Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles

doi:10.1016/j.procbio.2017.03.025

Process Biochemistry

Volume 57, June 2017, Pages 95-104

https://doi.org/10.1016/j.procbio.2017.03.025 Get rights and content

Highlights

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hCA I purified from human erythrocytes.
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hCA I covalently immobilized on APTES modified silica coated magnetic nanoparticle by glutaraldehyde.
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The structure of modified magnetic nanoparticles was characterized by XRD, FT-IR, VSM, and TEM techniques.
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Optimum immobilization conditions, temperature and pH and kinetic parameters of free and immobilized hCA I were determined.
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Immobilized enzyme showed 61% of its initial activity after thirteen repeated uses.

Abstract

In this study, we synthesized magnetic nanoparticles (MNPs) by co-precipitation method. After that, silica coating with tetraethyl orthosilicate (TEOS) (SMNPs), amine functionalization of silica coated MNPs (ASMNPs) by using 3-aminopropyltriethoxysilane (APTES) were performed, respectively. After activation with glutaraldehyde (GA) of ASMNPs, human carbonic anhydrase (hCA I) was immobilized on ASMNPs. The characterization of nanoparticles was performed by transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and vibrating sample magnetometer (VSM). The immobilization conditions such as GA concentration, activation time of support with GA, enzyme amount, enzyme immobilization time were optimized. In addition of that, optimum conditions for activity, kinetic parameters (K_m, V_max, k_cat, kcat/K_m), thermal stability, storage stability and reusability of immobilized enzyme were determined.

The immobilized enzyme activity was optimum at pH 8.0 and 25 °C. The K_m value of the immobilized enzyme (1.02 mM) was higher than the free hCA I (0.48 mM). After 40 days incubation at 4 °C and 25 °C, the immobilized hCA I sustained 89% and 85% of its activity, respectively. Also, it sustained 61% of its initial activity after 13 cycles. Such results revealed good potential of immobilized enzyme for various applications.

Graphical abstract

Introduction

Global warming is one of the most serious environmental issues that the world is facing today. Since the industrial revolution, the increasing concentration of carbon dioxide (CO₂) in the atmosphere from the combustion of fossil fuels has been regarded as the major contributor to global warming [1], [2]. In nature, chemical fixation of CO₂ is safe and eco-friendly but, it requires high pressure and temperature and takes a long time [3]. Hence, using of biological catalysts is particularly attractive for biomimetic CO₂ sequestration.

Carbonic anhydrase (CA, E.C. 4.2.1.1) catalyzes the reversible hydration of CO₂ into carbonate in the cells: CO₂ + H₂O ↔ H⁺ + HCO₃⁻. Its catalytic activity is common zinc-hydroxide mechanism, which involves the nucleophilic attack of a zinc-bound OH⁻ ion on a CO₂ molecule to form a metal-bound HCO₃⁻ ion that is displaced by a water molecule. Enzyme is inactive while the zinc ion coordinated with water. For regeneration of OH⁻ ions, H⁺ ion is given from zinc-bound water molecule to the bulk solution. [4], [5], [6].

Effect of CA on CO₂ also provides its using in various applications such as chemical conversion, biosensor, and bioremediation [7]. Previously, biosensors based on the CA were developed by Cammoroto et al. [8] for making the measurement of CO₂ easier and more reliable and by Fierke et al. [9] for zinc biosensing. CA can also be used for CO₂ sequestration, biofuel production in industrial applications and artificial lungs and drug delivery systems in medical applications [10]. However, high cost, poor thermal and storage stability of enzyme seriously limit their practical applications [11]. One of the successful ways to overcome these limitations is immobilization of enzymes onto insoluble support [12], [13], [14], [15].

There are several methods used to immobilize the enzymes onto supports [12]. Choosing of suitable immobilization systems is necessary to take advantages of the immobilization techniques [16]. Because these systems can improve the enzyme stability by generating a favorable enzyme environment, by avoiding the subunit dissociation of multimeric proteins or by increasing the enzyme rigidity [17], [18]. Multipoint covalent attachment is one of these systems [19].

The multipoint covalent immobilization requires the interaction of several residues of the same enzyme with active groups on the support [20]. For obtain maximum degree of attachment, it needs enough time and pH conditions [21], [22]. The overall three-dimensional structure of enzyme can become more rigid [20]. In this way, it can prevent conformational changes induced by heat, organic solvents or any other distorting agent [18], [23]. For multipoint attachment, amine groups of enzyme and aldehyde groups of support are a good choice. The amount of covalent bonds between the support and the enzyme depends activation degree of the support (concentration of aldehyde groups on the support surface) and the concentration of amine groups in the enzyme molecule. Activation of support with glutaraldehyde is one of the most popular technique and quite simple and efficient [20], [24]. After activation, supports are generally very versatile because of the presence of hidrophobic groups, ionic groups and covalent moieties for the enzyme to interact [25].

