Electrospun Polycaprolactone–Chitosan Nanofiber Scaffolds for Covalent Immobilization of Xylanase: Structural Characterization and Enzyme Performance

Electrospun Polycaprolactone–Chitosan Nanofiber Scaffolds for Covalent Immobilization of Xylanase: Structural Characterization and Enzyme Performance | 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 Electrospun Polycaprolactone–Chitosan Nanofiber Scaffolds for Covalent Immobilization of Xylanase: Structural Characterization and Enzyme Performance Tuğba Doğan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7775653/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Xylanases are critical enzymes that release xylose and its derivatives through the hydrolysis of xylan, the primary component of hemicellulose, and are of great importance to many industrial fields. However, there are several factors that limit their use in free form. Enzyme immobilization stands out as an important strategy to overcome these limitations. In this study, PCL/CHI nanofibers were synthesized by electrospinning, and an innovative nanocarrier platform was developed for Xylanase immobilization. A solution containing 10% PCL and 0.5% CHI was converted into nanofibers by electrospinning under optimized conditions (22 kV, 1 mL/h, 18 cm) and then cross-linked with glutaraldehyde to make them suitable for enzyme immobilization. SEM, EDX, XRD, and FT-IR analyses confirmed the morphological and structural integrity of the nanofibers. When comparing free and immobilized xylenese, the optimum temperature was determined as 50°C for both forms, while the optimum pH was determined as 6 for the free form and 5 for the immobilized form. The decrease in activation energy from 21.46 kJ/mol to 1.17 kJ/mol in the immobilized form indicated that the reaction occurred with a lower energy barrier. Furthermore, the decrease in Km value revealed that immobilization enhanced enzyme-substrate interaction, while reusability tests showed that the immobilized enzyme retained 45% of its initial activity after five cycles. The fact that the immobilized form maintained its high catalytic performance in the presence of metal ions highlights the system's potential for adaptation to industrial conditions. In conclusion, this developed platform has been demonstrated to be a promising approach for sustainable and economical solutions in enzyme technologies. Xylanases Enzyme Immobilization Electrospinning Nanofibers Biocatalysts Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction The Xylanase (endo-1,4-β-Xylanase; EC 3.2.1.8) are enzymes classified primarily in glycoside hydrolase (GH) families GH10 and GH11 that hydrolyze the β-1,4 glycosidic bonds of xylan, the major component of plant cell wall hemicellulose, to form xylo-oligosaccharides and xylose; this classification is associated with differences in catalytic architecture and substrate specificity [ 1 ]. Xylan is a complex polymer that interacts with cellulose and lignin, exhibiting branching and substituent diversity in the arabinoxylan structure, particularly in cereal straws and woody tissues. This structural heterogeneity contributes to the recalcitrance of biomass and determines the sensitivity of Xylanase activity to processing conditions [ 2 ]. The industrial applications, Xylanase play an important role in reducing chemical consumption and environmental burden by increasing the efficiency of biological pre-bleaching and bleaching agents in the paper/pulp industry; in improving pulp rheology, juice clarification[ 3 ] and optimizing fiber functionality in food technology; in increasing digestive efficiency by degrading non-starch polysaccharides in feed additives; and in converting lignocellulosic residues to sugars in biorefinery/biofuel lines [ 4 ]. However, like other enzymes, their protein structure can be easily affected by environmental conditions. Furthermore, if used in free form, they cannot be reused, making their applications extremely costly for industrial use. Therefore, researchers place significant emphasis on enzyme immobilization strategies. Enzyme immobilization is the binding of an enzyme to a solid carrier support through physical interactions or chemical bonds. Any nano or microcarrier can be used as a solid support for immobilization [ 5 ]. Among these, nanoparticles, metal organic frameworks, and nanofibers are the main ones. For example, Dik et al. [ 6 ] synthesized starch nanoparticles and immobilized xylenease onto these nanoparticles. They observed a significant improvement in the enzyme's catalytic performance, with the immobilized enzyme retaining approximately 62.8% of its initial activity even after seven cycles of use. On the other hand, in a study conducted by Bakar et al. [ 7 ], Xylanase enzyme was immobilized on ZIF-67 and manganese-doped ZIF-67 MOFs. Both material groups exhibited higher environmental resistance and higher reuse potential compared to the free enzyme. As can be seen, there are ongoing studies, particularly these, aimed at improving Xylanase immobilization. Nanofibers obtained by electrospinning offer suitable structures for high-capacity enzyme loading due to their high surface area-to-volume