Activity Of Immobilized Enzyme Research Articles

In Situ Biomineralization and Citric Acid Etching Strategy for Enhancing Activity of Immobilized Acetylcholinesterase.

Enhancing the structural stability of an enzyme and maintaining its catalytic activity are effective ways to improve enzyme utilization and reduce the cost of drug screening. However, immobilized enzyme activity tends to decrease in existing immobilization techniques due to conformational changes and microenvironmental restrictions. In this paper, we present a facile approach to prepare immobilized acetylcholinesterase (AChE) with high activity by a ZIF-8 in situ immobilization and citric acid (CA) etching strategy. CA breaks the coordination bond of ZIF-8 and produces defects, expanding the pore space, improving substrate accessibility, and fully exposing the active site of the enzyme. The enhancement of the catalytic activity of AChE@ZIF-8-CA was about 6.10-fold compared with the free enzyme. In addition, AChE@ZIF-8-CA exhibited an excellent encapsulation efficiency and good tolerance to temperature, pH, and organic solvents. The relative activity remains at the initial 83.77% even in five repeated experiments. The strategy provides a novel and efficient way to quickly construct highly active immobilized enzymes under mild conditions.

Enhanced Immobilization of Enzymes on Plasma Micro-Nanotextured Surfaces and Microfluidics: Application to HRP

The enhanced and direct immobilization of the enzyme horseradish peroxidase on poly(methyl methacrylate) (PMMA) microchannel surfaces to create a miniaturized enzymatic reactor for the biocatalytic oxidation of phenols is demonstrated. Enzyme immobilization occurs by physical adsorption after oxygen plasma treatment, which micro-nanotextures the PMMA surfaces. A five-fold enhancement in immobilized enzyme activity was observed, attributed to the increased surface area and, therefore, to a higher quantity of immobilized enzymes compared to an untreated PMMA surface. The enzymatic reaction yield reached 75% using a flow rate of 2.0 μL/min for the reaction mixture. Additionally, the developed microreactor was reused more than 16 times without affecting the enzymatic conversion yield. These results demonstrate the potential of microchannels with plasma micro/nanotextured surfaces for the rapid and facile fabrication of microfluidic enzymatic microreactors with enhanced catalytic activity and stability.

Integrating sodium cholate-modified MOF hybrid lipase and solubilization of hydrophobic candidates into a step for liganding fishing lipase inhibitors from Nelumbinis Folium

Enzyme immobilization by metal organic frameworks (MOFs) is an efficient way for screening active constituents in natural products. However, the enzyme’s biocatalysis activity is usually decreased due to unfavorable conformational changes during the immobilization process. In this study, sodium cholate was firstly used as the modifier for zeolitic imidazolate framework-8 (ZIF-8) immobilized lipase to increase both the stability and activity. More importantly, with the help of solubilization of sodium cholate, a total of 3 flavonoids and 6 alkaloids candidate compounds were fished out. Their structures were identified and the enzyme inhibitory activities were verified. In addition, the binding information between the candidate compound and the enzyme was displayed by molecular docking. This study provides valuable information for the improvement of immobilized enzyme activity and functional active ingredients in complicated medicinal plant extracts.

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.