The selection of the convenient support, the immobilization conditions and the reactive groups on enzyme are key points for the preparation of enzyme biocatalysts improved their stability with multipoint covalent attachment [26].

In recent years, using of MNPs as insoluble support has great interest due to their low mass transfer resistance and easy operation. Modification of MNPs has been performed with some reactive functional groups such as amine, hydroxyl, carboxyl and epoxy to achieve perfect performance of immobilized enzyme [11]. Many approaches have been employed for modification of nanoparticles including coating macromolecules, monomer co-polymerization, activated swelling, and silanization. Among them, silane coupling agents have provided the advantage of a high density of surface functional groups and simple operation by directly coated onto the surface of MNPs [27], [28]. Using of APTES with a terminal amine group ( single bond NH₂), a silane coupling agents, can be useful for covalent coupling of proteins to the surface of the support [29].

Previously, CA has immobilized on Fe₃O₄/SiO₂ that amine-grafted by using 3-chloropropyltrimethoxysilane and octa(aminophenyl)silsesquioxane and also, on Fe₃O₄ nanoparticles functionalized with carboxyl group [30], [31]. Another study, hCA was immobilized onto gold nanoparticles assembled over amine functionalized with APTES mesoporous SBA-15 [32].

To the best of our knowledgement, the covalent immobilization of hCA I on Fe₃O₄/SiO₂ nanoparticles amine-grafted with APTES was firstly performed via GA in this study. We evaluated the effects of immobilization conditions on the activity of hCA I. In addition, the optimum pH and temperature values, kinetic parameters (K_m, V_max, k_cat, k_cat/K_m), thermal stability, storage stability, and reusability of immobilized hCA I were determined.

Section snippets

Materials and methods

Iron(III) chloride hexahydrate (FeCl₃·6H₂O), hydrochloric acid (HCl, 35%), L-Tyrosine, sulfanylamide, 3-(aminopropyl)triethoxysilane (APTES), and glutaraldehyde were purchased from Merck, cyanogen bromide activated Sepharose 4B, p-nitrophenyl acetate (p-NPA) and aqueous ammonia solution (NH₃·H₂O, 25%) were purchased from Sigma, and tetraethyl orthosilicate (TEOS) were purchased from Alfa Aesear. All the other chemicals used in the study were of analytical grade.

Purification and characterization of hCA I

Table 1 summarizes the steps which are hemolysate and Sepharose^®4B-l-tyrosine-p-aminobenzene sulfonamide affinity chromatography for purifying hCA I from human erythrocytes. The specific activity of hCA I after purification with affinity column was 1320.07 ± 4.45 EU/mg and a yield of 36.04%. Purification fold was 91.35-fold. Enzyme concentration was 0.41 ± 0.08 mg/mL.

After purification, the enzyme preparation was loaded onto SDS-PAGE. The purified enzyme displayed similar band with standard hCA I on

Conclusions

In summary, a layer of SiO₂ was coated on MNPs, and then amine functionalization with APTES were successfully performed for hCA I immobilization. The average size of the obtained ASMNPs was 13.6 ± 3.7 nm. The immobilization conditions of hCA I were optimized to improve enzyme activity. The value of the K_m, k_cat and k_cat/K_m of the free hCA I was determined as 0.48 mM, 1.53 μmol min⁻¹ mL⁻¹, 0.92 s⁻¹ and 1917 M⁻¹ s⁻¹, while that of the immobilized CA was determined as 1.02 mM, 0.098 μmol min⁻¹ mL⁻¹ 0,06 s⁻¹ and

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Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials and methods

Purification and characterization of hCA I

Conclusions

J. Biosci. Bioeng.

J. Mol. Catal. B: Enzym.

Comp. Biochem. Physiol. A Mol. Integr. Physiol.

Polymer

Sens. Actuators B

Process Biochem.

Enzyme Microb. Technol.

Process Biochem.

Enzyme Microb. Technol.

J. Mol. Catal. B: Enzym.

Process Biochem.

Enzyme Microb. Technol.

Enzyme Microb. Technol.

J. Mol. Catal. B: Enzym.

J. Magn. Magn. Mater.

Int. J. Biol. Macromol.

J. Biol. Chem.

Anal. Biochem.

J. Chem. Eng.

J. Mol. Catal. B: Enzym.

Colloids Surf. A.

Colloids Surf. B.

J. Mol. Catal. B: Enzym.

Appl. Surf. Sci.

Biomass Bioenerg.

Process Biochem.

Biochem. Eng. J.

Process Biochem.

Process Biochem.

Enzyme Microb. Technol.

Biochem. Eng. J.

Food Chem.

Process Biochem.

J. Mol. Catal. B: Enzym.