ratio, adjustable porosity, and ease of functionalization [ 8 , 9 ]. Poly(ε-caprolactone) (PCL), one of the materials widely used in electrospinning, stands out with its biodegradability, mechanical strength, and biocompatibility [ 10 ]. However, PCL's hydrophobic structure and limited functional groups are insufficient for enzyme binding. Therefore, PCL is often combined with natural polymers such as chitosan (CHI). Chitosan, a valuable component derived from chitin, improves the biochemical properties of the support material through its reactive amino groups, hydrophilic structure, and antimicrobial properties, while also providing suitable surfaces for covalent binding [ 11 , 12 ]. Herein, PCL/CHI nanofibers were first synthesized via electrospinning (Fig. 1 ). For this synthesis, 10% PCL and 0.5% CHI were prepared with 10 mL of acetic acid: formic acid (6:4) and stirred for 4 hours. Following the stirring period, electrospinning parameters were investigated, and experiments determined that 22 kV, 1 mL/h flow rate, and 18 cm spinning were the optimum electrospinning conditions. These nanofibers were then cut at a specific surface area and treated with glutaraldehyde. The glutaraldehyde solution was removed from the fibers, and they were washed once with distilled water to remove impurities. Following this process, 150 IU of Xylanase enzyme solution was added to each fiber and stirred overnight at + 4°C. Following this process, the supernatants were separated and stored at + 4°C for subsequent experiments. They were then washed once with distilled water to remove impurities. The structural and morphological properties of the resulting PCL/CHI and PCL/CHI@XyL nanofibers were then investigated using various analysis techniques, including scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDX), X-ray diffraction analysis (XRD), and Fourier Transform Infrared Spectroscopy (FT-IR). Then, the optimum pH value for Free XyL and the optimum temperature values ​​for both enzyme forms were investigated. Additionally, activation energies (E a ) were calculated based on Arrhenius curves, and a significant decrease in E a value was recorded after the immobilization process. Also, the reusability of PCL/CHI@XyL was investigated, and it was observed that it retained approximately 45% of its initial activity even after 5 usage cycles. On the other hand, the effect of metal ions on the activity of both enzyme forms was investigated, and it was determined that the immobilized form exhibited quite good catalytic performance. Consequently, a promising nanocarrier platform for the immobilization of various enzymes was successfully obtained. Figure 1 . Schematic diagram of PCL/CHI Nanofibers fabrication and enzyme immobilization. 2. Materials and Methods 2.1. Material All materials used in this study were of analytical grade and were used without additional purification unless otherwise stated. Xylanase enzyme from Trichoderma viride (Purity: 100,000 U/g) was purchased from Bostonchem, and trichloroacetic acid (TCA, ≥ 99.0%) was purchased from Sigma-Aldrich (St. Louis, MO). Polycaprolactone (PCL, 99%) and chitosan were also supplied by Sigma-Aldrich. Potassium sodium tartrate tetrahydrate (99%), sodium hydroxide (NaOH, ≥ 98%), and sodium dihydrogen phosphate (NaH₂PO₄·H₂O, ≥ 98%) were purchased from Merck. Substrate solutions were prepared fresh on each experimental day and used on the same day. 2.2. Fabrication of PCL/CHI Nanofibers via Electrospinning Preparation of nanofibers was carried out as described in the literature by Balcıoğlu [ 13 ]. First, 10% PCL and 0.5% CHI were prepared in 6 mL of formic acid and 4 mL of cetic acid. This solution was thoroughly mixed for 4 hours to ensure homogeneity. Electrospinning was then performed using a voltage of 22 kV, a distance of 18 cm, and a flow rate of 1 mL/h. The resulting nanofibers were deposited on an aluminum foil on a collector plate. They were then stored at room temperature for subsequent analyses. 2.3. Covalent Immobilization of XyL onto PCL/CHI Nanofibers The resulting nanofibers were cut onto a collector plate to measure 1 cm x 1 cm. A 0.5% glutaraldehyde solution was prepared in pH 7.4 PBS buffer, and 1 mL was added to each cut nanofiber. The glutaraldehyde-added nanofibers were incubated on a shaker at 40°C for 1.5 hours. Following the incubation process, the glutaraldehyde solution was removed from the fibers, and they were washed once with distilled water to remove potential impurities. Then, 1 mL of a 150 IU enzyme solution prepared in distilled water was added to each nanofiber and allowed to stir overnight at + 4°C. Following this mixing process, the supernatants were removed from the nanofibers and stored at + 4°C for use in later experiments. The nanofibers were then washed once with distilled water to remove any remaining impurities and unbound enzymes and allowed to dry. 2.4. Determination of enzyme activity XyL activity was determined according to the method reported in the literature [ 6 ]. Here, it was determined by measuring the amount of reducing sugars (xylose equivalents) produced from xylan hydrolysis using the DNS method. First, 1% xylan solution was prepared with a pH of 5 in a sodium acetate solution. Then, 1 mL of the prepared xylan solution was added to free Xylanase (0.4 U) and 1 cm X 1 cm PCL/CHI@XyL nanofibers and incubated at 50°C for 30 min. After the incubation period, 100 µL of 1.5 M TCA was added to the free enzymes. Both free XyL and PCL/CHI@XyL were then centrifuged, and after centrifugation, 500 µL of the supernatant was taken to determine the activity of each sample, and 500 µL was added and left in boiling water for 5 min. At the end of 5 min, the samples were taken to room temperature, and absorbance measurements of the released reducing sugars at 540 nm were performed on an Eon Microplate Reader controlled by Gen5 2.0 Data Analysis Software (BioTek Instruments, Inc., Winooski, VT, USA). 