Immobilization Of Catalase Research Articles

Silaffin-3-derived pentalysine cluster as a new fusion tag for one-step immobilization and purification of recombinant Bacillus subtilis catalase on bare silica particles

Bio-catalysis by enzymes on solid surfaces has been implemented in several practical applications. However, the current methods for efficient enzyme immobilization with retained activity need further development. Herein, a simple, rapid, and economical, bio-affinity-based approach was developed for the direct immobilization with high activity recovery of the Bacillus subtilis catalase (CAT), recombinantly expressed in Escherichia coli. Silaffin-3-derived pentalysine cluster (Sil3K) from Thalassiosira pseudonana and its mutant variant (penta-arginine peptide; Sil3R) were used for the first time in the non-covalent immobilization of the recombinant enzyme on silica particles. The fusion proteins CAT-Sil3K and CAT-Sil3R were selectively loaded from the cell lysates onto the silica surface. Unexpectedly, the Lys-based tag (Sil3K) was the superior to Arg-based tag (Sil3R) or tag-less system for the high recovery of CAT activity upon immobilization; an 8.4-fold and 1.5-fold increase in the catalytic activity was observed for CAT-Sil3K compared with the tag-less CAT and CAT-Sil3R, respectively. Furthermore, the CAT-Sil3K immobilized on silica particles exhibited improved thermal, pH and storage stabilities, and retained 72% of the initial activity after five reaction cycles. Moreover, CAT-Sil3K was released with approximately 85% recovery and 91% purity, in a biologically active form when free lysine solution was used as the eluent. Our data proved that Sil3K-tag, 12-mer peptide, can be a highly promising silica-affinity tag for effective enzyme immobilization with preserved activity. Additionally, the novel findings obtained here may open a new route not only for cost-effective enzyme immobilization approaches but also for high recovery of enzyme activity.

Improving catalase stability by its immobilization on grass carp (Ctenopharyngodon idella) scale collagen self-assembly films.

We extracted collagen (CL) from the scales of Ctenopharyngodon idella and fabricated a CL self-assembly film. Catalase (CAT) was immobilized on the films using cross-linking, adsorption, and embedding methods. The activity and operational stability of immobilized CAT were investigated, along with the influence of the concentration of glutaraldehyde (GTA) and of the initial concentration of embedded CAT. The results showed that the CL triple helix remained intact. Differential scanning calorimetry data showed that the thermal stability of CL was significantly improved by neutral salt-induced self-aggregation and GTA cross-linking. The immobilized enzyme had high activity and good operational stability. When the enzyme concentration reached 0.5 mg/mL, immobilized enzyme activity of grass carp scales reached a maximum of 2596 U/g, and after twenty-two uses, enzyme activity remained above 50%, and it could be reused >45 times (CAT = 0.5 mg/mL, GTA = 5%, Temperature = 30 °C, pH = 7). Moreover, the optimum temperature and pH of immobilized CAT were 35 °C and 7, respectively, while the same for free CAT was 30 °C and 7, respectively. This indicated that immobilization of CAT has a protective effect.

In situ immobilization of glucose oxidase and catalase in a hybrid interpenetrating polymer network by 3D bioprinting and its application

In bio-catalysis applications, the non-catalytic properties of immobilized matrices have high importance. In this work, we prepared a sodium alginate/polyacrylamide/hydroxyapatite (SA/PAM/HA) hybrid interpenetrating polymer network (HIPN) hydrogel, which we then proved by SEM and FTIR. The HIPN hydrogel exhibited markedly improved mechanical properties. The compression modulus of the HIPN hydrogel (7.16 MPa) was 14 times higher than that of the interpenetrating polymer network (IPN) hydrogel, with the max storage modulus of the HIPN hydrogel being 31,960 Pa. We then employed an extrusion-based bioprinting (3D printing) technique to carry out a one-pot preparation of the immobilized enzymes. Next, we used different types of immobilized glucose oxidase/catalase to synthesize gluconic acid. There was an increase in the catalytic efficiency with the more porous of the immobilized enzymes. Furthermore, the immobilized enzymes exhibited operational stability. Finally, after reuse in 4 batches, a high conversion of 97% was maintained. These results illustrated that the preparation of one-pot immobilized enzymes by 3D bioprinting has great potential for future application.