In-situ honeycomb spheres for enhanced enzyme immobilization and stability

This paper presents a promising heterogeneous biocatalyst-immobilized cellulase system, which can facilitate the targeted catalytic conversion of biomass to reducing sugars. Magnetic Zeolite Imidazole Framework-8/chitosan (Fe3O4/ZIF-8/CS, FZC) hybrid honeycomb microspheres were synthesized to immobilize cellulase using an in-situ growth strategy. The enhancement of immobilization efficiency induced by each constituent of the composite carrier was explored. This exploration was grounded in varying combinations of components within the honeycomb composite spheres, revealing the indispensability of ZIF-8. Through in-situ assembly, the composite material presents a microsphere with an internal honeycomb structure and multilevel pores. Effective immobilization, temperature-responsive conformational change, and cyclic stability were achieved due to the scaling effect of the channel, extensive specific surface area, and abundant amino group interactions enable immobilized enzymes to maintain excellent performance under challenging operating conditions, demonstrating an enhanced structure-performance relationship. With the obtained immobilized cellulase, we achieved a high load (461.67 mg/g) of cellulase and a significant reducing sugar yield (328.39 mg/g) from corncob waste. In addition, we investigated the enhancement effect of thermal activation on the immobilized enzyme activity of composite carrier. The results showed that the appropriate heat treatment was conducive to the activation of FZC immobilized enzyme with a reducing sugar yield 1.61 times higher than that of the control. The strategy provided in this study promotes the development of immobilized biocatalysts and provides a useful reference for the further design of biocatalytic systems.

Artificial antibody-antigen-directed immobilization of lipase for consecutive catalytic synthesis of ester: Benzyl acetate case study

A strategy based on artificial antibody-antigen recognition was proposed for the specific directed immobilization of lipase. The artificial antibody was synthesized using catechol as a template, α-methacrylic acid as a functional monomer, and Fe3O4 as the matrix material. Lipase was modified with 3,4-dihydroxybenzaldehyde as an artificial antigen. The artificial antibody can specifically recognize catechol fragment in the enzyme structure to achieve the immobilization of lipase. The immobilization amount, yield, specific activity, and immobilized enzyme activity were 13.2 ± 0.2 mg/g, 78.9 ± 0.4 %, 7.9 ± 0.2 U/mgprotein, and 104.6 ± 1.7 U/gcarrier, respectively. Moreover, the immobilized lipase exhibited strong reusability and regeneration ability. Additionally, the immobilized lipase successfully catalyzed the synthesis of benzyl acetate and demonstrated robust continuous catalytic activity. These results fully demonstrate the feasibility of the proposed artificial antibody-antigen-directed immobilization of lipase.

Preparation and characterization of AChE immobilized magnetic bio-nanocomposites (Fe3O4@Cht/Au) for pesticide detection

Free enzymes cause difficulties in many applications due to their insufficient stability, loss of activity in a short time, and most importantly, although they are costly, they are used only once in reactions, lose their effect and cannot be recovered from the environment. Magnetic nanoparticles coated with biocompatible polymeric material are potential candidates for promising enzyme carriers due to their multifunctional pore surfaces, easy removal from the environment provided by the magnetization, ability to main stability under various harsh conditions. This study prepared a biosensor candidate based on the inhibiting acetylcholinesterase enzyme by organophosphate pesticides from chitosan-coated magnetic nanoparticles doped with gold. Transmission electron microscopy, scanning electron microscopy, X-ray diffraction diffractometry, and Fourier transform infrared spectroscopy analysis confirmed the structure of synthesized nanocomposites. Magnetic characteristics of the nanocomposites were assessed using VSM. Bio-nanocomposite (Fe3O4@Cht/Au/AChE) was used to determine environmental pollutants qualitatively. Remediation of organophosphate-containing wastewater is an essential issue for environmental sustainability. In this work, Dichlorvos and Chlorpyrifos were selected as organic pollutants to assess the enzymatic activity of immobilized Fe3O4@Cht/Au/AChE. Optimum conditions for AChE enzyme were immobilized nanostructures (Fe3O4@Cht/Au/AChE) were determined. The optimum pH for the immobilized enzyme was found to be 8, and the optimum temperature was found to be 60 °C. Retained immobilized enzyme activity is found to be around 50% for the 20th reuse. In the presence of 150 µL pesticide, retained immobilized enzyme activity is found to be around 25%. Method validation was performed for pesticides. When using immobilized AChE, the LOD (limit of detection)-LOQ (limit of quantitation) values for Dichlorovos and Chlorpyrifos was obtained in the range of 0.0087–0.029 nM and 0.0014–0.0046 nM, respectively. The relative standard deviation (RSD%) values, which are indicators of precision, were found to be below 2%.

