Improved stability of Aspergillus niger inulinase (ANI) by covalent immobilization using glutaraldehyde on carboxylated multiwalled carbon nanotubes (c-MWCNTs)

Improved stability of Aspergillus niger inulinase (ANI) by covalent immobilization using glutaraldehyde on carboxylated multiwalled carbon nanotubes (c-MWCNTs) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Improved stability of Aspergillus niger inulinase (ANI) by covalent immobilization using glutaraldehyde on carboxylated multiwalled carbon nanotubes (c-MWCNTs) İpek Alper, Yakup Aslan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6305427/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In this study, ANI was immobilized on c-MWCNTs via the crosslinker reagent glutaraldehyde via a covalent bonding method with 100% binding yield and 82.6% activity yield. While immobilization did not change the optimum pH range (5.5–6.5) or optimum temperature range (55–65°C) of the enzyme, it improved the pH and thermal stability of the enzyme. The V max values obtained for the free and immobilized enzymes were 671.1 µmol/mg/min and 568.2 µmol/mg/min, respectively, and the K m values were 662.3 g/L and 699.3 g/L, respectively. The initial activity of the immobilized enzyme was maintained under optimum activity conditions for 20 consecutive uses and for 30 days under optimum storage conditions. Using immobilized ANI, FOS syrup was obtained at a concentration of 546.9 g/L from 600 g/L inulin solution with a 91.15% conversion ratio. Consequently, the immobilized ANI enzyme obtained in this study can be used for the production of FOS from inulin in industry. Aspergillus niger inulin inulinase covalent immobilization enzyme fructooligosaccharides Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction FOSs are a group of prebiotics that are formed by the linking of 3 to 10 monosaccharides (the last glucose) with β-(2 − 1) glycosidic bonds [1] and are mainly short-chain oligomers, which are 1-lestose (GF 2 ), nystosis (GF 3 ) and fructofuranosyl nystosis (GF 4 ) [2]. FOSs are produced by the hydrolysis of inulin by the endoinulinase enzyme. Inulin Glu α- (1–2) [β-Fru (1–2)] n n > 10 is a polymer consisting of fructose units [3]. In general, FOS is found in large amounts in plants such as Jerusalem artichoke, onion, garlic, beets, apples, yams, bananas, wheat, artichokes and tomatoes [1, 4]. Its solubility is high, and its sweetness is approximately 30–50% that of sucrose. FOS can be used as a noncaloric sweetener in all products when sucrose is used [2]. FOS had positive effects on product properties when used in the production of ice cream, chocolate and low-calorie foods [5]. Although FOS is produced industrially by bacteria and yeast cells immobilized from inulin or sucrose, it is also produced by the hydrolytic effect of soluble or immobilized endo-inulinases from inulin from sucrose via the transferase effect of fructofuranosidase enzymes [6]. In other words, FOS production occurs in two different ways: first, a batch system using soluble enzymes and second, a continuous system using whole-cell or immobilized enzymes [7]. Enzymes are mostly used in immobilized form for industrial applications. Because separating soluble enzymes from a product is very difficult and expensive, a particular soluble enzyme sample can be used only once in an industrial application [8]. In addition, in applications that require low or high pH and high temperatures, they generally do not maintain their stability and activity for long periods, and they can be used only in batch processes, as they are not suitable for continuous production processes. Since enzymes are generally expensive products, the use of soluble enzymes in industrial applications increases the product cost. Except for those used for medical applications, the number of enzymes used in industry is approximately € 1.5 billion [9]. The most effective way to overcome these disadvantages of soluble enzymes is to use immobilized enzymes. Enzyme immobilization can be defined as limiting the movement of enzyme molecules by attaching them to a solid support surface with chemical bonds or by trapping them in a gel matrix mesh capsule [10]. The major advantage of immobilization is that it significantly improves the stability of biomolecules under various reaction conditions and increases the reusability of biomolecules over sequential catalytic cycles [11]. Moreover, after binding the enzyme molecules, the catalysts are converted from the homogeneous to the heterogeneous form, which facilitates the easy separation of the biocatalytic system from the reaction mixture, resulting in higher purity products [12, 13]. The classical methods used for enzyme immobilization are generally divided into five classes: adsorption, covalent bonding, entrapment, encapsulation, and cross-linking [9]. Upon the development of immobilization techniques, these four classes have been subdivided [10, 14]). One of the most important and widely used techniques for enzyme immobilization is covalent bonding, in which enzymes are attached to a solid carrier that is insoluble in the reaction medium [15]. Covalent bond formation between the enzyme and the matrix occurs between the functional groups in the side chain of the amino acid residues of the enzyme and the reactive groups in the matrix [16]. However, the presence of functional groups on amino acid residues in the active site of enzyme molecules leads to a decrease in the activity of the immobilized enzyme. Therefore, active immobilized enzyme activities can be protected by blocking the functional groups of active site amino acid residues through the addition of the enzyme's substrate [17]. The covalent immobilization method is used when the absence of an enzyme in the product is absolute [18]. Enzyme molecules are attached directly to reactive groups (e.g., amino, amide, carboxyl, and hydroxyl groups) on the support or by a spacer arm that is artificially attached to the matrix through various chemical reactions (e.g., diazotization, Schiff base, and imine bond formation) [19, 20]. Although covalent immobilization generally has several disadvantages, such as limited enzyme mobility, reduced enzyme activity, nonrenewable and less effective for cell immobilization, it has several advantages, such as strong binding, high heat stability, longer storability and reusability hundreds of times [21]. Since the first study on carbon nanotubes (CNTs) was published [21], they have attracted the attention of scientists working in different fields, including enzyme immobilization. As shown in Fig. 2.4, CNTs can be classified into single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs) [22–24]. SWCTs consist of tubes with an outer diameter of 0.7 nm and a single atomic thickness of a single sheet of extruded graphene, while MWCNTs consist of many SWCNTs stacked inside one another, and MWCTs are less than 15 nm in outer diameter and tens of micrometres in length [25]. The large surface area of SWCNTs is more advantageous for their enzyme loading capacity, but MWCNTs are preferred because of their high dispersibility and low cost [26]. Enzymes were immobilized on CNTs via adsorption or covalent binding methods. The optimum enzyme conformations required for activity can be maintained in adsorption, but their durability and activity losses upon separation of enzymes from the matrix are still a concern for industrial applications. Covalent enzyme immobilization can increase stability and activity [24]. Because CNTs have high natural affinity for diverse proteins [25], they are very suitable matrices for enzyme immobilization. The advantages of the use of CNTs as an immobilization matrix can be listed as follows: higher enzyme binding capacity, less diffusion restriction for macromolecular substrates or products, improved thermal stability and increased activity, greater reuse and longer storage stability, high mechanical stability and renewability. Many studies on the use of c-MWCNTs in enzyme immobilization have been published in the literature. The immobilization by adsorption method is carried out by mixing the enzyme mixture with the buffer mixture containing the matrix and incubating it for an appropriate time [22]. In covalent immobilization, carboxyl groups (-COOH) on c-MWCNTs are first activated with reagents such as N-ethyl-N'-(3-dimethyl amino propyl) carbodiimide hydrochloride (EDAC). Then, through these groups, the enzyme molecules are covalently attached to the matrix [27]. Numerous studies on the immobilization of ANI by different supports and methods have been published in the literature [1, 22, 28–34]. ANI has already been immobilized on c-MWCNTs by Garlet et al. [23] and Temkov et al. [36]. However, the dimensions of the c-MWCNTs (88% purity, 50–80 nm length and 10–20 µm length) used by Garlet et al. [23] are different from those of the c-MWCNTs (99% purity, 28–48 nm outer diameter and 0.5–2 µm length) used in this study. Additionally, Temkow et al. [36] immobilized ANI on a nanocomposite (MWCNT/Ppy/PEG) produced by using c-MWCNTs, polypyrrole (PPy), and polyethylene glycol (PEG) not directly on c-MWCNTs. Therefore, the results of the present study differ. Since a 5-fold and even a 12-fold increase in activity was reported in enzyme immobilization studies conducted with C-MWCNTs, the goal was to obtain as high an activity yield as possible by immobilizing the ANI enzyme via the covalent binding method on c-MWCNTs. This study is unique because the ANI enzyme was not immobilized by covalent binding via c-MWCNTs. The goals of this study were to achieve 100% immobilization efficiency, the highest possible activity yield, and the highest possible reusability and storage stability by optimizing the immobilization conditions through the covalent attachment of the ANI enzyme to the commercial immobilization matrix c-MWCNTs. 2. Materials and Methods 2.1. Materials ANI was supplied as a free sample from BIO-CAT Inc. (Troy, VA, USA), and c-MWCNTs (99% purity, 28–48 nm outer diameter and 0.5–2 µm in length) were purchased from Nanografi Co. Ltd. (Ankara, Türkiye). Nitrocellulose membrane filters (pore diameter 0.45 µm, membrane diameter 47 mm) were purchased from ISO-LAB (Wertheim, Germany). Inulin from chicory root (DP = ⁓10–15) was purchased from abcr GmbH (Karlsruhe, Germany). Bovine serum albumin (BSA), sodium hydroxide, sodium dihydrogen phosphate, hydrochloric acid, 3,5-dinitrosalicylic acid (DNSA), sodium potassium tartrate, sodium azide, glutaraldehyde (25%) and Bradford dye were obtained from Sigma‒Aldrich (Taufkirchen, Germany). 2.2. Methods 2.2.1. Functionalization of c-MWCNTs Fifty milligrams of c-MWCNTs were functionalized with glutaraldehyde via incubation in 5 mL of 25 mM sodium phosphate buffer (pH 6.0) containing different concentrations of glutaraldehyde for 0.5 hours at a shaking speed of 150 rpm and labelled F-MWCNTs for further studies. The F-MWCNTs were washed three times with 5 mL of distilled water and 5 mL of sodium phosphate buffer solution under vacuum and filtered through a nitrocellulose membrane filter. 2.2.1.2. Effect of buffer solution pH on functionalization efficiency Fifty milligrams of c-MWCNTs were functionalized with glutaraldehyde by incubation for 2 hours at room temperature (25°C) in a heated and orbital shaker incubator with shaking at 150 rpm in 5 mL of 25 mM sodium phosphate buffer solution at different pH values containing 0.5 M glutaraldehyde. Fc-MWCNTs were filtered through a nitrocellulose membrane filter, washed thrice with 5 mL of distilled water and thrice with 5 mL of sodium phosphate buffer solution under vacuum. 2.2.1.3. Effect of c-MWCNT and glutaraldehyde reaction times on functionalization efficiency Fifty milligrams of c-MWCNTs were functionalized with glutaraldehyde via incubation for different durations, heated to room temperature (25°C) and shaken at 150 rpm in an incubator containing 5 mL of 25 mM sodium phosphate buffer (pH = 6.0) containing 0.5 M glutaraldehyde. Fc-MWCNTs were filtered through a nitrocellulose membrane filter and washed three times with 5 mL of distilled water and three times with 5 mL of sodium phosphate buffer solution under vacuum. 