Catalase immobilization onto magnetic multi-walled carbon nanotubes: optimization of crucial parameters using response surface methodology

In this study catalase (CAT) immobilization onto magnetic multi-walled carbon nanotubes (mMWCNTs) was undertaken and response surface methodology (RSM) employed to determine the optimum immobilization conditions.

Covalently Entrapping Catalase into Calcium Alginate Worm Pieces Using EDC Carbodiimide as a Crosslinker.

Catalase is a biocatalyst used to break down toxic hydrogen peroxide into water and oxygen in industries such as cheese and textiles. Improving the efficiency of catalase would help us to make some industrial products, such as cheese, less expensively. The best way to maintain catalase’s conformation, and thus enhance its activity, is to immobilize it. The primary goal of this study was to find a new way of immobilizing catalase. There are many ways to immobilize an enzyme, one of the foremost being entrapment in calcium alginate. In past literature, researchers used calcium alginate as a bead, and an enzyme was physically entrapped into it. This form of immobilization has been proven inefficient because the enzyme is loosely attached to the material. To address this issue, we used calcium alginate as worm pieces with a carbodiimide as a crosslinker. Worm pieces here refer to small uniform pieces of cylindrical calcium alginate made by cutting long strings (worms) into small pieces. This research set out to understand whether or not EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) can increase the immobilization of catalase into the calcium alginate. We found that EDC is able to triple the amount of catalase immobilized in calcium alginate compared to only calcium alginate entrapment. This research devised a new type of covalent entrapment that can be used to immobilize many other enzymes that also contain lysine residues. The result suggests that catalase can be better covalently entrapped into the calcium alginate worm pieces using EDC.

Chitosan Co-polymeric nanostructures for catalase immobilization

Catalase is an industrial enzyme used in different areas including medical, textile, food and pharmaceutic industries. In the study, synthetic-natural copolymer in nanosize was synthesized using 2-hydroxyethyl methacrylate (HEMA) as a synthetic material and chitosan (CTS) as a natural material. Immobilized metal-chelate affinity chromatography (IMAC) material was prepared by chelating Cu(II) ions onto p(HEMA-CTS) nanostructures. Characterization studies such as Zeta sizer analysis, Fourier transform infra-red (FTIR) spectroscopy, scanning electron microscopy (SEM), and thermal gravimetric analysis (TGA) were performed during the preparation of IMAC nanostructures. The effects of some parameters (time, polymer amount, pH, initial catalase concentration, ionic strength and temperature) were identified and optimum immobilization conditions were determined as 75 min, 0.1 mg p(HEMA-CTS)-Cu, pH 6.0, and 25 °C. Maximum amount of immobilized catalase was detected as 11.29 g/g p(HEMA-CTS)-Cu. Immobilized catalase was desorbed successfully and it was detected that p(HEMA-CTS)-Cu nanostructures can be used repeatedly in catalase immobilization. Immobilization changed optimum temperature value from 37 °C to 25 °C while it did not change the optimum pH for catalase activity. Furthermore, it was detected that kinetic parameters of catalase changed by immobilization. Finally, operational and storage stabilities of immobilized catalase were investigated.

Effect of immobilization on the activity of catalase carried by poly(HEMA-GMA) cryogels