Tailored chitosan integration in diatomaceous earth particles as a scaffold for fructosyltransferase immobilization in fructo-oligosaccharide production.

Fructo-oligosaccharide (FOS) belongs to the group of short inulin-type fructans and is one of the most important non-digestible bifid-oligosaccharides capable of biotransforming sucrose using fructosyltransferase (FTase). However, there are no immobilized FTase products that can be successfully used industrially. In this study, diatomite was subjected to extrusion, sintering and granulation to form diatomaceous earth particles that were further modified via chitosan aminomethylation for modification. FTase derived from Aspergillus oryzae was successfully immobilized on the modified support via covalent binding. The immobilized enzyme activity was 503 IU g-1 at an enzyme concentration of 0.6 mg mL-1, immobilization pH of 7.0 and contact time of 3 h. Additionally, the immobilization yield was 56.91%. Notably, the immobilized enzyme was more stable under acidic conditions. Moreover, the half-life of the immobilized enzyme was 20.80 and 10.96 times as long as that of the free enzyme at 45 and 60 °C, respectively. The results show good reusability, as evidenced by the 84.77% retention of original enzyme activity after eight cycles. Additionally, the column transit time of the substrate was 35.56 min when the immobilized enzyme was applied in a packed-bed reactor. Furthermore, a consistently high FOS production yield of 60.68% was achieved and maintained over the 15-day monitoring period. Our results suggest that immobilized FTase is a viable candidate for continuous FOS production on an industrial scale. © 2024 Society of Chemical Industry.

Pretreatment of shrimp shells with an acidic deep eutectic solvent system for chitin extraction and its enhanced performance as a carrier for immobilized lipase

Existing methods for chitin extraction usually produce substantial waste, which poses ecological hazards. Natural deep eutectic solvent (NADES) offers a promising one-step pretreatment alternative, replacing the resource-intensive demineralization (DM) and deproteinization (DP) process. Hence, in this study, the influence of various acidic NADES, on achieving a simplified one-step DM and DP in the chitin extraction process was investigated. The study yielded chitin with 87.73 % purity, and microstructural analysis showed that NADES pretreatment minimally affected chitin quality without deacetylation. In addition, chitin extracted using choline chloride-oxalic acid as a carrier displayed excellent performance in the immobilization of Geobacillus thermocatenulatus lipase 2 (GTL2) because of obvious Ca2+ activation effect. This process contributed to enhancement of immobilized enzyme activity. The immobilized GTL2 showed excellent hydrolytic capabilities, with its highest activity reaching 547.80 ± 20.62 U/mg, significantly better than the five commercial lipases that exhibited <40 % of the enzyme activity. Furthermore, the hydrolytic capacity of immobilized GTL2 was notably high for 4-nitrophenyl butyrate, measuring 935.47 ± 51.60 U/mg. This study provided a constructive approach for the one-step pretreatment of shrimp shells with organic acid-based NADES to isolate and purify chitin and its potential application as an immobilized carrier to enhance enzyme activity.

Construction and Application of a Pepsin-Functionalized Covalent Organic Framework with Prominent Chiral Recognition Ability.

The stable Pepsin@covalent organic framework (Pepsin@COF) were constructed base on matching COF pore diameter to pepsin dimension. It exhibits excellent chiral recognition capabilities (e. e. % up to 62.63 %) and potential for enantioseparation. Furthermore, a positive correlation between the immobilized enzyme activity and chiral recognition was revealed, offering insights for the design of biocatalytic nanosystems in chiral separation.

Enhanced catalytic performance of penicillin G acylase by covalent immobilization onto functionally-modified magnetic Ni0.4Cu0.5Zn0.1Fe2O4 nanoparticles.

