Free Enzyme Research Articles

Detection of insecticides by Tetronarce californica acetylcholinesterase via expression and in silico analysis.

The acetylcholinesterase (AChE) is involved in termination of synaptic transmission at cholinergic synapses and plays a vital role in the insecticide detection and inhibitor screening. Here, we report the heterologous expression of an AChE from Tetronarce californica (TcA) in Escherichia coli (E. coli)as a soluble active protein. TcA was immobilized in calcium alginate beads; the morphology, biochemical properties, and insecticide detection performance of free and immobilized TcA were characterized. Moreover, we used sequence, structure-based approaches, and molecular docking to investigate structural and functional characterization of TcA. The results showed that TcA exhibited a specific activity of 102 U/mg, with optimal activity at pH 8.0 and 30 °C. Immobilized TcA demonstrated superior thermal stability, pH stability, and storage stability compared to the free enzyme. The highest sensitivity of free TcA was observed with trichlorfon, whereas immobilized TcA showed reduced IC50 values towards tested insecticides by 3 to 180-fold. Molecular docking analysis revealed the interaction of trichlorfon, acephate, isoprocarb, λ-cyhalothrin, and fenpropathrin in the active site gorge of TcA, particularly mediated through the formation of hydrogen bonds and π-π stacking. Therefore, TcA expressed heterologously in E. coli is a promising candidate for applications in food safety and environmental analysis. KEY POINTS: • T. californica AChE was expressed solubly in prokaryotic system. • The biochemical properties of free/immobilized enzyme were characterized. • The sensitivity of enzyme to insecticides was evaluated in vitro and in silico.

Novel co-immobilization of thermostable isomerase and aspartase on NH2-UiO66 for efficient biosynthesis of L-aspartate from maleate

L-aspartate is an important chemical in the food and pharmaceutical industries. Presently, it is meaningful to realize a co-immobilized multi-enzyme biocatalyst that can synthesize L-aspartate in vitro through multi-step cascade reaction. Herein, the immobilization method was first employed to investigate the ability of immobilized double enzymes, isomerase AaMaiA and aspartase AspB, to systhesize L-aspartate. The optimal temperature (50 ℃) of AaMaiA-immobilized NH2-UiO66, a metal organic framework material, was significantly superior to that of its free counterpart. Compared to free enzymes, AaMaiA@NH2-UiO66 retained 57.8% of enzyme activity and exhibited a half-life that was improved 2.59-fold at 50 ℃. The co-immobilized AaMaiA/AspB@NH2-UiO66 (AaMaiA: AspB = 6: 1, v/v) showed high protein loading (27.899 ± 1.945 mg/g) and 84.4% recovery of enzyme activity after 8 consecutive cycles. Compared to free AaMaiA/AspB, the novel AaMaiA/AspB@NH2-UiO66 exhibited far superior tolerance to organic solvents. For the first time, the prepared AaMaiA/AspB@NH2-UiO66 was successfully applied as an efficient biocatalyst and converted 1 M maleate to L-aspartate with a yield of 86.8% within 1 h. Remarkably, AaMaiA/AspB@NH2-UiO66 exhibited great potential for efficient biosynthesis of L-aspartate.

Film-shaped reusable smart polymer to produce lactose-free milk by simple immersion

In this study, we report the synthesis and characterization of a highly manageable polyacrylic film material for enzyme immobilization, using β-galactosidase (β-gal) as a model enzyme. The material is based on commercially available monomers and achieves efficient immobilization of β-gal through the formation of azo linkages between amino styrene groups in the polyacrylic material and the enzyme. The immobilized enzyme demonstrates superior performance compared to free enzyme in lactose hydrolysis of UHT milk, achieving lactose concentrations below 0.1% (<1 mg/mL), indicating its potential for lactose hydrolysis in dairy products. The film-shaped material is designed for easy submersion and removal, similar to a smart card, and offers reusability, with the ability to be reused at least 10 times without loss of enzymatic activity. Characterization of the immobilized enzyme on the polymeric material was performed using various techniques, including scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), and Raman spectroscopy. Protein release studies confirmed the stability of the immobilized enzyme during prolonged incubation in aqueous solution without significant enzyme leakage. Overall, the polyacrylic film material demonstrates promise as a simple and efficient approach for enzyme immobilization, with potential applications in various industries, including the food industry.