Hydrogen peroxide is converted by catalase to molecular oxygen and water to remove oxidative stress. In this study, catalase immobilization was performed using poly(2-hydroxyethyl methacrylate-glycidyl methacrylate) (poly(HEMA-GMA)) cryogels with different amounts of GMA. Catalase adsorption capacity of 298.7 ± 9.9 mg/g was achieved at the end of 9 h using the poly(HEMA-GMA)-250 cryogel. Kinetic parameters and the inhibitory effects of pesticides such as 4,4′-DDE and 4,4′-DDT on the activity of free and immobilized catalase enzyme were investigated. While the Vmax value of the immobilized enzyme was reduced 4-fold compared to the free enzyme, in the case of the comparison of the KM values, the affinity of the immobilized enzyme was increased by 1.94 times against the substrate. The inhibitory effect of 4,4′-DDT pesticide was found to be higher for the immobilized and free enzyme. NaCl (1 M, pH: 7.0) solution was used for desorption of the adsorbed catalase enzyme. A desorption ratio of 96.45% was achieved. The technique used in this study is promising regarding for the immobilization of catalase enzyme to increase the operational activity. Therefore, poly(HEMA-GMA) cryogels have the potential to be used for immobilization of catalase enzyme in the fields of biology and biochemistry.

Green synthesis of porous graphene and its application for sensitive detection of hydrogen peroxide and 2,4-dichlorophenoxyacetic acid

This work presented a facile and green method to prepare porous graphene (PGR) using a combination of sodium citrate treatment, hydrothermal reduction and lyophilization. The structure of the obtained PGR was analyzed by X-ray diffraction spectroscopy (XRD), X-ray photoelectron spectroscopy (XPS), Brunauer-Emmett-Teller (BET), and the morphology was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Furthermore, this PGR was used as the support for the immobilization of catalase. Fourier-transform infrared spectroscopy (FT-IR) spectra indicated that the immobilized catalase kept its structure without any change. Electrochemical impedance spectroscopy and cyclic voltammetry indicated the fast electron transfer between the immobilized catalase and the glassy carbon electrode (GCE). This biosensor showed a linearity in the 1.0 × 10−7 ∼ 7.7 × 10−6 mol L−1 concentration range with a low detection limit of 83.0 nmol L−1 (3δ/S) for H2O2. In addition, the biosensor was also applied to measure 2,4-dichlorophenoxyacetic acid (2,4-D) concentration based on catalase enzymatic inhibition. Under the optimized conditions, two linear relationships of the inhibition ratios vs. the 2,4-D concentrations ranging from 2.4 × 10−9 ∼ 2.6 × 10−8 mol L−1 and 2.6 × 10−8 ∼ 3.6 × 10−7 mol L−1 were found. The detection limit for 2,4-D was down to 1.5 nmol L−1 at 3δ/S. The inhibition constants (Ki) of 2,4-D to the immobilized catalase was calculated to be 6.50 × 10−8 mol L−1. This biosensor was applied for the determination of H2O2 and 2,4-D in real samples with satisfactory results.

Immobilization of catalase onto chitosan and chitosan–bentonite complex: A comparative study

The immobilization of catalase onto chitosan and chitosan–bentonite was investigated and immobilization yield of 95.91 and 95.26 was obtained respectively. The optimum pH and temperature were found as 7.5 and 8.0 at 40 °C for free and immobilized enzyme. The value of Vmax decreased by 33,000–26,300, 24,500 μmol (min mg protein)−1 and Km increased by 12.5–25 and 20 mM for free and immobilized on chitosan and chitosan–bentonite respectively. The thermal stability, half life, FTIR analyses of the beads was also performed in order to characterise the structural differences. The remaining immobilized catalase onto chitosan and chitosan–bentonite activity was 50% and 70% after 20 cycles respectively. The storage stability were found as 22%, 60%, and 70% from its original activity in case of free enzyme and immobilization of chitosan, chitosan–bentonite beads respectively after 60 days.

Direct Electrochemistry of Catalase Immobilized at Polymerized-SnO2 Multiporous Modified Electrode for an Amperometric H2O2 Biosensor

A novel amperometric H2O2 biosensor based on immobilization of catalase with polymerized MPNFs of SnO2 onto glassy carbon electrode with chitosan have been proposed in this work. Multiporous nanofibers of SnO2 were synthesized by electrospinning method from the tin precursor by controlling the concentration followed by polymerized with aniline. Catalase was then co-immobilized with the polymerized nanofibers on the surface of glassy carbon electrode by using chitosan. The polymerized MPNFs of SnO2 play a significant role in facilitating the electron exchange between the electroactive center of catalase and the electrode surface. Cyclic Voltammetry and amperometry were used to study and optimize the performance of the fabricated H2O2 biosensor. The PANI/SnO2-NFs/Catalase/Ch/GCE biosensor displayed a linear amperometric response towards the H2O2 concentration range from 10 to 120M with a detection limit of 0.6M (based on S/N=3). Furthermore, the biosensor reported in this work exhibited acceptable stability, reproducibility, and repeatability.