With the emergence of penicillin resistance, the development of novel antibiotics has become an urgent necessity. Semi-synthetic penicillin has emerged as a promising alternative to traditional penicillin. The demand for the crucial intermediate, 6-aminopicillanic acid (6-APA), is on the rise. Enzyme catalysis is the primary method employed for its production. However, due to certain limitations, the strategy of enzyme immobilization has also gained prominence. The magnetic Ni0.4Cu0.5Zn0.1Fe2O4 nanoparticles were successfully prepared by a rapid-combustion method. Sodium silicate was used to modify the surface of the Ni0.4Cu0.5Zn0.1Fe2O4 nanoparticles to obtain silica-coated nanoparticles (Ni0.4Cu0.5Zn0.1Fe2O4-SiO2). Subsequently, in order to better crosslink PGA, the nanoparticles were modified again with glutaraldehyde to obtain glutaraldehyde crosslinked Ni0.4Cu0.5Zn0.1Fe2O4-SiO2-GA nanoparticles which could immobilize the PGA. The structure of the PGA protein was analyzed by the PyMol program and the immobilization strategy was determined. The conditions of PGA immobilization were investigated, including immobilization time and PGA concentration. Finally, the enzymological properties of the immobilized and free PGA were compared. The optimum catalytic pH of immobilized and free PGA was 8.0, and the optimum catalytic temperature of immobilized PGA was 50°C, 5°C higher than that of free PGA. Immobilized PGA in a certain pH and temperature range showed better catalytic stability. Vmax and Km of immobilized PGA were 0.3727 μmol·min-1 and 0.0436 mol·L-1, and the corresponding free PGA were 0.7325 μmol·min-1 and 0.0227 mol·L-1. After five cycles, the immobilized enzyme activity was still higher than 25%.

Immobilization of Candida rugosa lipase on optimized polyamidoamine dendrimer functionalized magnetic multiwalled carbon nanotubes for green manufacture of butyl butyrate ester

Mineral acid catalysis in Fisher-Speier esterification is hazardous and have other disadvantages. The objective of the study was immobilization of Candida rugosa lipase onto iron oxide (Fe3O4) impregnated magnetic mutiwalled carbon naotubes (MMWCNTs) using amine functionalized polyamidoamine (PAMAM) dendrimers for manufacture of butyl butyrate ester. Dendrimers were synthesized from reaction of ethyl methacrylate with varying chain length of diamines 1, 4 diaminobutane (DAB) / 1, 3 diaminopropane (DAP) / ethylene diamine (ED). The effect of aliphatic alkyl chain length (CH2) of functionalizing agents ED, DAP and DAB, and dendrimer generations on amount of terminal NH2 group, surface charge, protein loading and immobilized enzyme activities were analysed. Magnetic MWCNTs support surface modified with third generation PAMAM using ethylene diamine showed highest protein load 220.1 mg g−1 and enzymatic activity 8,276.21 U g−1 support. The immobilized enzyme particles were characterized by HRTEM, FTIR, XRD, TGA, VSM and Zeta potential analysis. The immobilized enzyme showed improvement in the dispersion, activity, stability and reusability characteristics. Maximum esterification yield 92.67 % and reuse upto 11 cycles with 65.2 % yield obtained. The immobilized preparation showed 2.3 folds esterification than free enzyme. Developed nanobiocatalyst expects to possess potential for various other applications of lipase catalysed reactions.