2.2.1. Immobilization of ANI with Fc-MWCNTs Immobilizations were carried out by incubating 100 µL of ANI solution and wet Fc-MWCNTs in 5 mL of sodium phosphate buffer and shaking at 150 rpm in an incubator at room temperature (25°C) for certain times according to the covalent bonding method. At the end of the immobilization, the immobilized enzymes were filtered under vacuum using nitrocellulose membrane filters. After washing three times with 5 mL of 0.1 M phosphate buffer and 3 times with 5 mL of distilled water, the amount of protein in the immobilization buffer and the filtrates was determined. The immobilization yields were calculated from the determined protein amounts. Activity yields were calculated from the relative activities of free and immobilized enzymes determined according to the standard activity determination method. 2.2.2.4. Effect of the pH of the immobilization buffer on the immobilization efficiency Immobilizations were performed by incubating 316 mg wet Fc-MWCNTs (dry weight of 50 mg, average wet‒dry weight ratio of 6.32) with 100 µL free ANI solutions in 5 mL 25 mM sodium phosphate buffer solutions with different pH values (4.0–5.0–6.0–7.0) at room temperature (25°C) in an incubator at 150 rpm with shaking for 60 minutes. 2.2.2.5. Effect of the immobilization buffer concentration on the immobilization efficiency Immobilizations were performed by incubating 316 mg of wet Fc-MWCNTs with 100 µL of free ANI solutions in 5 mL of sodium phosphate buffer solutions of different concentrations with a pH of 8.0 at room temperature and shaking at 150 rpm for one hour. 2.2.2.6. Effect of the amount of c-MWCNTs on immobilization efficiency Different amounts of wet Fc-MWCNTs were incubated with 100 µL of free ANI solution in 5 mL of 100 mM sodium phosphate buffer (pH 8.0) at 150 rpm at room temperature for one hour while shaking. 2.2.8.7. Effect of immobilization time on immobilization efficiency Immobilizations were performed by incubating 632 mg of wet Fc-MWCNTs with 100 µL of free ANI solution in 5 mL of 100 mM sodium phosphate buffer (pH 8.0) at room temperature for different durations while shaking at 150 rpm. 2.2.8.8. Confirmation of covalent bonding by the desorption method Wet Fc-MWCNTs (632 mg) were incubated in 5 mL of 100 mM sodium chloride solution for 4 hours at room temperature with shaking at 150 rpm. At the end of the incubation, protein determination was performed on the filtrates according to Bradford’s method (1976). Wet immobilized ANIs were used for activity determination. 2.2.2. Protein (Enzyme) determination Protein concentrations were determined according to a previously described method [35] before and after immobilization. 2.2.3. Determination of reducing sugars The reducing sugars released or formed were determined via the DNS method [36]. 2.2.4. Determination of ANI activity ANI activity was determined by reactions of 100 µL of free ANI solution or 632 mg of wet immobilized ANI with 5 mL of 2% (w/v) inulin solutions under standard conditions. One IU of ANI activity was defined as the amount of enzyme (mg) that produces a reducing sugar equivalent to 1 µmol D-glucose in 1 min in 5 mL of 2% (w/v) inulin solution under standard conditions. ANI activity was calculated via Eq. ( 2.1 ). $$\:Activity=\:\frac{Reducing\:sugars\:\left(\mu\:mol\right)}{Ebzyme\:used\:\left(mg\right)x\:reaction\:duration\:\left(min\right)}$$ 2.1 2.2.5. Calculation of immobilization and activity efficiencies The immobilization yield was calculated via Eq. ( 2.2 ) from the enzyme amounts in the immobilization solutions before and after immobilization, and the activity yield was calculated via Eq. ( 2.3 ) from the current activities of the free and immobilized enzymes. $$\:Immobilization\:Yield=\:\frac{A-B}{A}\:X\:100$$ 2.2 A Free enzyme used for immobilization (mg) B Free enzyme remaining in the filtrate (mg) $$\:Activity\:Yield=\:\frac{Ai\:\left(IU\right)}{As\:\left(IU\right)}\:x\:100$$ 2.3 Ai Current activity of the immobilized enzyme As Current activity of free enzyme 2.2.6. Quantitative carbohydrate determination by high-performance liquid chromatography (HPLC) The carbohydrate content was determined via high-pressure liquid chromatography (HPLC), a refractive index detector, an amino column, a mobile phase consisting of 70/30% acetonitrile and water by volume and samples with a 20 µL volume [37]. 2.2.7. Characterization of free and immobilized ANIs Free and immobilized ANI were characterized by determining their optimum pH, optimum temperature, pH stability, thermal stability and kinetic constants, respectively [38]. 2.2.7.1. Optimum pH The optimum pH was determined by incubating 100 µL of free ANI and 632 mg of wet immobilized ANI with 5 mL of 25 mM inulin solutions prepared in sodium phosphate buffer at different pH values (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0) at 60°C for 15 min with shaking at 150 rpm. 2.2.7.2. Optimum temperature The optimum temperature ranges of the free ANI and the immobilized ANI were determined under standard activity conditions (pH 6.0, duration of 15 min, shaking speed of 150 rpm), with the exception of temperature. Activity determination reactions were performed in the range of 30–80°C. 2.2.7.3. pH stability The pH stabilities of the free ANI and the immobilized ANI were determined by incubating the enzyme with sodium phosphate buffer at different pH values (3.0–8.0) at 60°C for 15 min prior to activity determination. 2.2.7.4. Thermal stability The thermal stabilities of the free ANI and the immobilized ANI were determined by incubating the enzyme in sodium phosphate buffer (pH 6.0) for 15 min at different temperatures (30–80°C) prior to activity determination. 2.2.7.5. Kinetic constants The initial activities of the free ANI and the immobilized ANI were determined via incubation in 5 mL of inulin solution (pH 6.0) at different concentrations (5–600 g/L) at 60°C for 15 minutes in an incubator with shaking at 150 rpm. V max and K m constants were calculated from Lineweaver‒Burk plots. 2.2.8. Reusability of immobilized ANI The stability of the use of immobilized ANI was determined via a graph drawn using relative activities determined after 20 consecutive uses under standard conditions. Prior to each use, the immobilized ANI was washed with 5 mL of 0.1 M phosphate buffer and with 5 mL of distilled water three times under vacuum using nitrocellulose membrane filters. 2.2.9. Storage stability of immobilized ANI The storage stability of the immobilized ANI was determined via a graph of the relative activities measured according to the standard activity assay method every two days for 20 days. The immobilized enzyme was washed with plenty of distilled water after each use and stored in 5 mL of 0.1 M phosphate buffer solution (pH 6.0) in a refrigerator at + 4°C. 2.3. FOS production using immobilized ANI Since the amount of immobilized ANI, inulin concentration and reaction time affect the yield of FOS production, these parameters were changed sequentially, and their effects were determined. 2.3.1. Effect of the amount of immobilized ANI on FOS production Different amounts (158, 316, 474, 632, 948, and 1264 mg) of wet immobilized ANI were reacted with 5 mL of 20 g/L inulin solution (pH 6.0) for 15 min at 60°C with shaking at 150 rpm. The FOS and glucose concentrations produced in the reactions were determined via HPLC. 2.3.2. Effect of inulin concentration on FOS production Five milliliters of inulin solutions (pH 6.0) at different concentrations (5-50-100-200-400-600 g/L) were reacted with 948 mg wet immobilized ANIs at 60°C with shaking at 150 rpm. The FOS and glucose concentrations produced at the end of the reactions were determined via an HPLC device. 2.3.3. Effect of reaction time on FOS production Five milliliters of inulin solution (pH 6.0) at a concentration of 600 g/L was reacted with 948 mg wet immobilized ANIs at 60°C for different durations by shaking at 150 rpm. The FOS and glucose concentrations produced at the end of the reactions were determined via an HPLC device. 2.4. Statistical analysis and creation of graphics The arithmetic means and standard errors of the experiments repeated three times were calculated via the Microsoft Office Excel 2007 program. Graphs and error bars on the graphs were created via the OriginPro-2019 graphics program. 3. Results and Discussions 3.1. Protein (Enzyme) Determination The calculations were performed according to the correct equation of the standard BSA graph, which revealed that 1.17 mg of enzyme was present in the 5.1 mL reaction mixture. Since 0.1 mL of free enzyme (liquid enzyme preparation) was used in the reaction, the enzyme concentration of the liquid enzyme preparation was 11.7 mg/mL. 3.2. Determination of ANI activity As a result of the calculations performed according to the correct equation of the standard D-glucose standard graph, 38001.79 µg D-glucose was calculated to have formed in the 5.1 mL reaction mixture. When we divided this value by 180 g, which is the molar mass of glucose, the resulting D-glucose content was 211.12 µmol. According to Eq. (2.1), the free ANI activity was calculated as 140.75 IU/mL. Since there is 11.7 mg of free ANI in 1 mL, the specific activity of free ANI can also be expressed as 12.03 IU/mg. Accordingly, the amount of free instant ANI with 1 IU of activity was found to be 0.083 mg. 3.3. Optimization of Functionalization Conditions 3.3.1. Effect of glutaraldehyde concentration on functionalization efficiency As shown in Table 1, the activity yield decreased while the immobilization efficiency increased as the GA concentration increased. The increase in immobilization yield with increasing GA concentration can be explained by the fact that more ANI molecules are attached to the matrix. It is well known that at high GA concentrations, the binding of enzyme molecules to the matrix at multiple points causes a loss of activity by disrupting the three-dimensional structure of the enzyme [11, 39–42]. In addition, as a result of the crowding of the immobilized enzyme molecules on the matrix, the enzyme molecules prevent the substrate from reaching the active site of the enzyme [43]. Since the highest activity efficiency was obtained when 0.5 M GA was used, the most suitable glutaraldehyde concentration for functionalization was 0.5 M. Similarly, in another study, the highest efficiency in the immobilization of K. lactis β-galactosidase with MWCNTs was obtained when 0.5 M GA was used [44]. 