Oxime-functionalized cryogel disks for catalase immobilization

Catalase is a protective enzyme against oxidative stress and converts hydrogen peroxide into water and molecular oxygen. In the current study, catalase immobilization was applied onto the oxime-functionalized cryogel disks. Cryogel disks were produced by free radical polymerization. After cutting as circular disks, oxime ligand (4-biphenylchloroglyoxime, BPCGO) was attached and oxime-functionalized cryogel disks were obtained. After optimization of several immobilization parameters such as pH, initial catalase concentration, temperature and ionic strength, maximum catalase load was detected as 261.7 ± 11.2mg/g for cryogel disk at pH5.0. Activity studies indicated that immobilization enhanced the enzyme activity in basic pH region, the temperature range of 15–35°C and at ionic strengths between 0.2 and 1.0M NaCl. Km was detected as 9.9 and 11.0mM and Vmax was 357.1 and 769.2μmol min−1 for free and immobilized catalase, respectively. kcat and Km/kcat values showed that immobilization enhanced the catalytic efficiency. Storage stability experiments demonstrated that immobilization increased the usability period. Furthermore, catalase desorption was achieved by 1.0M NaSCN at pH8.0 successfully and catalase adsorption capacity of oxime-functionalized cryogel disk was decreased by 9.9% at the end of 5 adsorption-desorption cycle.

Development of novel delivery system for nanoencapsulation of catalase: formulation, characterization, and in vivo evaluation using oxidative skin injury model

One of the main challenges for successful pharmaceutical application of Catalase (CAT) is maintaining its stability. Physical immobilization of CAT through nano-encapsulation was proposed to resolve this challenge. CAT encapsulating niosomes (e-CAT) were prepared using Brij® 30, 52, 76, 92, and 97 in the presence of cholesterol (Ch) by thin film hydration method. Niosomes were characterized for encapsulation efficiency % (EE), size, poly-dispersity index (PI), and morphology. Kinetic parameters, pH optimum, thermal stability, and reusability of CAT were determined. The influence of optimized e-CAT dispersion onto thermally injured rat skin was evaluated. Results revealed that encapsulation enhanced CAT catalytic efficiency (Vmax/Km). Free CAT and e-CAT had pH optimum at 7.0. e-CAT exhibited improved thermal stability where it retained 50% residual activity at 60 °C. Free CAT lost its activity after three consecutive operational cycles; however, e-CAT retained 60% of its initial activity following 12 cycles. After 24 h of topical application on thermal injury, a significant difference in lesion size was observed with e-CAT compared with the control group. Based on these encouraging results, CAT immobilization demonstrated a promising novel delivery system that enhances its operational stability. In addition, nano-encapsulated CAT can be anticipated to be beneficial in skin oxidative injury.

Hierarchically macro/mesoporous silica spheres for catalase immobilization and catalysis

In this study, hierarchically macro/mesoporous silica sphere (HMMS) with structural macropores and skeleton mesopores was fabricated and adopted as an ideal carrier for immobilization of catalase (CAT). Compared with a commercial mesoporous silica (CMS) carrier, HMMS can immobilize much more CAT (270–300mg/g carrier) than the CMS (90–120mg/g carrier). Moreover, the immobilized CAT on HMMS exhibits comparable catalytic efficiency to the carrier-free CAT, and possesses appreciable reusability. These superiorities make HMMS a promising carrier for enzyme immobilization.

