Facile Immobilization of Enzyme via Co-Electrospinning: A Simple

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Facile Immobilization of Enzyme via Co-Electrospinning: A Simple Method for Enhancing Enzyme Reusability and Monitoring an Activity-Based Organic Semiconductor Musab M. Aldhahri,*,†,‡ Yaaser Q. Almulaiky,§ Reda M. El-Shishtawy,∥,⊥ Waleed Al-Shawafi,‡ Ahmed Alngadh,# and Rayan Maghrabi¶ †

Center of Nanotechnology, King Abdulaziz University, P.O. Box 80216, Jeddah 21589, Saudi Arabia Department of Biochemistry, ∥Department of Chemistry, and ¶Department of Biochemistry, King Abdulaziz University, P. O. Box 80200, Jeddah 21589, Saudi Arabia § Department of Biochemistry, Faculty of Science, University of Jeddah, P.O.Box 80203, Jeddah 21589, Saudi Arabia ⊥ Department of Dyeing, Printing and Textile Auxiliaries, National Research Centre, Dokki, 71516 Cairo, Egypt # King Abdulaziz City for Science and Technology, P.O. Box 6086, Riyadh 11442, Saudi Arabia

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‡

S Supporting Information *

ABSTRACT: The stability, reusability, and monitoring of enzyme activity have been investigated to improve their efficiency for successful utilization in a broad range of industrial and medical applications. Herein, we present a simple method for fabricating an electrospun fiber/enzyme scaffold via coelectrospinning. The characterization of soluble and immobilized α-amylases with regard to pH, thermal stability, and reusability were studied. An organic light emitting material tris(8-hydroxyquinoline)aluminum was incorporated to monitor the enzyme activity for several reuses.

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INTRODUCTION The tremendous potential of enzymes as green versatile biocatalysts has been previously reported.1 Enzymes consist of a complex three-dimensional structure of proteins with a high degree of specificity. This specificity, as well as the capability, energy efficiency, and cost-effective properties of enzymes, allow them to perform as catalysts.2 This function results in their use in a wide range of applications including pharmaceutical synthesis, biosensing, food processing, and bioremediation.3,4 However, biocatalysts that are composed of free enzymes may exhibit structural changes because of processing conditions, such as pH, temperature, chemical agents, and ionic strength.5−7 These process variables can limit the activity, stability, and reusability of the enzymes. To overcome these deficiencies, various methodologies have been employed to improve biocatalyst functionality, stability, and reusability.8 Enzyme immobilization has emerged as a promising and viable method for achieving better performance with little or no limitations.9 The results of immobilization primarily depend on the composition, structure, and type of immobilizing substrates.10 Different nanomaterials have been used as substrates, including porous silica, nanotubes, and magnetite nanoparticles.11,12 These nanostructures tend to be good supports because of their high aspect ratio. These © 2018 American Chemical Society

characteristic features of nanomaterials will result in efficient immobilization. However, some of these nanostructures are hampered by their morphology, which affects the enzymatic process. For example, enzyme that was immobilized on porous silica reduced its internal surface, which affected the diffusion of the enzymatic reaction and resulted in lower enzyme activity. In addition, the dispersion and reusability of nanoparticles and nanotubes can be difficult, which adversely affects their use as immobilizing substrates.10 In recent years, the use of the electrospinning technique has received considerable attention for fabricating nanofibers with well-defined lengths, diameters, and compositions.12 These predesigned nanofibers would ensure their applications in various fields, such as reinforcement,13 filtration,14,15 biomedical, and pharmaceutical applications16 as well as electronic and optical devices.17,18 In biocatalysis, electrospun nanofibers have attracted considerable interest because of its distinctive characteristics and superiority in many aspects. The advantages of this method include the multiple points of attachment to the electrospun fiber support, which can restrict undesirable Received: March 1, 2018 Accepted: June 4, 2018 Published: June 13, 2018 6346

DOI: 10.1021/acsomega.8b00366 ACS Omega 2018, 3, 6346−6350

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Figure 1. (a) Schematic representation of the fabrication process. (b) FESEM images of (i) pristine Carbothane, (ii) Carbothane/enzyme blend, and (iii) Carbothane/enzyme/Alq3.