3.3.2. Effect of buffer solution pH on functionalization efficiency As shown in Table 2, the immobilization efficiency increased with increasing pH. This result shows that with increasing pH, more glutaraldehyde binds to the matrix, and as a result, more enzyme molecules are immobilized. According to the table, the activity yield increases up to pH 6 and decreases at higher pH values. This decrease is probably due to the degradation of the three-dimensional structure of the enzyme molecules as a result of binding at multiple points with increasing GA concentration in the matrix [39, 45]. Table 2 Effect of buffer solution pH on functionalization efficiency Buffer Solution pH Immobilization Efficiency (%) Activity Yield (%) 4.0 25.63 ± 0.02 1.31 ± 0.05 5.0 31.36 ± 0.05 1.61 ± 0.04 6.0 36.12 ± 0.04 3.06 ± 0.02 7.0 40.13 ± 0.06 1.48 ± 0.05 8.0 43.56 ± 0.03 1.19 ± 0.03 3.3.3. Effect of c-MWCNT and glutaraldehyde reaction times on functionalization efficiency As shown in Table 3, as the reaction time increased, the immobilization efficiency and activity efficiency increased. This result is quite natural because as time progresses, more GAs are connected. Table 3 Effect of c-MWCNT and glutaraldehyde reaction times on functionalization efficiency Reaction Time (Hours) Immobilization Yied (%) Activity Yield (%) 2.0 38.39 ± 0.04 3.06 ± 0.02 4.0 54.89 ± 0.04 4.54 ± 0.02 6.0 57.35 ± 0.03 9.13 ± 0.04 8.0 60.12 ± 0.02 10.88 ± 0.05 3.4. Optimization of the Immobilization Conditions 3.4.1. Effect of buffer solution pH on immobilization efficiency Table 4 shows that as the pH of the immobilization solution increased, the immobilization efficiency and activity efficiency increased. This result shows that with increasing pH, amino groups in the side groups of amino acids in the structure of the enzyme shift more into the unprotonated form (NH 2 ), which is suitable for covalent bonding. GA primarily reacts with amino groups, especially at moderate pH values [46, 47]. The increase in immobilization efficiency from acidic pH to moderate pH confirms this information. Table 4 Effect of buffer solution pH on immobilization efficiency Buffer pH Immobilization Yield (%) Activity Yield (%) 4.0 42.22 ± 0.02 4.14 ± 0.05 5.0 50.24 ± 0.05 5.04 ± 0.04 6.0 56.55 ± 0.04 9.49 ± 0.02 7.0 62.64 ± 0.06 11.60 ± 0.05 8.0 67.26 ± 0.03 13.84 ± 0.03 3.4.2. Effect of buffer solution concentration on immobilization efficiency According to Table 5, buffer solution concentrations higher than 50 mM did not affect the immobilization efficiency but increased the activity efficiency. According to this result, while the amount of enzyme bound to the matrix does not change with increasing concentration, the increase in activity efficiency shows that increasing the concentration of salt ions helps to preserve the three-dimensional structures of the enzyme molecules attached to the matrix. The three-dimensional structures of enzymes are highly dependent on the concentration of salt ions in the environment [39, 48]. Table 5 Effect of buffer solution concentration on immobilization efficiency Buffer Concentration (mM) Immobilization Yield (%) Activity Yield (%) 25 67.26 ± 0.03 13.84 ± 0.03 50 76.66 ± 0.04 15.98 ± 0.02 75 76.66 ± 0.03 17.55 ± 0.03 100 76.66 ± 0.05 34.33 ± 0.04 3.4.3. Effect of wet Fc-MWCNT amount on immobilization efficiency As shown in Table 6, the immobilization efficiency and activity efficiency slightly increased when the amount of wet Fc-MWCNTs was doubled. However, when higher amounts of Fc-MWCNTs were used, the immobilization efficiency slightly increased, and the activity efficiency decreased. The reason for this may be the destruction of the three-dimensional structures of enzyme molecules, which are necessary for activity, as a result of multipoint attachment of the enzyme molecules to the matrix by increased GA groups on the matrix [39, 45]. Table 6 Effect of wet Fc-MWCNT amount on immobilization efficiency Wet Fc-MWCNT Amount (mg) Immobilization Yield (%) Activity Yield (%) 316 76.66 ± 0.05 34.33 ± 0.04 632 81.88 ± 0.04 37.99 ± 0.02 948 85.53 ± 0.03 33.67 ± 0.04 1264 86.16 ± 0.02 30.75 ± 0.05 3.4.4. Effect of immobilization time on immobilization efficiency As shown in Table 7, the immobilization efficiency increased with increasing immobilization time. After four hours, 100% immobilization and 82.60% activity efficiency were obtained. The activity yields obtained in previous studies with different matrices and methods were 66% [28], 145% [29], 83% [31], 81% [49], 400% [30], 100% [32], 92% [33], and 60.7% [50]. The results obtained in our study are greater than those of other studies, although they are below the results of de Oliveira Kuhn et al. [30] and de Oliveira Kuhn et al. [31]. Since the activity efficiency of the covalent bonding method is generally less than 100%, the activity yield we obtained in this study is sufficient for industrial production. Table 7 Effect of immobilization time on immobilization efficiency Duration (Hour) Immobilization Yield (%) Activity Yield (%) 1 81.88 ± 0.04 37.99 ± 0.02 2 83.22 ± 0.04 42.70 ± 0.02 3 94.09 ± 0.04 51.19 ± 0.02 4 100.00 ± 0.03 82.60 ± 0.04 3.5. Confirmation of covalent bonding by the desorption method In accordance with previous methods [51], 632 mg of immobilized ANI was incubated in 5 mL of 100 mM sodium chloride solution for the immobilization period (4 hours) at room temperature (25°C) in a heated incubator with shaking at 150 rpm. At the end of the incubation, the immobilized enzymes were washed thrice with 5 mL of distilled water and thrice with 5 mL of sodium phosphate buffer solution under vacuum and filtered through a nitrocellulose membrane filter. As shown in Table 8, the absence of protein in the filtrates and the absence of any decrease in the activities of the immobilized enzymes indicate that immobilization is achieved by covalent bonding. Table 8 Confirmation of covalent bonding by desorption Desorption Immobilization Yield (%) Activity Yield (%) Before 100.00 ± 0.03 82.60 ± 0.04 After 100.00 ± 0.03 82.60 ± 0.04 3.6. Characterization of Free and Immobilized ANI 3.6.1. Optimum pH As shown in Fig. 1, immobilization did not change the optimum pH range (5.5–6.5) of ANI. In addition, the activity of the immobilized ANI was greater than that of its free counterpart at acidic and alkaline pH values. For example, at pH 3 and 8, free ANI has only 60% of its activity, whereas immobilized ANI has 70% and 75% of its activity, respectively, at the same pH. Singh et al. (2019) reported that the optimum pH (5.0) did not affect the immobilization of ANI. This result can be attributed to the stability of the carbon nanotubes, which is not affected by the changing ionic microenvironment [50]. Furthermore, the unchanged optimum pH indicates that no significant conformational changes occur in the enzyme molecules after immobilization [52]. Moreover, the higher activity of immobilized ANI at acidic and alkaline pH values is probably the result of increased stability resulting from multipoint attachment during immobilization [53–55]. On the other hand, immobilization protects the three-dimensional structure of the enzyme responsible for the activity and prevents the loss of activity under severe conditions [14, 56]. 3.6.2. Optimum temperature Figure 2 shows that the optimum temperature range (55–65°C) was not affected by immobilization. The immobilized ANI clearly has higher activity than its free counterpart over the entire temperature range tested. The optimum temperature of ANI agrees with previous studies [28, 37, 50]. It is well known that immobilized enzymes exhibit greater activity than free enzymes at high temperatures due to the limited conformational mobility of molecules following immobilization [55] as a result of interactions between the enzyme and the support [57]. 3.6.3. pH stability As shown in Fig. 3, immobilized ANI has greater stability over a wider range than free ANI. Free ANI is most stable from pH 5.0–6.5, whereas immobilized ANI has the highest stability in the range of 4.5–7.0. In addition, compared with free ANI, immobilized ANI appears to have greater stability in the low and high pH ranges tested. For example, free ANI lost 22.5% and 25% of its activity at pH 3.0 and 8.0, respectively, whereas immobilized ANI lost only 20% of its activity at the same pH. The increase in the pH stability of ANI after immobilization may be due to the strong covalent interaction between the carbon nanotubes and the enzyme moiety. It is important for industrial processes that an enzyme has a stable-integral conformation over a wide pH range [50]. 3.6.4. Thermal stability As shown in Fig. 4, immobilization increased the thermal stability of ANI. While free ANI lost more than 10% of its activity at 70°C, immobilized ANI retained its entire activity at the same temperature. Additionally, while free ANI lost 80% of its activity at 80°C, immobilized ANI retained more than 60% of its activity at the same temperature. The increase in the thermal stability of inulinase after immobilization is a result of the good interaction between the carbon nanotubes and inulinase [50]. Carbon nanotubes themselves can withstand a longer period of time at elevated temperatures without any change in their tensile strength or elastic modulus. Therefore, the biocatalyst immobilized on these materials eventually also achieves this thermal stability property. Although an increase in the thermal stability of inulinases up to 60°C has been reported for other matrices, such as kaolin clay [58], chitin [59], and nonwoven fabrics [60], the reported increases are lower than those of ANI immobilized on c-MWCNTs in our present study. 3.6.5. Kinetic constants A Lineveawer–Burk plot of free and immobilized ANI is shown in Fig. 5. According to the data obtained from the graph, the maximum velocities (V max ) obtained for free and immobilized ANI are 671.1 µmol/mg.min and 568.2 µmol/mg*min, respectively. The Michaelis–Menten constant (km) was also calculated as 662.3 g/L and 699.3 g, respectively. As K m increases, the enzyme's affinity for the substrate decreases [61]. These results confirm the data obtained during immobilization. An activity efficiency of 82.6% was obtained via immobilization. The ratio of the maximum speeds is approximately the same (82.4%). The decrease in the maximum rate is due to the decreased affinity of ANI for its substrate inulin. The reason for this may be the partial disruption of the conformation of the catalytic groups in the active site of the enzyme, which is suitable for activity, as a result of the formation of strong covalent bonds between the enzyme molecules and the matrix. 3.6.6 Reusability of immobilized ANI As shown in Fig. 6, immobilized ANI did not lose its activity during 20 repeated uses. The stability of immobilized enzymes is very important for industrial applications. The product cost decreases inversely with the number of reuses [14, 56]. According to previous studies on the immobilization of ANI, ANI immobilized on c-MWCNTs by the covalent bonding method in this study clearly has the best usage stability. Karimi et al. [33], in their immobilization study of ANI with 50-, 100- and 200-nm amino functional silica NPs, reported that immobilized ANI lost 17% of its initial activity after 7 uses. de Oliveira Kuhn et al. (2016) reported that ANI immobilized in polyurethane foam retained approximately 49% of its initial activity after 24 uses. In another study, ANI 12 covalently immobilized on Fe3O4 magnetic nanoparticles functionalized with wheat gluten hydrolysates (WGHs), by Torabizadeh and Mahmoudi (2018), retained 70% of its initial activity after use. Finally, ANI, which was covalently immobilized on hydroxylated MWCNTs activated with 3-aminopropyl-triethoxysilane (APTES) by Singh et al. (2019), retained only 28% of its initial activity after 10 consecutive uses. 3.6.7. Storage stability of immobilized ANI The immobilized ANI maintained its activity for 30 days under storage conditions (in 0.1 M sodium phosphate buffer with pH 6.0 at + 4°C) (Fig. 7). When this result is compared with previous results in the literature, ANI immobilized on c-MWCNTs by the covalent bonding method in this study has a very good advantage for industrial applications. In the study by Yewale et al. (2013), immobilized ANI retained its initial activity for 6 days. The initial activity of ANI immobilized by the adsorption method on c-MWCNTs (purity 88%, diameter 50–80 nm, length 100–200 nm) was preserved for five weeks at room temperature [22]. In another study, ANI immobilized in polyurethane foam retained only 49% of its initial activity for 42 days [30]. Finally, ANI immobilized on micro polyhydroxy butyrate (PHB) fibres retained its initial activity for 3 months when stored in dry conditions [49]. Compared with these results, the immobilized ANI obtained in this study clearly has very high storage stability. 3.7. Optimization of the FOS Production Conditions 3.7.1 Effects of the amount of immobilized ANI on FOS production Figure 8 shows that as the amount of immobilized ANI increased, the total FOS also increased. The concentrations of the FOS components kestose, nystose and frucofuranosyl nystose and glucose, which are byproducts, first increased linearly and then remained constant. For example, when the amount of immobilized ANI was 158 mg, the total FOS concentration was approximately 6 g/L; when the amount of immobilized ANI was increased to 948 mg, the FOS concentration increased to approximately 18 g/L. Additionally, when 1264 mg of immobilized ANI was used, the FOS concentration did not change. According to these results, the optimum amount of immobilized enzyme for FOS production was 948 mg, and this amount of immobilized enzyme was used in subsequent experiments. 