Self-supporting biocatalytic polyelectrolyte complex hollow fiber membranes via salt-dilution induced phase separation

The potential use of biocatalytic membranes in industries such as water treatment, pharmaceuticals, and medicine has attracted much interest in recent years. However, the use of organic solvents in traditional membrane fabrication techniques presents a challenge in the direct integration of enzymes during the membrane production. Thus, enzyme immobilization relies on consecutive modification steps (coating, binding, activation). Yet, recent progress in membrane fabrication using all-aqueous phase inversion of polyelectrolyte complexes (PEC) enables the direct incorporation of enzymes in the polymer solution. We present the fabrication of free-standing, defect-free and mechanically stable biocatalytic PEC hollow fiber membranes via the salt-dilution induced phase separation method. A polymer solution containing poly(sodium-4-styrenesulfonate) (PSS), poly(diallyldimethylammonium chloride) (PDADMAC), KBr, and the enzyme alkaline phosphatase (ALP) was extruded through a spinneret with a mixture of water and glycerol as lumen fluid into a water coagulation bath at 0 °C. The mechanical stability of the fibers is tuned by subsequent photo-induced cross-linking using 4,4’-diazidostilbene-2,2’-difulfonate tetrahydrate (DAS) and UV light. The resulting membranes have a molecular weight cut-off of 400Da, a pure water permeance of 1.5LMH/bar, and a burst pressure of 7bar. The immobilization of ALP proves successful, as the immobilized shows similar catalytic activity to that of the free enzyme, and both storage and thermal stability are improved. Moreover, the enzymes retain 70% of their initial activity after UV cross-linking with DAS and maintain their catalytic activity after repeated wash and filtration cycles. This work highlights the versatility of the fabrication of polyelectrolyte complex hollow fiber membranes via the salt-dilution induced phase separation, serving as a platform technology for the fabrication of porous, selective, and bioactive membranes.

Improved Laccase Encapsulation in Copper-Doped Zeolitic Imidazolate Framework-8 for Reactive Black 5 Decolorization

As the largest group of synthetic dyes, azo dyes can pose various health and environmental risks due to their widespread use and challenging degradation. Laccases are efficient green biocatalysts for the degradation of organic pollutants. Herein, we report the in situ packaging of laccase in copper-doped zeolitic imidazolate framework-8 (ZIF-8) for the decolorization of reactive black 5, which is a model azo dye. The immobilization support (Cu5/mZIF-8) was obtained via lowering the precursor ratio of ZIF-8 and incorporating copper ions during the synthesis process. Cu5/mZIF-8 were found to be nanospheres with an average diameter of around 150 nm. Laccase encapsulated in Cu5/mZIF-8 showed an activity recovery of 75.6%, which was 2.2 times higher than that of the laccase embedded in ZIF-8. Meanwhile, the immobilized laccase (Lac@Cu5/mZIF-8) showed a higher catalytic activity in organic solvents than that of the free enzyme. In the presence of a mediator, Lac@Cu5/mZIF-8 could remove 95.7% of reactive black 5 in 40 min. After four consecutive cycles, the dye decolorization efficiency declined to 28%. About four transformation products of reactive black 5 were identified via LC-MS analysis, and the potential decolorization mechanism was proposed. The results indicated that the immobilized laccase could be used as an efficient biocatalyst in dye decolorization.

Efficient biosynthesis of (R)− 1-[3,5-bis(trifluoromethyl)phenyl]ethanamine with self-assembled dual-enzyme clusters

Purified SpyCatcher-ATA117 (5 µM) and SpyTag-ADH (5 µM) were covalently combined to form self-assembled dual-enzyme clusters (DENCs) at 4 °C for 3 h. The successful assembly of DENCs was verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS). DENCs improved the storage stability by 36.2% and pH stability by 11.2% compared with those of free double enzymes. 56.89 g/L (R)− 1-[3,5-bis(trifluoromethyl)phenyl]ethanamine (R-BPA) was synthesized using DENCs with high yield (99.9%) and high enantiomeric excess (99.9%) at 45 °C and pH 9 for 24 h. Moreover, DENCs retained more than 78% of the initial activity after being reused in the preparation of R-BPA for five cycles. Thus, DENCs were successfully formed, and efficient amination was achieved, providing a new biocatalyst for efficient biosynthesis of chiral amines via asymmetric transamination cascade reaction.