under the electrospinning system fan (NANON, MECC, Japan) overnight to remove any remaining solvent prior to use. The as-prepared scaffold was irradiated with a UV lamp in dark environment to demonstrate the presence of Alq3. Figure 1b shows a detailed morphological view of the fabricated scaffold using a field emission scanning electron microscope (FESEM; JEOL-7600F). As shown in Figure 1b(i), the pure Carbothane fibers have a high surface area with a clear morphology and a semiuniform size. In comparison to Figure 1b(i), the surface of the produced fibers gradually changed from smooth and clear to rough because of the encapsulation of αamylase as shown in Figure 1b(ii). After the addition of Alq3, the fiber surface became rougher with a slightly large diameter and no beads, as shown in Figure 1b(iii). The activity of α-amylase was determined spectrophotometrically at 560 nm by measuring the released maltose from starch according to the Miller method (1959) with a slight modification. A mixture, which consisted of 125 μL of 2% soluble starch, 125 μL of 50 mM sodium acetate buffer (pH 6.5), an appropriate amount of enzyme solution, and distilled water to achieve 0.5 mL volume in the tubes was incubated at 37 °C for 30 min, followed by the addition of 0.5 mL of the dinitrosalicylic acid reagent to terminate the reaction. The immobilized enzyme was removed from the reaction mixture and washed with distilled water. Next, the reaction mixture was incubated in a boiling water bath for 10 min and cooled. Then, the absorption was measured. The reusability of the immobilized enzyme and the enzyme activity were evaluated after repeated enzyme assaying and washing with water to remove the substrate and products. As shown in Figure 2, the immobilized enzyme demonstrated 30% of its initial activity after 10 cycles, indicating that the immobilized α-amylase has suitable stability and can be reused. Similar enzyme reusability results have been reported in previous studies.26,27 It is important to note that the applied electrical current (19 kV) had no effect on the catalytic activity of α-amylase enzyme. Moreover, a similar study reported that a direct electric current exhibited less effect on the laccase enzyme, and the laccase and electric field exhibited a better response to practical environment changes.28 Figure 3a shows the recorded photoluminescence (PL) emission spectra of Alq3 after nine cycles in the reusability

conformational changes in the biocatalytic enzymes. Enzyme immobilization on electrospun fibers can promote and maintain the natural catalytic activity and allow enzyme separation from the reaction medium for recycling.19 Several studies have reported different approaches for immobilizing enzymes on electrospun nanofibers. These approaches include the enzyme binding onto the surface of nanofibers as well as nanofiber−enzyme encapsulation.11,20 Covalent binding of an enzyme with nanofibers requires fabricating nanofibers that contain reactive groups. However, the resulting immobilized enzyme exhibits a reduced enzyme activity over time. This phenomenon may be due to the interaction between the immobilized enzyme and the supporting materials.21 In addition, the covalent attachment of enzymes onto nanofibers results in the formation of a monolayer on the fiber surface, which limits enzyme loading.22 In contrast, the co-electrospinning method offers an easy route for the formation of enzyme encapsulation into nanofibers with good efficiency.23 However, several concerns regarding this method remain (e.g., the aqueous solubility of the supporting materials, conformational change of the enzymes due to applied voltage, and organic solvent effects24,25). In this study, the α-amylase enzyme encapsulation approach via co-electrospinning was applied, and an organic light emitting material (Alq3) was incorporated during the electrospinning process Figure 1. This method enables low enzyme loading, high enzyme activity, and reusability. Alq3 fluorescence was used to monitor the enzyme activity for several cycles.

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RESULTS AND DISCUSSION Figure 1a shows a schematic representation of the initial preparation process for the enzyme and Alq3 scaffold substrate. 1 wt % of the α-amylase powder was mixed with Carbothane polymer in ethanol. 1 wt % of the Alq3 powder was dispersed in ethanol using ultrasonic irradiation. The two solutions were mixed to prepare a homogenous mixture prior to electrospinning. The final solution was placed in a 5 mL syringe. An electrical field of 19.5 kV over a distance of 15 cm was applied. The flow rate of the copolymer composite was set to 0.9 mL/h. The composite was electrospun for 3 h to achieve a thickness of approximately 450−500 μm. The electrospun sheet was dried 6347

