employed. The Langmuir isotherm well‐described the adsorption of MB on SNPs. High immobilization yield for the enzymes was obtained, and the storage stability of SNP‐alkaline protease and SNP‐α‐amylase was good, reaching 65% and 85% of their initial activities after 6 weeks of storage at 4°C, respectively. The immobilized enzymes could be reused for 7 cycles. Additionally, the immobilized enzymes retained residual activity to a greater extent than free enzymes in simulated basic detergent solutions. SNPs containing adsorbed alkaline protease and α‐amylase were mixed with a basic detergent solution, and the washing efficiency of some proteinous and starchy stains was examined through Hunter Lab spectrophotometry. The latter experiments demonstrated that the immobilized enzymes performed well during the washing process.

Hierarchically-structured laccase@Ni3(PO4)2 hybrid nanoflowers for antibiotic degradation: Application in real wastewater effluent and toxicity evaluation

Laccase@Ni3(PO4)2 hybrid nanoflowers (HNFs) were prepared by the anisotropic growth of biomineralized nickel phosphate. The immobilization yield was 77.5 ± 3.6 %, and the immobilized enzyme retained 50 % of its initial activity after 18 reusability cycles. The immobilized and free enzymes lost 80 % of their activity after 18 and 6 h incubation in municipal wastewater effluent (MWWE), respectively. The increase in α-helix content (8 %) following immobilization led to a more rigid enzyme structure, potentially contributing to its improved stability. The removal of ciprofloxacin from MWWE by laccase@Ni3(PO4)2·HNFs/p-coumaric acid oxidation system was optimized using a Box-Behnken design. Under the optimized conditions [initial laccase activity (0.05 U mL−1), the concentration of p-coumaric acid (2.9 mM), and treatment time (4.9 h)], the biocatalyst removed 90 % of ciprofloxacin (10 mg L−1) from MWWE. The toxicity of ciprofloxacin against some G+ and G− bacteria was reduced by 35–70 %, depending on their strain. The EC50 of ciprofloxacin for the alga Raphidocelis subcapitata reduced from 3.08 to 1.07 mg L−1 (p-value <0.05) after the bioremoval. Also, the acute and chronic toxicity of identified biodegradation products was lower than ciprofloxacin at three trophic levels, as predicted by ECOSAR software.

Effects of Lipase Immobilization Conditions and Support Materials for the Production of Structured Triacylglycerols

AbstractStructured triacylglycerols (STAG) with desired properties can be synthesized by transesterification using immobilized lipases. Herein, the effect of immobilization conditions and support material on the immobilization yield, specific activity and regioselectivity of lipases from Rhizomucor miehei (RML) and Rhizopus oryzae (ROL) in the production of STAG, are evaluated. Four different support materials utilizing adsorption and one with covalent binding are investigated. Ammonium sulfate is found to significantly increase the activity‐based immobilization yield (12% and 38% for RML and ROL, respectively) and specific activity on Accurel MP1000 (MP1000) when used as the immobilization buffer. Furthermore, the immobilization principle and support material influenced both the activity and regiospecificity. Immobilization by adsorption is found to result in higher catalytic activity, while covalent binding resulted in lipase inactivation. For RML, the highest specific activity of 43 µmol STAG min−1 g−1 (U) is obtained on MP1000, while ROL, which exhibited higher activities in general, results in a maximum activity of 120 U on Lifetech ECR8806. The obtained specific activites are comparable to the commercial preparations Novozym 40086 (45 U) and Lipase DF “Amano” IM (147 U) while the regiospecificity of the developed preparations is even higher, forming at least 64% less byproduct.Practical applications: There is an increasing need for lipids with specific nutritional and physical properties in health, nutrition, and food applications. In this context, STAG are highly promising products due to the possibility to tailor the composition to obtain the desired properties. Immobilized lipases are the catalyst of choice for the production of STAG, in which activity and regioselectivity are particularly important parameters for the process performance. In this study, it is shown that these parameters can be affected by the immobilization conditions and support material. Immobilized preparations with high activity and excellent regiospecificity are created on commercially available supports. This shows the possibility of STAG synthesis with high purity and beneficial properties.