Magnetic core-shell cellulose system for the oriented immobilization of a recombinant β-galactosidase with a protein tag

The objective of this study was to immobilize a recombinant β-galactosidase (Gal) tagged with a cellulose-binding domain (CBD) onto a magnetic core-shell (CS) cellulose system. After 30 min of reaction, 4 U/capsule were immobilized (CS@Gal), resulting in levels of yield and efficiency exceeding 80 %. The optimal temperature for β-galactosidase-CBD activity increased from 40 to 50 °C following oriented immobilization. The inhibitory effect of galactose decreased in the enzyme reactions catalyzed by CS@Gal, and Mg2+ increased the immobilized enzyme activity by 40 % in the magnetic CS cellulose system. The relative enzyme activity of the CS@Gal was 20 % higher than that of the soluble enzyme activity after 20 min at 50 °C. The CS support and CS@Gal capsules exhibited an average size of 8 ± 1 mm, with the structure of the shell (alginate-pectin-cellulose) enveloping and isolating the magnetic core. The immobilized β-galactosidase-CBD within the magnetic CS cellulose system retained ∼80 % of its capacity to hydrolyze lactose from skim milk after 10 reuse cycles. This study unveils a novel and promising support for the oriented immobilization of recombinant β-galactosidase using a magnetic CS system and a CBD tag. This support facilitates β-galactosidase reuse and efficient separation, consequently enhancing the catalytic properties of the enzyme.

Improved anti-biofilm activity and long-lasting effects of novel serratiopeptidase immobilized on cellulose nanofibers.

Today, enzymatic treatment is a progressive field in combating biofilm producing pathogens. In this regard, serratiopeptidase, a medicinally important metalloprotease, has been recently highlighted as an enzyme with proved anti-biofilm activity. In the present study, in order to increase the long-lasting effects of the enzyme, serratiopeptidase and the novel engineered forms with enhanced anti-biofilm activity were immobilized on the surface of cellulose nanofibers (CNFs) as a natural polymer with eminent properties. For this, recombinant serratiopeptidases including the native and previously designed enzymes were produced, purified and conjugated to the CNF by chemical and physical methods. Immobilization was confirmed using different scanning and microscopic methods. The enzyme activity was assessed using casein hydrolysis test. Enzyme release analysis was performed using dialysis tube method. Anti-biofilm activity of free and immobilized enzymes has been examined on Staphylococcus aureus and Pseudomonas aeruginosa strains. Finally, cytotoxicity of enzyme-conjugated CNFs was performed by MTT assay. The casein hydrolysis results confirmed fixation of all recombinant enzymes on CNFs by chemical method; however, inadequate fixation of these enzymes was found using cold atmospheric plasma (CAP). The AFM, FTIR, and SEM analysis confirmed appropriate conjugation of enzymes on the surface of CNFs. Immobilization of enzymes on CNFs improved the anti-biofilm activity of serratiopeptidase enzymes. Interestingly, the novel engineered serratiopeptidase (T344 [8-339ss]) exhibited the highest anti-biofilm activity in both conjugated and non-conjugated forms. In conclusion, incorporation of serratiopeptidases into CNFs improves their anti-biofilm activities without baring any cytotoxicity. KEY POINTS: • Enzymes were successfully immobilized on cellulose nanofibers using chemical method. • Immobilization of enzymes on CNFs improved their anti-biofilm activity. • T344 [8-339ss] exhibited the highest anti-biofilm activity in both conjugated and non-conjugated forms.

Rapid prediction method of ZIF-8 immobilized Candida rugosa lipase activity by near-infrared spectroscopy