DOI: 10.1021/acsomega.8b00366 ACS Omega 2018, 3, 6346−6350

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the effect of the reaction products in the quenching process (see Supporting Information Figure S1). Panel (b) shows different confocal images of the scaffold after each measurement. As shown in the images, the PL emission intensity of Alq3 was gradually quenched after each reusability experiment. Panel (c) shows a plot of the relationship between the Alq3 PL emission and the number of repeated enzyme experiments. The Alq3 PL retains 70% of its PL intensity after the first reaction experiment. The Alq3 PL intensity gradually decreased based on the number of enzyme reusability experiments. Figure 4 shows assessments of the pH effect on the free and immobilized enzyme range from 4.0 to 9.0. The pH of free and Figure 2. Determination of α-amylase activity and reusability.

experiments. A confocal microscope (Carl-Zeiss LSM 780) was used for this experiment. Organometallic derivatives of 8hydroxyquinoline, such as Alq3, are highly fluorescent materials due to the metal ion, which changes the energy levels and causes charge transfer from the metal ion to the aromatic rings.29 Therefore, any changes in the order of Alq3 and/or its interaction with surrounding molecules may increase the nonradiative process with PL quenching. The Alq3 peak position was observed at approximately 519 nm in all recorded samples. The emission spectrum of Alq3 results from its 8hydroxyquinoline ligands.30 The position of this peak is close to that previously reported.31,32 The corresponding PL emission intensity of Alq3 quenches after repeating the enzyme reactions. The PL emission spectrum of Alq3 in the absence of the enzyme was compared with the current experiment. In this case, the PL emission of Alq3 was nearly stable with minor fluctuations during all repeated measurements. Because the pH of the experiment was adjusted at 6.5, the quenching of Alq3 may be attributed to the hydrolyzed reaction products (glucose and maltose) that were produced during the enzymatic process. Glucose and/or maltose may penetrate the scaffold and become adhered to the Alq3 particles causing disorder and changes the energy levels. To confirm this hypothesis, the prepared scaffold was individually placed in two different solutions (glucose and maltose). As expected, the Alq3 PL was quenched, indicating

Figure 4. Optimum pH of free and immobilized α-amylase. Each point represents the average of two experiments.

immobilized α-amylase was shifted from 6.0 to 6.5, respectively. In the immobilized sample, the α-amylase enzyme by virtue of its functional groups could be physically bonded within the scaffold through multiple connections.33 These bonds created between α-amylase enzyme and the scaffold lead to different environmental changes in response to the effect of pH compared with the free enzyme.34 Figure 5 illustrates the effect of temperature ranges from 30 to 80 °C for the free and immobilized enzymes. The optimum temperature for the free and immobilized α-amylase was 40 and 50 °C, respectively. The free α-amylase enzyme exhibited around 40% of the activity, whereas immobilized α-amylase

Figure 3. (a) Schematic representation of the fabrication process. (b) FESEM images of (i) pristine Carbothane, (ii) Carbothane/enzyme blend, and (iii) Carbothane/enzyme/Alq3. 6348

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Figure 5. Effect of temperature on free and immobilized α-amylase. Each point represents the average of two experiments.

retained 60% of the activity at 70 °C. Results having the similar changes with optimum temperature changes were reported in previous studies.35 Our results highlight the potential of using the coelectrospinning approach as a feasible method for enzyme immobilization. This method appears to preserve the enzyme activity. Alq3, which is a molecular organic semiconductor that was incorporated inside the polymer scaffold, is capable of recognizing biochemical interactions. The PL intensity was quenched as the product concentration produced by the enzyme-catalyzed reaction increased. This study signifies a new approach for preparing organic light emitting materials for unprecedented biochemical sensing applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00366. PL spectrum of the prepared Alq3 scaffold after placing in glucose and maltose (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (M.M.A.). ORCID

Musab M. Aldhahri: 0000-0001-5216-3766 Reda M. El-Shishtawy: 0000-0002-7744-529X Author Contributions

M.M.A. and Y.Q.A. conceived and designed the experiments and analyzed the data; W.A.-S., M.A., Y.Q.A., and R.M. performed the experiments; R.M.E.-S., A.A. contributed reagents/materials; M.M.A., Y.Q.A. and R.M.E.-S. wrote the paper; All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the director of the Center of Nanotechnology at King Abdulaziz University for the great support and free access to all instruments.

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REFERENCES

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DOI: 10.1021/acsomega.8b00366 ACS Omega 2018, 3, 6346−6350