Preparation of Chitosan-Carbon Nanotubes Decorated with ZnO@laccase: Application in the Removal of Bisphenol A from Aqueous Solution

Bioremediation with immobilized enzymes has been established as the method for restoring contaminated ecosystems with the help of biological tools on a global scale. The purpose of this work is to immobilize the laccase enzyme for increasing the biocatalyst′s ability in order to remove bisphenol A (BPA). Initially, the nanocatalyst was prepared by immobilizing laccase on chitosan using carbon nanotubes decorated with zinc oxide. Subsequently, Fourier transformed infrared (FTIR) spectroscopy, X-ray diffraction (XRD), field emission scanning microscopy (FE-SEM), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDX), and dot mapping were employed to characterize the prepared nanocomposites. Laccase activity, immobilization yield (%), thermal and storage stability, and reusability of laccase immobilized on chitosan-coated carbon nanotubes (CNTs)/ZnO nanocomposites were also measured. The chitosan (CS)-CNTs/[email protected] nanocatalyst performance in different operational conditions (initial concentrations of bisphenol A, amount of nanobiocatalyst, pH, and irradiation time) on the photodegradation process of bisphenol A were also investigated and optimized using the response surface methodology (RSM)-central composite design (CCD) model. The RSM design and experiments demonstrated that 94.51% of BPA was removed under optimal conditions (initial BPA concentration of 100 mg L−1, nanocatalyst amount of 1 g L−1, pH=9, and 40 min of contact exposure). Laccase was a critical factor in this process, and after five recycling cycles, immobilized laccase retained 61% of its original activity, indicating that this nanobiocatalyst can be used in purification processes as a durable and robust catalyst.

(Magnetic) Cross-Linked Enzyme Aggregates of Cellulase from T. reesei: A Stable and Efficient Biocatalyst.

Cross-linked enzyme aggregates (CLEAs) represent an effective tool for carrier-free immobilization of enzymes. The present study promotes a successful application of functionalized magnetic nanoparticles (MNPs) for stabilization of cellulase CLEAs. Catalytically active CLEAs and magnetic cross-linked enzyme aggregates (mCLEAs) of cellulase from Trichoderma reesei were prepared using glutaraldehyde (GA) as a cross-linking agent and the catalytic activity and stability of the CLEAs/mCLEAs were investigated. The influence of precipitation agents, cross-linker concentration, concentration of enzyme, addition of bovine serum albumin (BSA), and addition of sodium cyanoborohydride (NaBH3CN) on expressed activity and immobilization yield of CLEAs/mCLEAs was studied. Particularly, reducing the unsaturated Schiff's base to form irreversible linkages is important and improved the activity of CLEAs (86%) and mCLEAs (91%). For increased applicability of CLEAs/mCLEAs, we enhanced the activity and stability at mild biochemical process conditions. The reusability after 10 cycles of both CLEAs and mCLEAs was investigated, which retained 72% and 65% of the initial activity, respectively. The thermal stability of CLEAs and mCLEAs in comparison with the non-immobilized enzyme was obtained at 30 °C (145.65% and 188.7%, respectively) and 50 °C (185.1% and 141.4%, respectively). Kinetic parameters were determined for CLEAs and mCLEAs, and the KM constant was found at 0.055 ± 0.0102 mM and 0.037 ± 0.0012 mM, respectively. The maximum velocity rate (Vmax) was calculated as 1.12 ± 0.0012 µmol/min for CLEA and 1.17 ± 0.0023 µmol/min for mCLEA. Structural characterization was studied using XRD, SEM, and FT-IR. Catalytical properties of immobilized enzyme were improved with the addition of reducent NaBH3CN by enhancing the activity of CLEAs and with addition of functionalized aminosilane MNPs by enhancing the activity of mCLEAs.