3.7.2 Effects of inulin concentration on FOS production Figure 9 shows that the relationship between FOS production and inulin concentration is in accordance with the classical Michaelis‒Menten graph. For example, when 5 g/L inulin solution was used, the total FOS concentration was approximately 2 g/L. Furthermore, when 200, 400 and 600 g/L inulin were used, the total FOS concentration also increased to 30 g/L, 32 g/L, and 36 g/L, respectively, and then remained stable at 36 g/L. According to these results, the most suitable inulin concentration was 600 g/L, and an inulin solution at this concentration was used in the next experiment. 3.7.3 Effects of reaction duration on FOS production When Fig. 10 is examined, all the FOS components increase linearly until the 16th hour, and then, the rate of increase slows down and stabilizes at the 20th hour. By optimizing the FOS production conditions, after 20 hours of reaction, FOS was obtained at a concentration of 546.9 g/L with a conversion rate of 91.15%. This ratio achieved in FOS production from inulin is the highest for industrial FOS production compared with the results obtained in previous studies (200 g/L–540 g/L) [1, 22, 28–33]. 4. Conclusion In this study, by optimizing the conditions, ANI was covalently immobilized onto c-MWCNTs with 100% binding yield and 82.6% activity yield. Immobilization did not change the optimum pH range (5.5–6.5) or the optimum temperature range (55–65°C) of ANI. Immobilization increased the km constant from 662.3 g/L to 699.3 g/L but decreased the Vmax from 671.1 µmol/mg/min to 568.2 µmol/mg/min. The initial activity of the immobilized enzyme did not decrease during 20 cycles of use or during storage for 30 days under optimum conditions, indicating high reusability and storage stability. By using immobilized ANI, FOS syrup at a concentration of 546.9 g/L was produced from 600 g/L inulin solution, with a conversion rate of 91.15% after 20 hours. Compared with previous studies in the literature, the FOS rate obtained in this study was the highest. Consequently, the immobilized ANI obtained in this study can be used in the industrial production of FOS syrup from inulin. Declarations Credit authorship contribution statement Yakup Aslan: Writing – review & editing, validation, supervision, resources, project administration, methodology, original draft, investigation, funding acquisition, and software conceptualization. İpek Alper: Visualization, Methodology, Investigation, Formal analysis, Data curation. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements Work was carried out within the scope of the project code 219O034, which was financially supported by The Scientific and Technological Research Council of Türkiye in 2020. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. References R.P. de Souza Oliveira, P. Perego, M.N. de Oliveira and A. Converti Journal of Food Engineering 107, 1 (2011) P. Sangeetha, M. Ramesh and S. Prapulla Trends in food science & technology 16, 10 (2005) G.R. Gibson Clinical Nutrition Supplements 1, 2 (2004) P.M. Rolim Food Science and Technology 35, (2015) K.R. Niness The Journal of nutrition 129, 7 (1999) J.W. 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Rastian, A.A. Khodadadi, F. Vahabzadeh, C. Bortolini, M. Dong, Y. Mortazavi, A. Mogharei, M.V. Naseh and Z. Guo Biochemical engineering journal 90, (2014) E.O. Garuba and A. Onilude Journal of Genetic Engineering and Biotechnology 16, 2 (2018) P.K. Gill, R.K. Manhas and P. Singh Journal of Food Engineering 76, 3 (2006) T.M. Mohamed, S.M.A. El-Souod, E.M. Ali, M.O. El-Badry, M.M. El-Keiy and A.S. Aly Journal of biosciences 39, (2014) I.H. Segel (No Title), (1976) Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6305427","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":434557563,"identity":"4f31dced-1341-44bf-aa5c-fa12b653adff","order_by":0,"name":"İpek Alper","email":"","orcid":"","institution":"Siirt University","correspondingAuthor":false,"prefix":"","firstName":"İpek","middleName":"","lastName":"Alper","suffix":""},{"id":434557564,"identity":"4ebf62db-4fc4-4706-860f-3c99be5c31b5","order_by":1,"name":"Yakup 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2","display":"","copyAsset":false,"role":"figure","size":118110,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of temperature on the activity of free and immobilized ANI\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6305427/v1/66a08c8739967c9be8caf5c0.png"},{"id":79334637,"identity":"79515b2a-55a0-4eb2-b892-17816d4b164e","added_by":"auto","created_at":"2025-03-27 07:36:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":102780,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of pH\u003cstrong\u003e \u003c/strong\u003eon the stability of free and immobilized \u003cem\u003eANI\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6305427/v1/b7f19174d6e58a9dd613c6a1.png"},{"id":79335335,"identity":"025095df-25b3-4739-bdd6-86e2569e5f0c","added_by":"auto","created_at":"2025-03-27 07:44:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":100273,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of temperature on the stability of free and immobilized \u003cem\u003eANI\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6305427/v1/bc45456c3e987d3b372bc7bb.png"},{"id":79336781,"identity":"41f4f363-a6c4-4c79-a5d7-dbf40466de5c","added_by":"auto","created_at":"2025-03-27 08:00:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":54798,"visible":true,"origin":"","legend":"\u003cp\u003eKinetic constants of free and immobilized \u003cem\u003eANI\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6305427/v1/6c91be40d357c7ecec9b4de0.png"},{"id":79335336,"identity":"8558ebfd-3dc6-435c-b0d9-6b956d404a6c","added_by":"auto","created_at":"2025-03-27 07:44:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":84095,"visible":true,"origin":"","legend":"\u003cp\u003eReusability of immobilized \u003cem\u003eANI\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-6305427/v1/0c8b29d20b944ac25663a523.png"},{"id":79335337,"identity":"39595b56-143e-4c72-89c4-576bd48afb61","added_by":"auto","created_at":"2025-03-27 07:44:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":86305,"visible":true,"origin":"","legend":"\u003cp\u003eStorage stability of immobilized ANI\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-6305427/v1/459f4cb183c7c9ddd24baca3.png"},{"id":79335652,"identity":"e515d114-0b45-4a87-8b6c-b3bc536d8aef","added_by":"auto","created_at":"2025-03-27 07:52:13","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":140477,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of the amount of immobilized ANI on FOS production.\u003c/p\u003e","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-6305427/v1/aa4244e47c959d067933ae1b.png"},{"id":79334662,"identity":"e70a757f-044e-4aed-bc72-7c38f1b5f743","added_by":"auto","created_at":"2025-03-27 07:36:13","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":133657,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of inulin concentration on FOS production.\u003c/p\u003e","description":"","filename":"floatimage17.png","url":"https://assets-eu.researchsquare.com/files/rs-6305427/v1/0b2d5e0e17463b6fa5a5a199.png"},{"id":79335339,"identity":"cac3bdf9-380e-4ead-83b6-9b80685ca3a2","added_by":"auto","created_at":"2025-03-27 07:44:13","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":154978,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of reaction time on FOS production\u003c/p\u003e","description":"","filename":"floatimage19.png","url":"https://assets-eu.researchsquare.com/files/rs-6305427/v1/0e43c15c2437ce8a5d07cb23.png"},{"id":80470908,"identity":"c6a8fa54-693b-4f7c-9c03-f05d4d894122","added_by":"auto","created_at":"2025-04-13 07:46:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2861019,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6305427/v1/d1034ef3-bcb5-409f-939f-cd04ffd200d5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Improved stability of Aspergillus niger inulinase (ANI) by covalent immobilization using glutaraldehyde on carboxylated multiwalled carbon nanotubes (c-MWCNTs)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFOSs are a group of prebiotics that are formed by the linking of 3 to 10 monosaccharides (the last glucose) with β-(2\u0026thinsp;\u0026minus;\u0026thinsp;1) glycosidic bonds [1] and are mainly short-chain oligomers, which are 1-lestose (GF\u003csub\u003e2\u003c/sub\u003e), nystosis (GF\u003csub\u003e3\u003c/sub\u003e) and fructofuranosyl nystosis (GF\u003csub\u003e4\u003c/sub\u003e) [2]. FOSs are produced by the hydrolysis of inulin by the endoinulinase enzyme. Inulin Glu α- (1\u0026ndash;2) [β-Fru (1\u0026ndash;2)]\u003csub\u003en\u003c/sub\u003e n\u0026thinsp;\u0026gt;\u0026thinsp;10 is a polymer consisting of fructose units [3].\u003c/p\u003e \u003cp\u003eIn general, FOS is found in large amounts in plants such as Jerusalem artichoke, onion, garlic, beets, apples, yams, bananas, wheat, artichokes and tomatoes [1, 4]. Its solubility is high, and its sweetness is approximately 30\u0026ndash;50% that of sucrose. FOS can be used as a noncaloric sweetener in all products when sucrose is used [2]. FOS had positive effects on product properties when used in the production of ice cream, chocolate and low-calorie foods [5].\u003c/p\u003e \u003cp\u003eAlthough FOS is produced industrially by bacteria and yeast cells immobilized from inulin or sucrose, it is also produced by the hydrolytic effect of soluble or immobilized endo-inulinases from inulin from sucrose via the transferase effect of fructofuranosidase enzymes [6]. In other words, FOS production occurs in two different ways: first, a batch system using soluble enzymes and second, a continuous system using whole-cell or immobilized enzymes [7].\u003c/p\u003e \u003cp\u003eEnzymes are mostly used in immobilized form for industrial applications. Because separating soluble enzymes from a product is very difficult and expensive, a particular soluble enzyme sample can be used only once in an industrial application [8]. In addition, in applications that require low or high pH and high temperatures, they generally do not maintain their stability and activity for long periods, and they can be used only in batch processes, as they are not suitable for continuous production processes. Since enzymes are generally expensive products, the use of soluble enzymes in industrial applications increases the product cost. Except for those used for medical applications, the number of enzymes used in industry is approximately \u0026euro; 1.5\u0026nbsp;billion [9]. The most effective way to overcome these disadvantages of soluble enzymes is to use immobilized enzymes.\u003c/p\u003e \u003cp\u003eEnzyme immobilization can be defined as limiting the movement of enzyme molecules by attaching them to a solid support surface with chemical bonds or by trapping them in a gel matrix mesh capsule [10]. The major advantage of immobilization is that it significantly improves the stability of biomolecules under various reaction conditions and increases the reusability of biomolecules over sequential catalytic cycles [11]. Moreover, after binding the enzyme molecules, the catalysts are converted from the homogeneous to the heterogeneous form, which facilitates the easy separation of the biocatalytic system from the reaction mixture, resulting in higher purity products [12, 13].\u003c/p\u003e \u003cp\u003eThe classical methods used for enzyme immobilization are generally divided into five classes: adsorption, covalent bonding, entrapment, encapsulation, and cross-linking [9]. Upon the development of immobilization techniques, these four classes have been subdivided [10, 14]).