Catalase immobilization on amino-activated Fe3O4@SiO2 nanoparticles: Loading density affected activity recovery of catalase

A blackberry-shaped Fe3O4@SiO2 nanoparticles were prepared, characterized and applied in covalently binding catalase. The enzyme loading decreased with the increase of pH, however, the activity recovery increased simultaneously. To elucidate the influence factor of the activity recovery, the enzyme loading was further regulated by changing the initial free enzyme content. The relationship between enzyme loading and activity recovery showed the consistent trend, whether the variation of enzyme loading was incurred by pH or by initial enzyme content. The simulated parameters showed the similar values according to the experiment at different conditions. It was concluded that activity recovery was dominated by protein density on surface, not by the orientation of the enzyme on surface, due to the negligible diffusion limit for H2O2 as the substrate of catalase. The immobilized catalase at pH 7.0 has a high activity recovery of 100% at 14.4 enzyme μg/mg nanoparticles. The Km and Vmax of the immobilized enzyme above are 0.215mol and 0.797mol/min, similar to 0.167mol and 0.727mol/min for the free enzyme, respectively.

Catalase immobilized-radiation grafted functional cellulose matrix: A novel biocatalytic system

The work highlights a unique, one step protocol for room temperature immobilization of enzyme catalase onto epoxy functionalised cellulose matrix developed via gamma radiation induced simultaneous irradiation grafting of Glycidylmethacrylate (GMA). Effect of grafting parameters, such as radiation dose, monomer concentration and solvent composition on the grafting yield, was studied in order to optimize the radiation grafting process. Poly(GMA)-g-cellulose matrices were characterized by grafting yield determination, FTIR, SEM and TGA techniques. The epoxy functionalized poly(GMA)-g-cellulose matrix was subsequently employed for covalent immobilization of an industrially relevant enzyme catalase. The catalytic activity of catalase-immobilized-poly(GMA)-g-cellulose was assayed by spectrophotometrically monitoring the enzymatic degradation of H2O2 at 240nm. Catalase-immobilized-poly(GMA)-g-cellulose was observed to be reusable for over 5 cycles within ten days. Catalase was observed to show improved activity at higher pH after immobilization. Thermal stability of Catalase-immobilized-poly(GMA)-g-cellulose was also enhanced in comparison to the free enzyme system.

Intensifying the O2-dependent heterogeneous biocatalysis: Superoxygenation of solid support from H2O2 by a catalase tailor-made for effective immobilization

Besides merely destroying H2O2, an important use of the catalase reaction, H2O2→1/2 O2+H2O, is to supply O2 to oxygenation reactions. Due to convenient spatiotemporal control over O2 release, oxygenation from H2O2 is useful in particular for enzymatic reactions confined to solid supports. Because commercial catalases are difficult to immobilize, we have developed a one-step procedure of purification and immobilization of Bordetella pertussis catalase, recombinantly produced in Escherichia coli. Fusion of the catalase to a positively charged binding module enabled effective immobilization of the chimeric enzyme on anionic support (Relisorb SP 400), giving a controllable activity loading of between 5000 and 100,000 units/g support. Use of the immobilized catalase in combination with H2O2 feeding provided O2 to the reaction of glucose oxidase in solution for a range of volumetric conversion rates (0.2–1.5mM/min). Using optical sensing to measure the O2 concentration in the liquid but also in the solid phase, we showed that internal superoxygenation of the support was made possible under these conditions, resulting in an inverted (that is, negative) O2 concentration gradient between the bulk and the particle and allowing the internal O2 concentration to exceed by up to 4-fold the limit of atmospheric-pressure air saturation in solution. By tailored immobilization of B. pertussis catalase, therefore, an efficient biocatalytic system for hydrogen peroxide conversion in porous solid support was developed. This could find application for bubble-free oxygenation of O2-dependent enzymes co-immobilized with the catalase whereby enhanced internal availability of O2 would contribute to biocatalytic reaction intensification.