Candida rugosa lipase (CRL, EC3.1.1.3) is one of the main enzymes synthesizing esters, and ZIF-8 was chosen as an immobilization carrier for lipase. Enzyme activity testing often requires expensive reagents as substrates, and the experiment processes are time-consuming and inconvenient. As a result, a novel approach based on near-infrared spectroscopy (NIRs) was developed for predicting CRL/ZIF-8 enzyme activity. The absorbance of the immobilized enzyme catalytic system was evaluated using UV–Vis spectroscopy to investigate the amount of CRL/ZIF-8 enzyme activity. The powdered samples' near-infrared spectra were obtained. The sample’s enzyme activity data were linked with each sample’s original NIR spectra to establish the NIR model. A partial least squares (PLS) model of immobilized enzyme activity was developed by coupling spectral preprocessing with a variable screening technique. The experiments were completed within 48 h to eliminate inaccuracies between the reduction in enzyme activity with increasing laying-aside time throughout the test and the NIRs modeling. The root-mean-square error of cross-validation (RMSECV), the correlation coefficient of validation set (R) value, and the ratio of prediction to deviation (RPD) value were employed as assessment model indicators. The near-infrared spectrum model was developed by merging the best 2nd derivative spectral preprocessing with the Competitive Adaptive Reweighted Sampling (CARS) variable screening method. This model's root-mean-square error of cross-validation (RMSECV) was 0.368 U/g, the correlation coefficient of calibration set (R_cv) value was 0.943, the root-mean-square error of prediction (RMSEP) set was 0.414 U/g, the correlation coefficient of validation set (R) value was 0.952, and the ratio of prediction to deviation (RPD) was 3.0. The model demonstrates that the fitting relationship between the predicted and the reference enzyme activity value of the NIRs is satisfactory. The findings revealed a strong relationship between NIRs and CRL/ZIF-8 enzyme activity. As a result, the established model could be implemented to quantify the enzyme activity of CRL/ZIF-8 quickly by including more variations of natural samples. The prediction method is simple, rapid, and adaptable to be the theoretical and practical basis for further studying other interdisciplinary research work in enzymology and spectroscopy.

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

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

Immobilization of Tyrosinase and Its Application

Immobilized enzymes are more robust and resistant to environmental changes than free enzymes in solution. More crucially, the immobilized enzyme systems' heterogeneity enables facile recovery of enzymes and products, multiple reuses, continuous enzymatic processes, quick reaction termination, and a more comprehensive range of bioreactor designs. This paper examines recent findings on enzyme immobilization using diverse approaches for various uses. The information gathered from the reactions catalyzed by the encapsulated tyrosinase provided a good view of hetero-biocatalysts in the phenol biosensor industries. This review proposes an effective method for immobilizing tyrosinase biomolecules into a silica aerogel matrix. Silica matrix has been utilized to encapsulate a wide range of biomolecules, mainly in sol-gel composites. We also discovered that silica aerogel synthesized from sol-gel method retains all the immobilized enzyme activity. The use of a silica matrix for enzyme immobilization, in conjunction with a moderate immobilization method, results in the successful retention of enzyme activity. Future studies should explore practical encapsulating approaches and inventively modified supports to enhance the commercialization of immobilized enzymes and offer fresh perspectives to the industrial sector.

The effect of ethanol solvent on thermostability and esterification activity of immobilized Candida rugosa lipase on celite

Lipase is an enzyme commonly applied as a catalyst in the food industry, the synthesis of bioplastics, and the production of biodiesel. Candida rugosa lipase (CRL) is one of the widely used lipases due to its high selectivity and activity. However, high operating temperatures can denature enzymes, affecting enzyme activity due to structural changes. An enzyme can be immobilized on a supporting matrix to improve stability. Ethanol as an immobilization solvent may also increase the thermostability and esterification activity. This study examines the effect of ethanol solvent on the thermostability of immobilized Candida rugosa lipase. Immobilization was carried out on the Celite-545 matrix in buffer/ethanol solution. Different operating conditions such as enzyme concentrations, immobilization temperatures, immobilization time, and immobilization pH were observed. The immobilized enzyme activity was determined by esterification reaction using butyric acid and 1-butanol as substrates and isooctane as reaction medium. The results showed that immobilization increased the esterification activity by 76% at 37°C. Thermostability of immobilized enzyme was also enhanced by 29-51%, depending on the esterification temperature.