\u003c/p\u003e \u003cp\u003eOne of the most important and widely used techniques for enzyme immobilization is covalent bonding, in which enzymes are attached to a solid carrier that is insoluble in the reaction medium [15]. Covalent bond formation between the enzyme and the matrix occurs between the functional groups in the side chain of the amino acid residues of the enzyme and the reactive groups in the matrix [16]. However, the presence of functional groups on amino acid residues in the active site of enzyme molecules leads to a decrease in the activity of the immobilized enzyme. Therefore, active immobilized enzyme activities can be protected by blocking the functional groups of active site amino acid residues through the addition of the enzyme's substrate [17]. The covalent immobilization method is used when the absence of an enzyme in the product is absolute [18]. Enzyme molecules are attached directly to reactive groups (e.g., amino, amide, carboxyl, and hydroxyl groups) on the support or by a spacer arm that is artificially attached to the matrix through various chemical reactions (e.g., diazotization, Schiff base, and imine bond formation) [19, 20]. Although covalent immobilization generally has several disadvantages, such as limited enzyme mobility, reduced enzyme activity, nonrenewable and less effective for cell immobilization, it has several advantages, such as strong binding, high heat stability, longer storability and reusability hundreds of times [21].\u003c/p\u003e \u003cp\u003eSince the first study on carbon nanotubes (CNTs) was published [21], they have attracted the attention of scientists working in different fields, including enzyme immobilization. As shown in Fig.\u0026nbsp;2.4, CNTs can be classified into single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs) [22\u0026ndash;24]. SWCTs consist of tubes with an outer diameter of 0.7 nm and a single atomic thickness of a single sheet of extruded graphene, while MWCNTs consist of many SWCNTs stacked inside one another, and MWCTs are less than 15 nm in outer diameter and tens of micrometres in length [25]. The large surface area of SWCNTs is more advantageous for their enzyme loading capacity, but MWCNTs are preferred because of their high dispersibility and low cost [26]. Enzymes were immobilized on CNTs via adsorption or covalent binding methods. The optimum enzyme conformations required for activity can be maintained in adsorption, but their durability and activity losses upon separation of enzymes from the matrix are still a concern for industrial applications. Covalent enzyme immobilization can increase stability and activity [24]. Because CNTs have high natural affinity for diverse proteins [25], they are very suitable matrices for enzyme immobilization. The advantages of the use of CNTs as an immobilization matrix can be listed as follows: higher enzyme binding capacity, less diffusion restriction for macromolecular substrates or products, improved thermal stability and increased activity, greater reuse and longer storage stability, high mechanical stability and renewability.\u003c/p\u003e \u003cp\u003eMany studies on the use of c-MWCNTs in enzyme immobilization have been published in the literature. The immobilization by adsorption method is carried out by mixing the enzyme mixture with the buffer mixture containing the matrix and incubating it for an appropriate time [22]. In covalent immobilization, carboxyl groups (-COOH) on c-MWCNTs are first activated with reagents such as N-ethyl-N'-(3-dimethyl amino propyl) carbodiimide hydrochloride (EDAC). Then, through these groups, the enzyme molecules are covalently attached to the matrix [27].\u003c/p\u003e \u003cp\u003eNumerous studies on the immobilization of ANI by different supports and methods have been published in the literature [1, 22, 28\u0026ndash;34]. ANI has already been immobilized on c-MWCNTs by Garlet et al. [23] and Temkov et al. [36]. However, the dimensions of the c-MWCNTs (88% purity, 50\u0026ndash;80 nm length and 10\u0026ndash;20 \u0026micro;m length) used by Garlet et al. [23] are different from those of the c-MWCNTs (99% purity, 28\u0026ndash;48 nm outer diameter and 0.5\u0026ndash;2 \u0026micro;m length) used in this study. Additionally, Temkow et al. [36] immobilized ANI on a nanocomposite (MWCNT/Ppy/PEG) produced by using c-MWCNTs, polypyrrole (PPy), and polyethylene glycol (PEG) not directly on c-MWCNTs. Therefore, the results of the present study differ. Since a 5-fold and even a 12-fold increase in activity was reported in enzyme immobilization studies conducted with C-MWCNTs, the goal was to obtain as high an activity yield as possible by immobilizing the ANI enzyme via the covalent binding method on c-MWCNTs. This study is unique because the ANI enzyme was not immobilized by covalent binding via c-MWCNTs. The goals of this study were to achieve 100% immobilization efficiency, the highest possible activity yield, and the highest possible reusability and storage stability by optimizing the immobilization conditions through the covalent attachment of the ANI enzyme to the commercial immobilization matrix c-MWCNTs.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eANI was supplied as a free sample from BIO-CAT Inc. (Troy, VA, USA), and c-MWCNTs (99% purity, 28\u0026ndash;48 nm outer diameter and 0.5\u0026ndash;2 \u0026micro;m in length) were purchased from Nanografi Co. Ltd. (Ankara, T\u0026uuml;rkiye). Nitrocellulose membrane filters (pore diameter 0.45 \u0026micro;m, membrane diameter 47 mm) were purchased from ISO-LAB (Wertheim, Germany). Inulin from chicory root (DP = ⁓10\u0026ndash;15) was purchased from abcr GmbH (Karlsruhe, Germany). Bovine serum albumin (BSA), sodium hydroxide, sodium dihydrogen phosphate, hydrochloric acid, 3,5-dinitrosalicylic acid (DNSA), sodium potassium tartrate, sodium azide, glutaraldehyde (25%) and Bradford dye were obtained from Sigma‒Aldrich (Taufkirchen, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Methods\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Functionalization of c-MWCNTs\u003c/h2\u003e \u003cp\u003eFifty milligrams of c-MWCNTs were functionalized with glutaraldehyde via incubation in 5 mL of 25 mM sodium phosphate buffer (pH 6.0) containing different concentrations of glutaraldehyde for 0.5 hours at a shaking speed of 150 rpm and labelled F-MWCNTs for further studies. The F-MWCNTs were washed three times with 5 mL of distilled water and 5 mL of sodium phosphate buffer solution under vacuum and filtered through a nitrocellulose membrane filter.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section4\"\u003e \u003ch2\u003e2.2.1.2. Effect of buffer solution pH on functionalization efficiency\u003c/h2\u003e \u003cp\u003eFifty milligrams of c-MWCNTs were functionalized with glutaraldehyde by incubation for 2 hours at room temperature (25\u0026deg;C) in a heated and orbital shaker incubator with shaking at 150 rpm in 5 mL of 25 mM sodium phosphate buffer solution at different pH values containing 0.5 M glutaraldehyde. Fc-MWCNTs were filtered through a nitrocellulose membrane filter, washed thrice with 5 mL of distilled water and thrice with 5 mL of sodium phosphate buffer solution under vacuum.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section4\"\u003e \u003ch2\u003e2.2.1.3. Effect of c-MWCNT and glutaraldehyde reaction times on functionalization efficiency\u003c/h2\u003e \u003cp\u003eFifty milligrams of c-MWCNTs were functionalized with glutaraldehyde via incubation for different durations, heated to room temperature (25\u0026deg;C) and shaken at 150 rpm in an incubator containing 5 mL of 25 mM sodium phosphate buffer (pH\u0026thinsp;=\u0026thinsp;6.0) containing 0.5 M glutaraldehyde. Fc-MWCNTs were filtered through a nitrocellulose membrane filter and washed three times with 5 mL of distilled water and three times with 5 mL of sodium phosphate buffer solution under vacuum.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Immobilization of ANI with Fc-MWCNTs\u003c/h2\u003e \u003cp\u003eImmobilizations were carried out by incubating 100 \u0026micro;L of ANI solution and wet Fc-MWCNTs in 5 mL of sodium phosphate buffer and shaking at 150 rpm in an incubator at room temperature (25\u0026deg;C) for certain times according to the covalent bonding method. At the end of the immobilization, the immobilized enzymes were filtered under vacuum using nitrocellulose membrane filters. After washing three times with 5 mL of 0.1 M phosphate buffer and 3 times with 5 mL of distilled water, the amount of protein in the immobilization buffer and the filtrates was determined. The immobilization yields were calculated from the determined protein amounts. Activity yields were calculated from the relative activities of free and immobilized enzymes determined according to the standard activity determination method.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.4. Effect of the pH of the immobilization buffer on the immobilization efficiency\u003c/h2\u003e \u003cp\u003eImmobilizations were performed by incubating 316 mg wet Fc-MWCNTs (dry weight of 50 mg, average wet‒dry weight ratio of 6.32) with 100 \u0026micro;L free ANI solutions in 5 mL 25 mM sodium phosphate buffer solutions with different pH values (4.0\u0026ndash;5.0\u0026ndash;6.0\u0026ndash;7.0) at room temperature (25\u0026deg;C) in an incubator at 150 rpm with shaking for 60 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.5. Effect of the immobilization buffer concentration on the immobilization efficiency\u003c/h2\u003e \u003cp\u003eImmobilizations were performed by incubating 316 mg of wet Fc-MWCNTs with 100 \u0026micro;L of free ANI solutions in 5 mL of sodium phosphate buffer solutions of different concentrations with a pH of 8.0 at room temperature and shaking at 150 rpm for one hour.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.6. Effect of the amount of c-MWCNTs on immobilization efficiency\u003c/h2\u003e \u003cp\u003eDifferent amounts of wet Fc-MWCNTs were incubated with 100 \u0026micro;L of free ANI solution in 5 mL of 100 mM sodium phosphate buffer (pH 8.0) at 150 rpm at room temperature for one hour while shaking.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section4\"\u003e \u003ch2\u003e2.2.8.7. Effect of immobilization time on immobilization efficiency\u003c/h2\u003e \u003cp\u003eImmobilizations were performed by incubating 632 mg of wet Fc-MWCNTs with 100 \u0026micro;L of free ANI solution in 5 mL of 100 mM sodium phosphate buffer (pH 8.0) at room temperature for different durations while shaking at 150 rpm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section4\"\u003e \u003ch2\u003e2.2.8.8. Confirmation of covalent bonding by the desorption method\u003c/h2\u003e \u003cp\u003eWet Fc-MWCNTs (632 mg) were incubated in 5 mL of 100 mM sodium chloride solution for 4 hours at room temperature with shaking at 150 rpm. At the end of the incubation, protein determination was performed on the filtrates according to Bradford\u0026rsquo;s method (1976). Wet immobilized ANIs were used for activity determination.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Protein (Enzyme) determination\u003c/h2\u003e \u003cp\u003eProtein concentrations were determined according to a previously described method [35] before and after immobilization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Determination of reducing sugars\u003c/h2\u003e \u003cp\u003eThe reducing sugars released or formed were determined via the DNS method [36].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4. Determination of ANI activity\u003c/h2\u003e \u003cp\u003eANI activity was determined by reactions of 100 \u0026micro;L of free ANI solution or 632 mg of wet immobilized ANI with 5 mL of 2% (w/v) inulin solutions under standard conditions. One IU of ANI activity was defined as the amount of enzyme (mg) that produces a reducing sugar equivalent to 1 \u0026micro;mol D-glucose in 1 min in 5 mL of 2% (w/v) inulin solution under standard conditions. ANI activity was calculated via Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e2.1\u003c/span\u003e).