Bio-inspired functional photocatalyst: Lipase enzyme functionalized TiO2 with excellent photocatalytic, enzymatic, and antimicrobial performance

Herein, we proposed a novel approach for the improvement of a photocatalytic material via functionalization with biocatalyst for successfully utilization in photocatalytic, enzymatic, and antimicrobial processes. For this, titanium dioxide (TiO2) was first modified using 3-aminopropyltriethoxysilane (APTES) to incorporate silane groups for the effective immobilization of Lipozyme TL 100 L enzyme. The as-synthesized samples were characterized by FT-IR, TGA, BET, SEM, XRD, XPS, UV–vis DRS and PL analyses in order to examine the thermal, structural, textural and optical features. The covalent immobilization yield of lipozyme on the modified TiO2 (TiO2@APTES) was found to be 97.5 % with 2,437.5 U/g of enzyme activity, and the immobilized enzyme (TiO2@LPZM) displayed excellent stability at different pH and temperatures when compared to its free form. The photocatalytic performance of TiO2@LPZM towards degradation of antibiotics significantly enhanced under both UV-A and visible light irradiation. The tetracycline and ciprofloxacin degradation rates of the bio-inspired photocatalyst were found 3.4- and 1.65-times higher than that of raw TiO2, respectively. The enhanced performance was ascribed to the attachment of target antibiotics by enzyme molecules, extended light absorption and reduced recombination rate of charge pairs. In addition, the enzyme immobilization increased the surface adsorbed oxygen species which boosted the photocatalytic reaction. The antimicrobial activity tests of the samples were examined against two types of bacterial species as E. coli and S. aureus and the enhanced antibacterial efficiencies were attributed to the presence of silanol, amino, and hydroxyl groups of modified TiO2. This research highlights the importance of enzyme immobilization on a photocatalytic support with synergistically improved surface features.

Synthesis and Characterization of Aminoamidine-Based Polyacrylonitrile Fibers for Lipase Immobilization with Effective Reusability and Storage Stability.

Lipases are extensively utilized industrial biocatalysts that play an important role in various industrial and biotechnological applications. Herein, polyacrylonitrile (PAN) was treated with hexamethylene diamine (HMDA) and activated by glutaraldehyde, then utilized as a carrier support for Candida rugosa lipase. In this regard, the morphological structure of modified PAN before and after the immobilization process was evaluated using FTIR and SEM analyses. The immobilized lipase exhibited the highest activity at pH 8.0, with an immobilization yield of 81% and an activity of 91%. The optimal pH and temperature for free lipase were 7.5 and 40 °C, while the immobilized lipase exhibited its optimal activity at a pH of 8.0 and a temperature of 50 °C. After recycling 10 times, the immobilized lipase maintained 76% of its activity and, after 15 reuses, it preserved 61% of its activity. The lipase stability was significantly improved after immobilization, as it maintained 76% of its initial activity after 60 days of storage. The calculated Km values were 4.07 and 6.16 mM for free and immobilized lipase, and the Vmax values were 74 and 77 μmol/mL/min, respectively. These results demonstrated that synthetically modified PAN is appropriate for immobilizing enzymes and has the potential for commercial applications.

Controlling the Adsorption of β-Glucosidase onto Wrinkled SiO2 Nanoparticles To Boost the Yield of Immobilization of an Efficient Biocatalyst.