Enzyme immobilized on magnetic fluorescent bifunctional nanoparticles for α-glucosidase inhibitors virtual screening from Agrimonia pilosa Ledeb extracts accompanied with molecular modeling

Agrimonia pilosa Ledeb (APL) is a significant source of inhibitors for α-glucosidase, which is an essential target enzyme for the treatment of type 2 diabetes, cancer and acquired immune deficiency syndrome. Ligand fishing is a suitable approach for the highly selective screening of bioactive substances in complex mixtures. Yet it is unable to conduct biomedical imaging screening, which is crucial for real-time identification. In this case, a bioanalytical platform combining magnetic fluorescent ligand fishing and in-situ imaging technique was established for the screening and identification of α-glucosidase inhibitors (AGIs) from APL crude extract, utilizing α-glucosidase coated CuInS2/ZnS-Fe3O4@SiO2 (AG-CIZSFS) nanocomposites as extracting material and fluorescent tracer. The AG-CIZSFS nanocomposites prepared through solvothermal and crosslinking methods displayed fast magnetic separation, excellent fluorescence performance and high enzyme activity. The tolerance of immobilized enzyme to temperature and pH was stronger than that of free enzyme. Prior to proof-of-concept with APL crude extract, a number essential parameters (glutaraldehyde concentration, immobilized time, enzyme amount, reaction solution pH, incubation temperature, incubation time, percentage of methanol in eluen, elution times and eluent volume) were optimized using an artificial test mixture. The fished ligands were identified by UPLC-MS/MS and their biological activities were preliminarily evaluated by real-time cellular morphological imaging of human colon carcinoma (HCT-116) cells based on confocal laser scanning microscope (CLSM). Their α-glucosidase inhibitory activities were further verified and studied by classical pNPG method and molecular docking. The isolated compounds exhibited significant α-glucosidase inhibitory activities with a IC50 value of 11.57 µg·mL−1. Six potential AGIs including tribuloside, ivorengenin A, tormentic acid, 1β, 2β, 3β, 19α-Tetra hydroxyurs-12-en-28-oic acid, corosolic acid and pomolic acid were ultimately screened out and identified from APL crude extracts. The proposed approach, which combined highly specific screening with in-situ visual imaging, provided a powerful platform for discovering bioactive components from multi-component and multi-target traditional Chinese medicine (TCM).

Relay run property of immobilized cholesterol oxidase on magnetic biochar for sterol removal

This study investigated the biocatalytic performance of immobilized cholesterol oxidase (CHOD) on magnetite-based carbon (MBC) for degrading cholesterol. The results showed that MBC-CHOD exhibited higher activity and good affinity towards substrate compared to free enzyme and other immobilized enzymes. Mass spectra analysis revealed that MBC-CHOD damaged the main structure of cholesterol, benefitting the further biological treatment. The study proposes a Fenton process mechanism by which H2O2 is transferred to free radicals such as ·OH under acidic conditions, promoting further substrate degradation. This suggests that MBC-CHOD has a relay run property leading to high degradation of cholesterol. Molecular docking indicates that cholesterol preferentially binds to TYR-28 residue and LYS-138 residue in CHOD through hydrogen bonds. Overall, MBC-CHOD proved to be a promising candidate for efficient and sustainable cholesterol degradation.

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.

Bioconversion of starch to Cyclodextrin using Cyclodextrin glycosyltransferase immobilized on metal organic framework

The superiority of a metal organic framework, namely, MIL-101 (Cr) over conventional support, zeolite Y, as immobilization support for Cyclodextrin glycosyltransferase (CGTase) was investigated. The adsorption capacity, stability, and secondary structures of CGTase upon immobilization were investigated. CGTase adsorption on MIL-101 and zeolite Y was best represented by the Langmuir isotherm with optimum adsorption capacities of 37.5 and 6.1 mg/g on two supports, respectively. The deconvolution of the amide I band of the FTIR spectrum indicated that free CGTase molecules predominantly contain β-sheets, which increased with the immobilization on MIL-101 and zeolite Y from 56% to 84.1% and 69.7%, respectively, suggesting the existence of CGTase agglomerates on the supports. The CGTase immobilized on MIL-101 showed relative activity of about 50% at a substrate concentration of up to 4 g/L (compared with free enzymes) and above 3 times higher selectivity towards the more valuable α-CD. A diffusion–reaction model was used to predict the behavior of the immobilized system, and reusability studies revealed that immobilized CGTase may be utilized for numerous reaction cycles, with possible improvements via covalent attachment using glutaraldehyde. Overall, the obtained results in this study provide insights into the usefulness of MIL-101 as a suitable support for CGTase.