Performance enhancement of white rot fungi extracellular enzymes via new hydrogel microenvironments for remediation of benzo[a]pyrene contaminated soil

Organic pollutants with low solubility and high ecotoxicity, mutagenicity, and carcinogenicity, are rapidly entering and accumulating in soil, resulting in soil pollution. Several methods have been investigated for remediation of organic contaminated soil, including enzymatic remediation approach. However, free enzymes are easily deactivated, which hinders their practical application in soil remediation. Immobilization of enzyme improves its stability and catalytic performance, but the immobilized material itself becomes secondary pollutants in soil. In this study, Trametes versicolor extracellular enzyme was immobilized on the degradable calcium alginate hydrogel microspheres. The laccase maintained a high activity. In addition, the addition of cellulose improved the strength of the carrier. Hydrogel microspheres solved the problems of easy inactivation of free enzyme and secondary contamination of immobilized materials. By a novel combination of extracellular enzymes and hydrogel microenvironments, immobilized enzymes not only demonstrate outstanding performance in thermal stability and pH adaptability, but also achieves a significant improvement in biocatalytic activity for benzo[a]pyrene contaminated soil. The thermal stability of immobilized enzyme was much higher than that of free enzyme. When the temperature increased to 50 °C, the activity of immobilized enzyme remained at 93.15% of the maximum enzyme activity, while the activity of free enzyme decreased to 63.76%. At pH 8, the immobilized enzyme activity maintained 74.84% of the maximum enzyme activity, while the free enzyme activity was only 11.86%. Immobilized enzymes can effectively remove 91.40% of benzo[a]pyrene from soil within 96 h. Furthermore, the catalytic oxidation of benzo[a]pyrene by enzymes that have been immobilized ultimately results in the production of 6,12-benzo[a]pyrene-dione. Molecular dynamics simulation showed that the catalytic degradation of benzo[a]pyrene was mainly through the interaction of amino acid residues PRO-391 with the Pi-alkyl of benzo[a]pyrene. This study presents an innovative strategy for designing and developing immobilized enzymes for use in biocatalytic applications related to eco-remediation of soil.

Acrylic fabric and nanomaterials to enhance α-amylase-based biocatalytic immobilized systems for industrial food applications

An innovative approach for immobilizing α-amylase was used in this investigation. The acrylic fabric was first treated with hexamethylene diamine (HMDA) and then coated with copper ions that were later reduced to copper nanoparticles (CuNPs). The corresponding materials obtained, Cu(II)@HMDA-TA and CuNPs@HMDA-TA, were employed as carriers for α-amylase, respectively. The structural and morphological characteristics of the produced support matrices before and after immobilization were assessed using various techniques, including FTIR, SEM, EDX, TG/DTG, DSC, and zeta potential. The immobilized α-amylase exhibited the highest level of activity at pH 7.0, with immobilization yields observed for CuNPs@HMDA-TA (81.7 %) (60 unit/g support) followed by Cu(II)@HMDA-TA (71.7 %) (49 unit/g support) and 75 % and 61 % of activity yields, and 91.7 % and 85 % of immobilization efficiency, respectively. Meanwhile, biochemical characterizations of the activity of the soluble and immobilized enzymes were carried out and compared. Optimal temperature, pH, kinetics, storage stability, and reusability parameters were optimized for immobilized enzyme activity. The optimal pH and temperature were recorded as 6.0 and 50 °C for soluble α-amylase while the two forms of immobilized α-amylase exhibit a broad pH of 6.0–7.0 and optimal temperature at 60 °C. After recycling 15 times, the immobilized α-amylase on CuNPs@HMDA-TA and Cu(II)@HMDA-TA preserved 63 % and 52 % of their activities, respectively. The two forms of immobilized α-amylase displayed high stability when stored for 6 weeks and preserved 85 % and 76 % of their activities, respectively. Km values were calculated as 1.22, 1.39, and 1.84 mg/mL for soluble, immobilized enzymes on CuNPs@HMDA-TA, and Cu(II)@HMDA-TA, and Vmax values were calculated as 36.25, 29.68, and 21.57 μmol/mL/min, respectively. The total phenolic contents of maize kernels improved 1.4 ± 0.01 fold after treatment by two immobilized α-amylases.