β-Glucosidase (BG) catalyzes the hydrolysis of cellobiose to glucose, a substrate for fermentation to produce the carbon-neutral fuel bioethanol. Enzyme thermal stability and reusability can be improved through immobilization onto insoluble supports. Moreover, nanoscaled matrixes allow for preserving high reaction rates. In this work, BG was physically immobilized onto wrinkled SiO2 nanoparticles (WSNs). The adsorption procedure was tuned by varying the BG:WSNs weight ratio to achieve the maximum controllability and maximize the yield of immobilization, while different times of immobilization were monitored. Results show that a BG:WSNs ratio equal to 1:6 wt/wt provides for the highest colloidal stability, whereas an immobilization time of 24 h results in the highest enzyme loading (135 mg/g of support) corresponding to 80% yield of immobilization. An enzyme corona is formed in 2 h, which gradually disappears as the protein diffuses within the pores. The adsorption into the silica structure causes little change in the protein secondary structure. Furthermore, supported enzyme exhibits a remarkable gain in thermal stability, retaining complete folding up to 90 °C. Catalytic tests assessed that immobilized BG achieves 100% cellobiose conversion. The improved adsorption protocol provides simultaneously high glucose production, enhanced yield of immobilization, and good reusability, resulting in considerable reduction of enzyme waste in the immobilization stage.

Lipase immobilization using scalable and biocompatible lignin-based material as a carrier

Lignin, derived from agricultural-waste corncob residue, was used to immobilize C. antarctica lipase B (CALB) by taking advantage of the mechanism that the surface hydrophobicity of lignin is positive to the immobilization of lipase by the interfacial activation between the carrier and lipase. The immobilization yield of 93.0 ± 0.2% and enzyme loading of 28.1 ± 0.3 mg/g were obtained under optimized process. The maximum transesterification yield of ethyl palmitate (80.7 ± 1.2%) was obtained within p-nitrophenol palmitate and ethanol as the substrate, transesterification time of 6 h, temperature of 42 ℃, and the organic phase of n-hexane. The recovered activity of immobilized enzyme reached 77.0 ± 0.6% after 20 days of storage, and maintained 88.6 ± 1.4% of its initial activity after 5 cycles application. These results demonstrate that the process presents a useful protocol to immobilize lipase on lignin, which is a fully enzyme-mediated green and sustainable biocatalytic system based exclusively on lignin.

Green synthesis of silica-coated magnetic nanocarriers for simultaneous purification-immobilization of β-1,3-xylanase

Tailoring magnetic nanocarriers with tunable properties is of great significance for the development of multifunctional candidate materials in numerous fields. Herein, we report a one-pot biomimetic silicification-based method for the synthesis of silica-coated magnetic nanoparticles. The synthesis process was mild, low cost, and highly efficient, which took only about 21 min compared with 4.5–120 h in other literature. Then, the carriers had been characterized by VSM, SEM, TEM, XRD, FT-IR, and EDS to confirm their function. To evaluate the usefulness of the carriers, they were adopted to couple the purification and immobilization of β-1,3-xylanase from the cell lysate in a single step with high immobilization yield (92.8 %) and high activity recovery (82.4 %). The immobilized enzyme also retained 58.4 % of the initial activity after 10 cycles and displayed good storage properties, and improved thermal stability, which would be promising in algae biomass bioconversion as well as other diverse applications.

Aspergillus Niger thermostable Cytosine deaminase-dextran conjugates with enhanced structure stability, proteolytic resistance, and Antiproliferative activity