Immobilization protects enzymes from plasma-mediated inactivation.

Non-thermal plasmas are used in various applications to inactivate biological agents or biomolecules. A complex cocktail of reactive species, (vacuum) UV radiation and in some cases exposure to an electric field together cause the detrimental effects. In contrast to this disruptive property of technical plasmas, we have shown previously that it is possible to use non-thermal plasma-generated species such as H2O2 as cosubstrates in biocatalytic reactions. One of the main limitations in plasma-driven biocatalysis is the relatively short enzyme lifetime under plasma-operating conditions. This challenge could be overcome by immobilizing the enzymes on inert carrier materials. Here, we tested whether immobilization is suited to protect proteins from inactivation by plasma. To this end, using a dielectric barrier discharge device (PlasmaDerm), plasma stability was tested for five enzymes immobilized on ten different carrier materials. A comparative analysis of the treatment times needed to reduce enzyme activity of immobilized and free enzyme by 30% showed a maximum increase by a factor of 44. Covalent immobilization on a partly hydrophobic carrier surface proved most effective. We conclude from the study, that immobilization universally protects enzymes under plasma-operating conditions, paving the way for new emerging applications.

Ultrathin 2D-MOFs for dual-enzyme cascade biocatalysis with sensitive glucose detection performances.

In recent years, two-dimensional nanosheet metal-organic frameworks (2D MOFs) have been widely considered as promising carriers for enzyme immobilization owing to their large surface area, designable and tunable structures, and other properties that enhance enzyme loading and modulate interactions with enzymes. In this study, a series of ultrathin 2D M-TCPP (M = Co, Ni, Zn, Cu) nanosheets were synthesized employing a surfactant-assisted bottom-up approach, and the effect of solvent ratio on the morphology and properties of 2D MOFs was explored. After systematic characterization, Cu-based 2D MOFs with large specific surface areas and excellent water stability was selected as the carrier for the co-immobilization of glucose oxidase (GOx) and horseradish peroxidase (HRP). The effects of adsorption and covalent immobilization strategies on bis-enzyme loading and enzyme activity, as well as their applications in rapid glucose detection, were systematically investigated. The results showed that A-CTGH and C-CTGH owned enzyme loadings of 187.9 and 249.1mg/g, respectively, and exhibited superior enzymatic activity when exposed to harsh environments compared to free enzymes. In addition, the covalently immobilized biocatalyst based on GOx demonstrated a more sensitive glucose detection performance, including a wide linear range from 5.0 to 16μM with a detection limit of 0.6μM.

Engineered Escherichia coli Consortia Function in a Programmable Pattern for Multiple Enzymatic Biosynthesis.

Coordinating microbial consortia to realize complex synthetic pathways is an area of great interest in the rapidly growing field of biomanufacturing. This work presents a programmable method for assembling living cells based on the surface display of affinity groups, enabling whole-cell catalysis with optimized catalytic efficiency through the rational arrangement of cell assemblies and enzymes. In the context of d-phenyllactic acid (d-PLA) synthesis, four enzymes were rationally arranged considering substrate channeling and protein expression levels. The production efficiencies of d-PLA catalyzed by engineered microbial consortia were 1.31- and 2.55-fold higher than those of biofilm and whole-cell catalysts, respectively. Notably, substrate channeling was identified between the coimmobilized rate-limiting enzymes, resulting in a 3.67-fold improvement in catalytic efficiency compared with hybrid catalysts (free enzymes coupled with whole-cell catalysts). The highest yield of d-PLA catalyzed by microbial consortia was 102.85 ± 3.39 mM with 140 mM benzaldehyde as the substrate. This study proposes a novel approach to cell enzyme assembly for coordinating microbial consortia in multiple enzymatic biosynthesis processes.