\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:Activity=\\:\\frac{Reducing\\:sugars\\:\\left(\\mu\\:mol\\right)}{Ebzyme\\:used\\:\\left(mg\\right)x\\:reaction\\:duration\\:\\left(min\\right)}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2.1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5. Calculation of immobilization and activity efficiencies\u003c/h2\u003e \u003cp\u003eThe immobilization yield was calculated via Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e) from the enzyme amounts in the immobilization solutions before and after immobilization, and the activity yield was calculated via Eq.\u0026nbsp;(\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e2.3\u003c/span\u003e) from the current activities of the free and immobilized enzymes.\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:Immobilization\\:Yield=\\:\\frac{A-B}{A}\\:X\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2.2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eA\u003c/strong\u003e \u003cp\u003eFree enzyme used for immobilization (mg)\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eB\u003c/strong\u003e \u003cp\u003eFree enzyme remaining in the filtrate (mg)\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv id=\"Equ3\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:Activity\\:Yield=\\:\\frac{Ai\\:\\left(IU\\right)}{As\\:\\left(IU\\right)}\\:x\\:100$$\u003c/div\u003e \u003cdiv class=\"EquationNumber\"\u003e2.3\u003c/div\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAi\u003c/strong\u003e \u003cp\u003eCurrent activity of the immobilized enzyme\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAs\u003c/strong\u003e \u003cp\u003eCurrent activity of free enzyme\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.2.6. Quantitative carbohydrate determination by high-performance liquid chromatography (HPLC)\u003c/h2\u003e \u003cp\u003eThe carbohydrate content was determined via high-pressure liquid chromatography (HPLC), a refractive index detector, an amino column, a mobile phase consisting of 70/30% acetonitrile and water by volume and samples with a 20 \u0026micro;L volume [37].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e2.2.7. Characterization of free and immobilized ANIs\u003c/h2\u003e \u003cp\u003eFree and immobilized ANI were characterized by determining their optimum pH, optimum temperature, pH stability, thermal stability and kinetic constants, respectively [38].\u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section4\"\u003e \u003ch2\u003e2.2.7.1. Optimum pH\u003c/h2\u003e \u003cp\u003eThe optimum pH was determined by incubating 100 \u0026micro;L of free ANI and 632 mg of wet immobilized ANI with 5 mL of 25 mM inulin solutions prepared in sodium phosphate buffer at different pH values (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0) at 60\u0026deg;C for 15 min with shaking at 150 rpm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section4\"\u003e \u003ch2\u003e2.2.7.2. Optimum temperature\u003c/h2\u003e \u003cp\u003eThe optimum temperature ranges of the free ANI and the immobilized ANI were determined under standard activity conditions (pH 6.0, duration of 15 min, shaking speed of 150 rpm), with the exception of temperature. Activity determination reactions were performed in the range of 30\u0026ndash;80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section4\"\u003e \u003ch2\u003e2.2.7.3. pH stability\u003c/h2\u003e \u003cp\u003e \u003cb\u003eThe\u003c/b\u003e pH stabilities of the free ANI and the immobilized ANI were determined by incubating the enzyme with sodium phosphate buffer at different pH values (3.0\u0026ndash;8.0) at 60\u0026deg;C for 15 min prior to activity determination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section4\"\u003e \u003ch2\u003e2.2.7.4. Thermal stability\u003c/h2\u003e \u003cp\u003eThe thermal stabilities of the free ANI and the immobilized ANI were determined by incubating the enzyme in sodium phosphate buffer (pH 6.0) for 15 min at different temperatures (30\u0026ndash;80\u0026deg;C) prior to activity determination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section4\"\u003e \u003ch2\u003e2.2.7.5. Kinetic constants\u003c/h2\u003e \u003cp\u003eThe initial activities of the free ANI and the immobilized ANI were determined via incubation in 5 mL of inulin solution (pH 6.0) at different concentrations (5\u0026ndash;600 g/L) at 60\u0026deg;C for 15 minutes in an incubator with shaking at 150 rpm. V\u003csub\u003emax\u003c/sub\u003e and K\u003csub\u003em\u003c/sub\u003e constants were calculated from Lineweaver‒Burk plots.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e2.2.8. Reusability of immobilized ANI\u003c/h2\u003e \u003cp\u003eThe stability of the use of immobilized ANI was determined via a graph drawn using relative activities determined after 20 consecutive uses under standard conditions. Prior to each use, the immobilized ANI was washed with 5 mL of 0.1 M phosphate buffer and with 5 mL of distilled water three times under vacuum using nitrocellulose membrane filters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e2.2.9. Storage stability of immobilized ANI\u003c/h2\u003e \u003cp\u003eThe storage stability of the immobilized ANI was determined via a graph of the relative activities measured according to the standard activity assay method every two days for 20 days. The immobilized enzyme was washed with plenty of distilled water after each use and stored in 5 mL of 0.1 M phosphate buffer solution (pH 6.0) in a refrigerator at +\u0026thinsp;4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e2.3. FOS production using immobilized ANI\u003c/h2\u003e \u003cp\u003eSince the amount of immobilized ANI, inulin concentration and reaction time affect the yield of FOS production, these parameters were changed sequentially, and their effects were determined.\u003c/p\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Effect of the amount of immobilized ANI on FOS production\u003c/h2\u003e \u003cp\u003eDifferent amounts (158, 316, 474, 632, 948, and 1264 mg) of wet immobilized ANI were reacted with 5 mL of 20 g/L inulin solution (pH 6.0) for 15 min at 60\u0026deg;C with shaking at 150 rpm. The FOS and glucose concentrations produced in the reactions were determined via HPLC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Effect of inulin concentration on FOS production\u003c/h2\u003e \u003cp\u003eFive milliliters of inulin solutions (pH 6.0) at different concentrations (5-50-100-200-400-600 g/L) were reacted with 948 mg wet immobilized ANIs at 60\u0026deg;C with shaking at 150 rpm. The FOS and glucose concentrations produced at the end of the reactions were determined via an HPLC device.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Effect of reaction time on FOS production\u003c/h2\u003e \u003cp\u003eFive milliliters of inulin solution (pH 6.0) at a concentration of 600 g/L was reacted with 948 mg wet immobilized ANIs at 60\u0026deg;C for different durations by shaking at 150 rpm. The FOS and glucose concentrations produced at the end of the reactions were determined via an HPLC device.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical analysis and creation of graphics\u003c/h2\u003e \u003cp\u003eThe arithmetic means and standard errors of the experiments repeated three times were calculated via the Microsoft Office Excel 2007 program. Graphs and error bars on the graphs were created via the OriginPro-2019 graphics program.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussions","content":"\u003cdiv id=\"Sec33\"\u003e\n \u003ch2\u003e3.1. Protein (Enzyme) Determination\u003c/h2\u003e\n \u003cp\u003eThe calculations were performed according to the correct equation of the standard BSA graph, which revealed that 1.17 mg of enzyme was present in the 5.1 mL reaction mixture. Since 0.1 mL of free enzyme (liquid enzyme preparation) was used in the reaction, the enzyme concentration of the liquid enzyme preparation was 11.7 mg/mL.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec34\"\u003e\n \u003ch2\u003e3.2. Determination of ANI activity\u003c/h2\u003e\n \u003cp\u003eAs a result of the calculations performed according to the correct equation of the standard D-glucose standard graph, 38001.79 µg D-glucose was calculated to have formed in the 5.1 mL reaction mixture. When we divided this value by 180 g, which is the molar mass of glucose, the resulting D-glucose content was 211.12 µmol. According to Eq.\u0026nbsp;(2.1), the free ANI activity was calculated as 140.75 IU/mL. Since there is 11.7 mg of free ANI in 1 mL, the specific activity of free ANI can also be expressed as 12.03 IU/mg. Accordingly, the amount of free instant ANI with 1 IU of activity was found to be 0.083 mg.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec35\"\u003e\n \u003ch2\u003e3.3. Optimization of Functionalization Conditions\u003c/h2\u003e\n \u003cdiv id=\"Sec36\"\u003e\n \u003ch2\u003e3.3.1. Effect of glutaraldehyde concentration on functionalization efficiency\u003c/h2\u003e\n \u003cp\u003eAs shown in Table\u0026nbsp;1, the activity yield decreased while the immobilization efficiency increased as the GA concentration increased. The increase in immobilization yield with increasing GA concentration can be explained by the fact that more ANI molecules are attached to the matrix. It is well known that at high GA concentrations, the binding of enzyme molecules to the matrix at multiple points causes a loss of activity by disrupting the three-dimensional structure of the enzyme [11, 39–42]. In addition, as a result of the crowding of the immobilized enzyme molecules on the matrix, the enzyme molecules prevent the substrate from reaching the active site of the enzyme [43]. Since the highest activity efficiency was obtained when 0.5 M GA was used, the most suitable glutaraldehyde concentration for functionalization was 0.5 M. Similarly, in another study, the highest efficiency in the immobilization of \u003cem\u003eK. lactis\u003c/em\u003e β-galactosidase with MWCNTs was obtained when 0.5 M GA was used [44].\u003c/p\u003e\n \u003cdiv\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec37\"\u003e\n \u003ch2\u003e3.3.2. Effect of buffer solution pH on functionalization efficiency\u003c/h2\u003e\n \u003cp\u003eAs shown in Table\u0026nbsp;2, the immobilization efficiency increased with increasing pH. This result shows that with increasing pH, more glutaraldehyde binds to the matrix, and as a result, more enzyme molecules are immobilized. According to the table, the activity yield increases up to pH 6 and decreases at higher pH values. This decrease is probably due to the degradation of the three-dimensional structure of the enzyme molecules as a result of binding at multiple points with increasing GA concentration in the matrix [39, 45].\u003c/p\u003e\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eEffect of buffer solution pH on functionalization efficiency\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBuffer Solution pH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eImmobilization Efficiency (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eActivity Yield (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.63 ± 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.31 ± 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.36 ± 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.61 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.12 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.06 ± 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.13 ± 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.48 ± 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.56 ± 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.19 ± 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec38\"\u003e\n \u003ch2\u003e3.3.3. Effect of c-MWCNT and glutaraldehyde reaction times on functionalization efficiency\u003c/h2\u003e\n \u003cp\u003eAs shown in Table\u0026nbsp;3, as the reaction time increased, the immobilization efficiency and activity efficiency increased. This result is quite natural because as time progresses, more GAs are connected.