Improved catalytic activity by catalase immobilization using γ‐cyclodextrin and electrospun PCL nanofibers

ABSTRACTNanofibrous structures are promising for biocatalyst immobilization due to their large surface area which facilitates the enzyme attachment, stability, ease of separation, and fine porous structure. There is limited research available on the change in enzyme activity following interaction with cyclodextrin. In this study, catalase enzyme was immobilized into nanofibrous structures by various techniques, with and without γ‐CD addition, and the enzymatic activity of catalase was evaluated. In addition, catalase‐γ‐CD complex containing PEO polymer solution was electrospun in between PCL nanofibrous layers as a newly developed technique. The enzyme immobilized nanofibrous structures were characterized by SEM, XRD, and FT‐IR analysis methods. Among all the activity tests, best enzyme activity was recorded with catalase‐γ‐CD physical mixture encapsulated PCL nanofibrous layers. Moreover, the test results indicated that the use of cyclodextrin in immobilization process considerably improves the catalytic activity of the enzyme. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 44404.

Immobilization of Genetically-Modified d-Amino Acid Oxidase and Catalase on Carbon Nanotubes to Improve the Catalytic Efficiency

d-amino acid oxidase (DAAO) and catalase (CAT) have been genetically modified by fusing them to an elastin-like polypeptide (ELP). ELP-DAAO and ELP-CAT have been separately immobilized on multi-walled carbon nanotubes (MWNTs). It has been found that the secondary structures of the enzymes have been preserved. ELP-DAAO catalyzed the oxidative deamination of d-alanine, and H2O2 was evolved continuously. When the MWNT-supported enzymes were used together, the generated hydrogen peroxide of ELP-DAAO could be decomposed in situ. The catalytic efficiency of the two immobilized enzymes was more than five times greater than that of free ELP-DAAO when the ratio of immobilized ELP-CAT to immobilized ELP-DAAO was larger than 1:1.

Catalase‐Modified Carbon Electrodes: Persuading Oxygen To Accept Four Electrons Rather Than Two

We successfully exploited the natural highly efficient activity of an enzyme (catalase) together with carbon electrodes to produce a hybrid electrode for oxygen reduction, very appropriate for energy transformation. Carbon electrodes, in principle, are cheap but poor oxygen reduction materials, because only two-electron reduction of oxygen occurs at low potentials, whereas four-electron reduction is key for energy-transformation technology. With the immobilization of catalase on the surface, the hydrogen peroxide produced electrochemically is decomposed back to oxygen by the enzyme; the enzyme natural activity on the surface regenerates oxygen, which is further reduced by the carbon electrode with no direct electron transfer between the enzyme and the electrode. Near full four-electron reduction of oxygen is realised on a carbon electrode, which is modified with ease by a commercially available enzyme. The value of such enzyme-modified electrode for energy-transformation devices is evident.

Covalent linkage of alkalothermophilic catalase onto functionalized cellulose

Catalase from a thermophilic bacterium belonging to the genus Geobacillus was purified and covalently immobilized onto a functionalized polymer via a spacer with an objective to improve its kinetic and biochemical properties. This is the first report on the purification and immobilization of catalase from the genus Geobacillus. The tetrameric catalase of about 221 kDa was successfully purified using a multistep purification strategy. A shift in pH and temperature optima from 8.0 to 9.0 and 55 °C to 60 °C, respectively was recorded after covalent binding of catalase onto the functionalized matrix. The kinetic constants i.e. Km, Vmax, Kcat and Kcat/Km were found to be 1.2 mM, 4.43 × 106 IU, 6.3 × 105 s−1 and 5.25 × 108 s−1 M−1 for free, and 1.8 mM, 4.01 × 106 IU, 5.9 × 105 s−1 and 3.20 × 108 s−1 M−1 for immobilized catalase, respectively. The ease of binding of alkalothermophilic catalase from a novel isolated bacterium G. extremocatsoochus sp. nov., MTCC 5873 onto a low cost activated cellulose support demonstrated enhanced pH and thermal stability as compared to its free counterpart. This immobilized catalase preparation with improved characteristics has good potential for diverse applications. The present findings provide valuable information on how to tailor enzymes and supported polymer matrices to improve the performance of a biocatalyst.