Creating a new biocatalytic complex with extremolipases and biocompatible ionic liquids for improved transesterification reactions

The ongoing energy crisis has spurred increased research into sustainable and more competitive methods for producing biofuels, including biodiesel. In this context, the focus of the current study is to underscore the viability of investing in a novel biocatalytic complex. This complex incorporates extremophilic lipases and biocompatible ionic liquids with the aim of achieving exceptionally high conversions in transesterification reactions without generating glycerol. Through a meticulous screening process encompassing various amino acid and dipeptide-based ionic liquids from the ammonium family, cholinium glycinate turned out to be the optimal choice. This selection was driven not only by its enhanced compatibility with a commercially available Candida antarctica lipase B (CaLB) but also with extremophilic enzymes synthesized in-house, derived from halophilic (Halomonas spLM1C) and thermophilic (Thermus thermophilus HB27) strains. Following rigorous testing of both free and immobilized enzymes, the ideal concentration of the ionic liquid in transesterification reactions was determined to be 1% relative to the sunflower oil content. Comparative analysis of conversion rates between immobilized thermophilic lipase and immobilized CaLB revealed the efficacy of the proposed approach. Maximum conversions were found to increase by 20%, with specific conversion rates soaring by approximately 180% when utilizing the immobilized thermophilic lipase. In conclusion, this research ushers in new prospects for advancing the competitiveness of biocatalytic solutions in glycerol-free transesterification reactions, underscoring its potential to revolutionize the landscape of sustainable energy production.

A dual-enzyme microreactor based on encapsulation and covalent bond for enzymatic electrocatalytic CO2 reduction

Enzymatic reduction of CO2 into high-value chemicals has become an emerging technology attributed to its high selectivity and catalytic efficiency under mild conditions. While, the low solubility of CO2 at room temperature, the expensive cofactors and the poor stability of free enzyme limit the application potential. To solve these problems, we used organic microcapsules together with MOF to protect formate dehydrogenase (FDH) by encapsulation and carbonic anhydrase (CA) by covalent bond with high CO2 capture ability to construct a dual-enzyme microreactor. Besides, the additional mesopores of the microreactor can ensure the efficient internal diffusion of the substrate and product. Electron mediator was used to enhance electrocatalytic NADH regeneration to efficiently reduce CO2 to formate in the enzyme electrocatalysis system. After optimization, the highest yield of formate in the electrocatalytic CO2 reduction system by the dual enzyme microreactor was 2.917 mM with a generation rate of 1.296 × 104 μmol gcat-1h−1, which was 8.9 times higher than free enzyme.

Characterization of diamine oxidase-based biosensors for biogenic amines detection assembled onto functionalized SiO₂ substrates using aliphatic and aromatic di-aldehydes as crosslinkers

ABSTRACT Development of two biosensors to detect biogenic amines was carried out using immobilized diamine oxidase from Pisum sativum on 3-APTMS-functionalized SiO2 substrates with either glutaraldehyde or terephthalaldehyde as crosslinkers. The assembly process was validated by FT-IR, SEM-EDX, and AFM. Biosensor responses to biogenic amines were c.a. 3-fold lower compared to the free enzyme kinetics as assessed by UV/VIS spectrophotometry. The calculated initial velocities (ΔAbs·min-1) for glutaraldehyde and terephthalaldehyde biosensors in the given order were (3.2 ± 0.68) × 10–2, (0.48 ± 0.09) × 10–2 for putrescine; (4.6 ± 1.2) × 10–2, (0.35 ± 0.47) × 10–2 for cadaverine; (5.8 ± 2.4) × 10–3, (19.0 ± 155) × 10–3 for spermidine, and (3.0 ± 2.0) × 10–4, (0.0 ± 7) × 10–4for histamine. The 2-way t-tests indicated that the crosslinker type affected the catalytic activity of immobilized diamine oxidase as the responses of glutaraldehyde biosensors to putrescine and cadaverine were significantly higher than those of the terephthalaldehyde. Molecular Docking simulations showed that glutaraldehyde has a stronger affinity for DAO’s lysine and arginine residues while terephthalaldehyde to phenylalanine.

Highly active and recyclable immobilized multiple enzymes for one-pot enantioselective cascade reactions: Synthesis of (R)- and (S)-α-amino acids from racemic α-hydroxy acids

Cascade biotransformation of racemic alcohols to enantiopure amines is highly wanted in pharmaceutical manufacturing but requires green and efficient solution. Here we developed the first immobilized enzyme-based approach for this transformation, being cleaner and more productive than whole-cells biocatalysis, and more economic and higher-yielding than using isolated enzymes. As demonstration, the conversion of racemic mandelic acids to high-value (R)- or (S)-phenylglycines was achieved by immobilized enantioselective alcohol oxidase, racemase, catalase, and (S)- or (R)-transaminase. Co-immobilization of the four enzymes on Ni-NTA functionalised carriers gave high enzyme loadings (192–195 mg enzyme/g carrier), loading efficiencies (97–98%), and free enzyme activities (107–113%). Immobilized enzymes transformed racemic mandelic acids to the corresponding four (S)-phenylglycines in 91–99% ee and 82–95% yields or five (R)-phenylglycines in 93–99% ee and 83–94% yields. These catalysts showed good recyclability, retaining 86–98% productivities in the fifth reaction cycles.