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eEffect of c-MWCNT and glutaraldehyde reaction times on functionalization efficiency\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReaction Time (Hours)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eImmobilization Yied (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eActivity Yield (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.39 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.06 ± 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54.89 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.54 ± 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57.35 ± 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.13 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60.12 ± 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.88 ± 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec39\"\u003e\n \u003ch2\u003e3.4. Optimization of the Immobilization Conditions\u003c/h2\u003e\n \u003cdiv id=\"Sec40\"\u003e\n \u003ch2\u003e3.4.1. Effect of buffer solution pH on immobilization efficiency\u003c/h2\u003e\n \u003cp\u003eTable\u0026nbsp;4 shows that as the pH of the immobilization solution increased, the immobilization efficiency and activity efficiency increased. This result shows that with increasing pH, amino groups in the side groups of amino acids in the structure of the enzyme shift more into the unprotonated form (NH\u003csub\u003e2\u003c/sub\u003e), which is suitable for covalent bonding. GA primarily reacts with amino groups, especially at moderate pH values [46, 47]. The increase in immobilization efficiency from acidic pH to moderate pH confirms this information.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 4\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eEffect of buffer solution pH on immobilization efficiency\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBuffer pH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eImmobilization Yield (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eActivity Yield (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.22 ± 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.14 ± 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.24 ± 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.04 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56.55 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.49 ± 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62.64 ± 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.60 ± 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.26 ± 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.84 ± 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec41\"\u003e\n \u003ch2\u003e3.4.2. Effect of buffer solution concentration on immobilization efficiency\u003c/h2\u003e\n \u003cp\u003eAccording to Table\u0026nbsp;5, buffer solution concentrations higher than 50 mM did not affect the immobilization efficiency but increased the activity efficiency. According to this result, while the amount of enzyme bound to the matrix does not change with increasing concentration, the increase in activity efficiency shows that increasing the concentration of salt ions helps to preserve the three-dimensional structures of the enzyme molecules attached to the matrix. The three-dimensional structures of enzymes are highly dependent on the concentration of salt ions in the environment [39, 48].\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 5\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eEffect of buffer solution concentration on immobilization efficiency\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBuffer Concentration (mM)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eImmobilization Yield (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eActivity Yield (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.26 ± 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.84 ± 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76.66 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.98 ± 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76.66 ± 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.55 ± 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76.66 ± 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.33 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec42\"\u003e\n \u003ch2\u003e3.4.3. Effect of wet Fc-MWCNT amount on immobilization efficiency\u003c/h2\u003e\n \u003cp\u003eAs shown in Table\u0026nbsp;6, the immobilization efficiency and activity efficiency slightly increased when the amount of wet Fc-MWCNTs was doubled. However, when higher amounts of Fc-MWCNTs were used, the immobilization efficiency slightly increased, and the activity efficiency decreased. The reason for this may be the destruction of the three-dimensional structures of enzyme molecules, which are necessary for activity, as a result of multipoint attachment of the enzyme molecules to the matrix by increased GA groups on the matrix [39, 45].\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 6\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eEffect of wet Fc-MWCNT amount on immobilization efficiency\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWet Fc-MWCNT Amount (mg)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eImmobilization Yield (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eActivity Yield (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e316\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76.66 ± 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.33 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e632\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81.88 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.99 ± 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e948\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85.53 ± 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.67 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1264\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.16 ± 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.75 ± 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec43\"\u003e\n \u003ch2\u003e3.4.4. Effect of immobilization time on immobilization efficiency\u003c/h2\u003e\n \u003cp\u003eAs shown in Table\u0026nbsp;7, the immobilization efficiency increased with increasing immobilization time. After four hours, 100% immobilization and 82.60% activity efficiency were obtained. The activity yields obtained in previous studies with different matrices and methods were 66% [28], 145% [29], 83% [31], 81% [49], 400% [30], 100% [32], 92% [33], and 60.7% [50]. The results obtained in our study are greater than those of other studies, although they are below the results of de Oliveira Kuhn et al. [30] and de Oliveira Kuhn et al. [31]. Since the activity efficiency of the covalent bonding method is generally less than 100%, the activity yield we obtained in this study is sufficient for industrial production.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab7\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 7\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eEffect of immobilization time on immobilization efficiency\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDuration (Hour)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eImmobilization Yield (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eActivity Yield (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81.88 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.99 ± 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.22 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.70 ± 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.09 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.19 ± 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.00 ± 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82.60 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec44\"\u003e\n \u003ch2\u003e3.5. Confirmation of covalent bonding by the desorption method\u003c/h2\u003e\n \u003cp\u003eIn accordance with previous methods [51], 632 mg of immobilized ANI was incubated in 5 mL of 100 mM sodium chloride solution for the immobilization period (4 hours) at room temperature (25°C) in a heated incubator with shaking at 150 rpm. At the end of the incubation, the immobilized enzymes were washed thrice with 5 mL of distilled water and thrice with 5 mL of sodium phosphate buffer solution under vacuum and filtered through a nitrocellulose membrane filter. As shown in Table\u0026nbsp;8, the absence of protein in the filtrates and the absence of any decrease in the activities of the immobilized enzymes indicate that immobilization is achieved by covalent bonding.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab8\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 8\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eConfirmation of covalent bonding by desorption\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDesorption\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eImmobilization Yield (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eActivity Yield (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBefore\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.00 ± 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82.60 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAfter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.00 ± 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82.60 ± 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec45\"\u003e\n \u003ch2\u003e3.6. Characterization of Free and Immobilized ANI\u003c/h2\u003e\n \u003cdiv id=\"Sec46\"\u003e\n \u003ch2\u003e3.6.1. Optimum pH\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig.\u0026nbsp;1, immobilization did not change the optimum pH range (5.5–6.5) of ANI. In addition, the activity of the immobilized ANI was greater than that of its free counterpart at acidic and alkaline pH values. For example, at pH 3 and 8, free ANI has only 60% of its activity, whereas immobilized ANI has 70% and 75% of its activity, respectively, at the same pH. Singh et al. (2019) reported that the optimum pH (5.0) did not affect the immobilization of ANI. This result can be attributed to the stability of the carbon nanotubes, which is not affected by the changing ionic microenvironment [50]. Furthermore, the unchanged optimum pH indicates that no significant conformational changes occur in the enzyme molecules after immobilization [52]. Moreover, the higher activity of immobilized ANI at acidic and alkaline pH values is probably the result of increased stability resulting from multipoint attachment during immobilization [53–55]. On the other hand, immobilization protects the three-dimensional structure of the enzyme responsible for the activity and prevents the loss of activity under severe conditions [14, 56].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec47\"\u003e\n \u003ch2\u003e3.6.2. Optimum temperature\u003c/h2\u003e\n \u003cp\u003eFigure\u0026nbsp;2 shows that the optimum temperature range (55–65°C) was not affected by immobilization. The immobilized ANI clearly has higher activity than its free counterpart over the entire temperature range tested. The optimum temperature of ANI agrees with previous studies [28, 37, 50]. It is well known that immobilized enzymes exhibit greater activity than free enzymes at high temperatures due to the limited conformational mobility of molecules following immobilization [55] as a result of interactions between the enzyme and the support [57].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec48\"\u003e\n \u003ch2\u003e3.6.3. pH stability\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig.\u0026nbsp;3, immobilized ANI has greater stability over a wider range than free ANI. Free ANI is most stable from pH 5.0–6.5, whereas immobilized ANI has the highest stability in the range of 4.5–7.0. In addition, compared with free ANI, immobilized ANI appears to have greater stability in the low and high pH ranges tested. For example, free ANI lost 22.5% and 25% of its activity at pH 3.0 and 8.0, respectively, whereas immobilized ANI lost only 20% of its activity at the same pH. The increase in the pH stability of ANI after immobilization may be due to the strong covalent interaction between the carbon nanotubes and the enzyme moiety. It is important for industrial processes that an enzyme has a stable-integral conformation over a wide pH range [50].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec49\"\u003e\n \u003ch2\u003e3.6.4. Thermal stability\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig.\u0026nbsp;4, immobilization increased the thermal stability of ANI. While free ANI lost more than 10% of its activity at 70°C, immobilized ANI retained its entire activity at the same temperature. Additionally, while free ANI lost 80% of its activity at 80°C, immobilized ANI retained more than 60% of its activity at the same temperature. The increase in the thermal stability of inulinase after immobilization is a result of the good interaction between the carbon nanotubes and inulinase [50]. Carbon nanotubes themselves can withstand a longer period of time at elevated temperatures without any change in their tensile strength or elastic modulus. Therefore, the biocatalyst immobilized on these materials eventually also achieves this thermal stability property. Although an increase in the thermal stability of inulinases up to 60°C has been reported for other matrices, such as kaolin clay [58], chitin [59], and nonwoven fabrics [60], the reported increases are lower than those of ANI immobilized on c-MWCNTs in our present study.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec50\"\u003e\n \u003ch2\u003e3.6.5. Kinetic constants\u003c/h2\u003e\n \u003cp\u003eA Lineveawer–Burk plot of free and immobilized ANI is shown in Fig.\u0026nbsp;5. According to the data obtained from the graph, the maximum velocities (V\u003csub\u003emax\u003c/sub\u003e) obtained for free and immobilized ANI are 671.1 µmol/mg.min and 568.2 µmol/mg*min, respectively. The Michaelis–Menten constant (km) was also calculated as 662.3 g/L and 699.3 g, respectively. As K\u003csub\u003em\u003c/sub\u003e increases, the enzyme's affinity for the substrate decreases [61]. These results confirm the data obtained during immobilization. An activity efficiency of 82.6% was obtained via immobilization. The ratio of the maximum speeds is approximately the same (82.4%). The decrease in the maximum rate is due to the decreased affinity of ANI for its substrate inulin. The reason for this may be the partial disruption of the conformation of the catalytic groups in the active site of the enzyme, which is suitable for activity, as a result of the formation of strong covalent bonds between the enzyme molecules and the matrix.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec51\"\u003e\n \u003ch2\u003e3.6.6 Reusability of immobilized ANI\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig.\u0026nbsp;6, immobilized ANI did not lose its activity during 20 repeated uses. The stability of immobilized enzymes is very important for industrial applications. The product cost decreases inversely with the number of reuses [14, 56]. According to previous studies on the immobilization of ANI, ANI immobilized on c-MWCNTs by the covalent bonding method in this study clearly has the best usage stability. Karimi et al. [33], in their immobilization study of ANI with 50-, 100- and 200-nm amino functional silica NPs, reported that immobilized ANI lost 17% of its initial activity after 7 uses. de Oliveira Kuhn et al. (2016) reported that ANI immobilized in polyurethane foam retained approximately 49% of its initial activity after 24 uses. In another study, ANI 12 covalently immobilized on Fe3O4 magnetic nanoparticles functionalized with wheat gluten hydrolysates (WGHs), by Torabizadeh and Mahmoudi (2018), retained 70% of its initial activity after use. Finally, ANI, which was covalently immobilized on hydroxylated MWCNTs activated with 3-aminopropyl-triethoxysilane (APTES) by Singh et al. (2019), retained only 28% of its initial activity after 10 consecutive uses.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec52\"\u003e\n \u003ch2\u003e3.6.7. Storage stability of immobilized ANI\u003c/h2\u003e\n \u003cp\u003eThe immobilized ANI maintained its activity for 30 days under storage conditions (in 0.1 M sodium phosphate buffer with pH 6.0 at + 4°C) (Fig.\u0026nbsp;7). When this result is compared with previous results in the literature, ANI immobilized on c-MWCNTs by the covalent bonding method in this study has a very good advantage for industrial applications. In the study by Yewale et al. (2013), immobilized ANI retained its initial activity for 6 days. The initial activity of ANI immobilized by the adsorption method on c-MWCNTs (purity 88%, diameter 50–80 nm, length 100–200 nm) was preserved for five weeks at room temperature [22]. In another study, ANI immobilized in polyurethane foam retained only 49% of its initial activity for 42 days [30]. Finally, ANI immobilized on micro polyhydroxy butyrate (PHB) fibres retained its initial activity for 3 months when stored in dry conditions [49]. Compared with these results, the immobilized ANI obtained in this study clearly has very high storage stability.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec53\"\u003e\n \u003ch2\u003e3.7. Optimization of the FOS Production Conditions\u003c/h2\u003e\n \u003cdiv id=\"Sec54\"\u003e\n \u003ch2\u003e3.7.1 Effects of the amount of immobilized ANI on FOS production\u003c/h2\u003e\n \u003cp\u003eFigure\u0026nbsp;8 shows that as the amount of immobilized ANI increased, the total FOS also increased. The concentrations of the FOS components kestose, nystose and frucofuranosyl nystose and glucose, which are byproducts, first increased linearly and then remained constant. For example, when the amount of immobilized ANI was 158 mg, the total FOS concentration was approximately 6 g/L; when the amount of immobilized ANI was increased to 948 mg, the FOS concentration increased to approximately 18 g/L. Additionally, when 1264 mg of immobilized ANI was used, the FOS concentration did not change. According to these results, the optimum amount of immobilized enzyme for FOS production was 948 mg, and this amount of immobilized enzyme was used in subsequent experiments.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec55\"\u003e\n \u003ch2\u003e3.7.2 Effects of inulin concentration on FOS production\u003c/h2\u003e\n \u003cp\u003eFigure\u0026nbsp;9 shows that the relationship between FOS production and inulin concentration is in accordance with the classical Michaelis‒Menten graph. For example, when 5 g/L inulin solution was used, the total FOS concentration was approximately 2 g/L. Furthermore, when 200, 400 and 600 g/L inulin were used, the total FOS concentration also increased to 30 g/L, 32 g/L, and 36 g/L, respectively, and then remained stable at 36 g/L. According to these results, the most suitable inulin concentration was 600 g/L, and an inulin solution at this concentration was used in the next experiment.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec56\"\u003e\n \u003ch2\u003e3.7.3 Effects of reaction duration on FOS production\u003c/h2\u003e\n \u003cp\u003eWhen Fig.\u0026nbsp;10 is examined, all the FOS components increase linearly until the 16th hour, and then, the rate of increase slows down and stabilizes at the 20th hour. By optimizing the FOS production conditions, after 20 hours of reaction, FOS was obtained at a concentration of 546.9 g/L with a conversion rate of 91.15%. This ratio achieved in FOS production from inulin is the highest for industrial FOS production compared with the results obtained in previous studies (200 g/L–540 g/L) [1, 22, 28–33].\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, by optimizing the conditions, ANI was covalently immobilized onto c-MWCNTs with 100% binding yield and 82.6% activity yield. Immobilization did not change the optimum pH range (5.5\u0026ndash;6.5) or the optimum temperature range (55\u0026ndash;65\u0026deg;C) of ANI. Immobilization increased the km constant from 662.3 g/L to 699.3 g/L but decreased the Vmax from 671.1 \u0026micro;mol/mg/min to 568.2 \u0026micro;mol/mg/min. The initial activity of the immobilized enzyme did not decrease during 20 cycles of use or during storage for 30 days under optimum conditions, indicating high reusability and storage stability. By using immobilized ANI, FOS syrup at a concentration of 546.9 g/L was produced from 600 g/L inulin solution, with a conversion rate of 91.15% after 20 hours. Compared with previous studies in the literature, the FOS rate obtained in this study was the highest. Consequently, the immobilized ANI obtained in this study can be used in the industrial production of FOS syrup from inulin.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCredit authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYakup Aslan: Writing \u0026ndash; review \u0026amp; editing, validation, supervision, resources, project administration, methodology, original draft, investigation, funding acquisition, and software conceptualization. İpek Alper: Visualization, Methodology, Investigation, Formal analysis, Data curation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWork was carried out within the scope of the project code 219O034, which was financially supported by The Scientific and Technological Research Council of T\u0026uuml;rkiye in 2020.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eR.P. de Souza Oliveira, P. 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Segel (No Title), (1976)\u003c/li\u003e\n\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aspergillus niger, inulin, inulinase, covalent immobilization, enzyme, fructooligosaccharides","lastPublishedDoi":"10.21203/rs.3.rs-6305427/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6305427/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, ANI was immobilized on c-MWCNTs via the crosslinker reagent glutaraldehyde via a covalent bonding method with 100% binding yield and 82.6% activity yield. While immobilization did not change the optimum pH range (5.5\u0026ndash;6.5) or optimum temperature range (55\u0026ndash;65\u0026deg;C) of the enzyme, it improved the pH and thermal stability of the enzyme. The V\u003csub\u003emax\u003c/sub\u003e values obtained for the free and immobilized enzymes were 671.1 \u0026micro;mol/mg/min and 568.2 \u0026micro;mol/mg/min, respectively, and the K\u003csub\u003em\u003c/sub\u003e values were 662.3 g/L and 699.3 g/L, respectively. The initial activity of the immobilized enzyme was maintained under optimum activity conditions for 20 consecutive uses and for 30 days under optimum storage conditions. Using immobilized ANI, FOS syrup was obtained at a concentration of 546.9 g/L from 600 g/L inulin solution with a 91.15% conversion ratio. Consequently, the immobilized ANI enzyme obtained in this study can be used for the production of FOS from inulin in industry.\u003c/p\u003e","manuscriptTitle":"Improved stability of Aspergillus niger inulinase (ANI) by covalent immobilization using glutaraldehyde on carboxylated multiwalled carbon nanotubes (c-MWCNTs)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-27 07:36:08","doi":"10.21203/rs.3.rs-6305427/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bcdaf07b-74f3-4232-b5f5-53258dc8d5f0","owner":[],"postedDate":"March 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-13T07:38:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-27 07:36:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6305427","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6305427","identity":"rs-6305427","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}