Cytosine deaminase (CDA) is a prodrug mediating enzyme converting 5-flurocytosine into 5-flurouracil with profound broad-range anticancer activity towards various cell lines. Availability, molecular stability, and catalytic efficiency are the main limiting factors halting the clinical applications of this enzyme on prodrug and gene therapies, thus, screening for CDA with unique biochemical and catalytic properties was the objective. Thermotolerant/ thermophilic fungi could be a distinctive repertoire for enzymes with affordable stability and catalytic efficiency. Among the recovered thermotolerant isolates, Aspergillus niger with optimal growth at 45 °C had the highest CDA productivity. The enzyme was purified, with purification 15.4 folds, molecular mass 48 kDa and 98 kDa, under denaturing and native PAGE, respectively. The purified CDA was covalently conjugated with dextran with the highest immobilization yield of 75%. The free and CDA-dextran conjugates have the same optimum pH 7.4, reaction temperature 37 °C, and pI 4.5, and similar response to the inhibitors and amino acids suicide analogues, ensuring the lack of effect of dextran conjugation on the CDA conformational structure. CDA-Dextran conjugates had more resistance to proteolysis in response to proteinase K and trypsin by 2.9 and 1.5 folds, respectively. CDA-Dextran conjugates displayed a dramatic structural and thermal stability than the free enzyme, authenticating the acquired structural and catalytic stability upon dextran conjugation. The thermal stability of CDA was increased by about 1.5 folds, upon dextran conjugation, as revealed from the half-life time (T1/2). The affinity of CDA-conjugates (Km 0.15 mM) and free CDA (Km 0.22 mM) to deaminate 5-fluorocytosine was increased by 1.5 folds. Upon dextran conjugation, the antiproliferative activity of the CDA towards the different cell lines “MDA-MB, HepG-2, and PC-3” was significantly increased by mediating the prodrug 5-FC. The CDA-dextran conjugates strongly reduce the tumor size and weight of the Ehrlich cells (EAC), dramatically increase the titers of Caspase-independent apoptotic markers PARP-1 and AIF, with no cellular cytotoxic activity, as revealed from the hematological and biochemical parameters.

Fabrication and Characterization of Dextranase Nano-Entrapped Enzymes in Polymeric Particles Using a Novel Ultrasonication–Microwave Approach

In the current study, a novel method to improve the nano-entrapment of enzymes into Ca-alginate gel was investigated to determine the synergistic effects of ultrasound combined with microwave shock (UMS). The effects of UMS treatment on dextranase enzymes’ loading effectiveness (LE) and immobilization yield (IY) were investigated. By using FT-IR spectra and SEM, the microstructure of the immobilized enzyme (IE) was characterized. Additionally, the free enzyme was used as a control to compare the reusability and enzyme-kinetics characteristics of IEs produced with and without UMS treatments. The results demonstrated that the highest LE and IY were obtained when the IE was produced with a US of 40 W at 25 kHz for 15 min combined with an MS of 60 W at a shock rate of 20 s/min for 20 min, increasing the LE and the IY by 97.32 and 78.25%, respectively, when compared with an immobilized enzyme prepared without UMS treatment. In comparison with the control, UMS treatment dramatically raised the Vmax, KM, catalytic, and specificity constant values for the IE. The outcomes suggested that a microwave shock and ultrasound combination would be an efficient way to improve the immobilization of enzymes in biopolymer gel.

Immobilization of the metagenomic lipase, LipG9, on porous pellets of poly-hydroxybutyrate produced by the double emulsion solvent evaporation technique.

This work aimed to produce porous poly-hydroxybutyrate (PHB) pellets in order to evaluate the pellets as a support for immobilization of the metagenomic lipase, LipG9. Four types of pelletized PHB particles with different morphological characteristics were obtained using the double emulsion and solvent evaporation technique (DESE). The micropores of these PHB pellets had similar average diameters (about 3nm), but the pellets had different specific surface areas: 11.7 m2 g-1 for the PHB powder, 8.4m2 g-1 for the control pellets (Ø<0.5mm, produced without the pore forming agent), 10.0m2 g-1 for the small pellets (Ø<0.5mm), 9.5m2 g-1 for the medium pellets (0.5 < Ø<0.8mm) and 8.4m2 g-1 for the large pellets (Ø>1.4mm). Purified LipG9 was immobilized by adsorption on these pellets, and the results were compared with those obtained with PHB powder. The highest immobilization yield (83%) was obtained for the medium PHB pellets, followed by large (76%) and small (55%) PHB pellets. The activity of LipG9 immobilized on the pellets, for the synthesis of ethyl oleate in n-hexane, was highest for the medium pellets (22Ug-1 ). The immobilization yield was high for PHB powder (99%) but the esterification activity was slightly lower (20Ug-1 ). These results show that pelletized PHBbeads can be used for the immobilization of lipases, with the advantage that pelletized PHB will perform better than PHB powder in large-scale enzyme bioreactors.