Isolation, purification, and characterization of carbonic anhydrase from Azadirachta indica (neem) leaves and its CO2 sequestration efficiency

Carbonic anhydrase (CA) has been purified, immobilized, and characterized from a novel plant source Azadirachta indica (neem) leaves. The CA enzyme was purified to homogeneity to 19-fold. By SDS-PAGE the molecular mass of the purified CA was obtained to be 42.49 kDa. The pI value of the purified CA was found to be 4.95. Immobilization study of the purified CA was carried out with chitosan beads. The pH and temperature optima for free and immobilized enzyme were found to be 8.0, 30 °C and 9.0, 40 °C, respectively. From Lineweaver burk plot the km and Vmax values of the free and immobilized CA were found to be 0.89 mM, 0.118 μmol/min and 1.04 mM, 0.083 μmol/min, respectively. The storage stability of the free and immobilized CA was studied for 30 days where the immobilized CA was found to show better stability. The immobilized CA also showed satisfactory reusable capacity. Both free and immobilized neem leaves CA exhibited good CO2 sequestration capacity, forming CaCO3 formation in presence of CaCl2·2H2O under optimized conditions. The formed CaCO3 were further characterized by SEM, XRD, and FTIR.

Covalent immobilization of recombinant L-asparaginase from Geobacillus kaustophilus on ReliZyme supports for mitigation of acrylamide

In this study, a new recombinant L-asparaginase from Geobacillus kaustophilus was covalently immobilized on ReliZyme EA403 (Relizyme/EA@GkASNase) and ReliZyme HA403 (Relizyme/HA@GkASNase) supports, and the free and immobilized L-asparaginases were used for their acrylamide mitigation performances in a food model system. The immobilization was confirmed by fourier-transform infrared spectroscopy, scanning electron microscopy, and transmission electron microscopy analysis. The optimum pH was determined as 8.5 for all the free and immobilized L-asparaginase samples. The optimum temperature was determined as 55 °C for the free enzyme and 60 °C for both the immobilized samples. The thermal stability of L-asparaginase was increased by 17.6 and 37.2 folds at 60 °C for Relizyme/EA@GkASNase and Relizyme/HA@GkASNase, respectively. Relizyme/EA@GkASNase and Relizyme/HA@GkASNase showed 16% and 43% of the catalytic efficiency of free GkASNase. The acrylamide mitigation performances of free and immobilized L-asparaginase samples were investigated using the L-asparagine–starch food model system and the formed acrylamide was completely mitigated in 1 h for all the L-asparaginase samples. Both the immobilized L-asparaginase samples retained at least 80% of their activities after five reuses. Hence, the immobilized GkASNase preparations can be potentially used in heat-treated food industries to remove acrylamide.

Edible Composite Coating of Chitosan and Curdlan Maintains Fruit Quality of Postharvest Cherry Tomatoes

Postharvest cherry tomatoes are prone to senescence, decay and nutrient loss during the storage period owing to microbial invasion and their own metabolism. In this work, postharvest cherry tomatoes were treated with a composite coating of 1% chitosan and 1% curdlan, and the characteristics of postharvest cherry tomatoes during storage were investigated. Compared to control samples, after 21 d of storage under ambient conditions, the cherry tomatoes treated with the chitosan and curdlan coatings showed less rottenness, less weight loss, a lower respiration rate, reduced ethylene production, lower malonaldehyde (MDA) content and reduced membrane permeability. After the samples were treated with the composite coating, the activities of free radical scavenging enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were maintained at higher levels; the activities of related disease-resistant enzymes such as chitinase (CHI) and glucanase (GLU) were also maintained at higher levels. The soluble solids, titratable acidity, firmness, vitamin C content, lycopene content and antioxidant activities of postharvest cherry tomatoes treated with the composite coating exhibited higher levels as well. The composite coating of chitosan and curdlan might be a potentially promising method for preserving postharvest cherry tomatoes and other fruits.