2.5. Determination of Optimal Temperature and Activation Energy To investigate the temperature effect on enzyme activity, a substrate solution prepared at the optimum pH for the immobilized enzyme was used. 1 mL of substrate solution was added to each PCL/CHI/@XyL sample and incubated for 30 minutes at different temperature conditions (30, 40, 50, 60, 70, and 80°C). Absorbance values ​​were measured at the end of the incubation period. The highest absorbance value obtained was accepted as 100% activity, and relative activities at other temperatures were calculated according to this value. The activation energies of the PCL/CHI/@XyL samples were determined using the Arrhenius equation given below (Eq. 1). In this calculation, the activation energy was calculated by plotting the logarithm of the relative activity against 1000/T (T: temperature in Kelvin). $$\:Slope=\frac{Ea}{R}$$ 2.6. Determination of Kinetic Parameters (K m and V max ) Here, to determine the kinetic parameters of PCL/CHI/@XyL samples, the enzyme was incubated with different substrate concentrations under optimal conditions. After incubation, absorbance values ​​were measured, and K m and V max were calculated using a Lineweaver-Burk plot. These values ​​were compared with data for free enzyme reported in the literature. 2.7. Effect of Metal Ions on Enzyme Activity A series of experiments was conducted to evaluate the effects of metal ions and organic solvents on the activity of the immobilized enzyme. First, metal ion solutions (Al³⁺, Cu²⁺, Ca²⁺, Co²⁺, Na⁺, Mg²⁺, K⁺) were prepared at a concentration of 5 mM. 200 µL of each solution was added to the nanofiber samples and incubated overnight. Then, substrate solution adjusted to pH 5 was added to the samples and incubated at 50°C for 30 min. After incubation, the absorbance of the samples was measured as previously described. The absorbance of the PCL/CHI@XyL sample that was not exposed to the metal solution was considered 100%, and the results of the other samples were expressed as relative activity. 2.8. Reusability of PCL/CHI@XyL Reusability is a key parameter that limits the use of free enzymes in industrial settings. For this purpose, the reusability of PCL/CHI/@Xyl was measured for five cycles under optimal conditions. After each measurement, the PCL/CHI@XyL nanofiber was filtered and washed with distilled water. Fresh substrate was then added to the immobilized enzyme to restart the reaction. The initial enzyme activity was considered 100%, and this process was repeated for five consecutive replicates. 2.9. Characterization techniques Characterization studies were conducted to compare the structural and morphological properties of free nanofibers and enzyme-immobilized nanofibers. FTIR spectra were obtained with a Perkin-Elmer spectrophotometer in the wavelength range of 400–4000 cm⁻¹. X-ray diffraction (XRD) analyses were performed using a Rigaku RadB-DMAX II powder XRD instrument (USA) with a 2θ angle between 10° and 80°. The surface morphology of the samples was examined using a scanning electron microscope (SEM) using an EVO 40-LEO instrument (LEO Ltd., Cambridge, UK). Elemental analyses were performed using a 125 eV resolution EDX detector manufactured by Bruker AXS (Berlin, Germany). 3. Results And Discussion 3.1. Characterization The FT-IR spectra given in Fig. 2 show the intermolecular interactions of PCL/CHI nanofibers and PCL/CHI@XyL nanofibers. In PCL/CHI nanofibers, the bands at 1725 cm⁻¹ (C = O) and 2950 cm⁻¹ (CH₂) confirm the presence of the PCL structure, while the band around 3450 cm⁻¹ corresponds to the –OH and –NH groups of chitosan. The significant broadening of the 3450 cm⁻¹ band after Xylanase immobilization indicates increased hydrogen bonding due to the hydroxyl and amine groups of the enzyme. In addition, the shift observed around 1590–1620 cm⁻¹ confirms the Schiff base (C = N) bonds formed through glutaraldehyde and the covalent binding of the enzyme to the nanofiber surface. Overall, these analyses demonstrate that the PCL/CHI nanofibers retain their structural integrity, successfully immobilize Xylanase, and achieve covalent binding via glutaraldehyde. The resulting composite structure has the potential to increase the enzyme's stability and reusability under industrial conditions [ 14 ]. Figure 2 . FT-IR spectra of PCL/CHI and PCL/CHI@XyL nanofibers The XRD patterns of PCL/CHI and PCL/CHI@XyL nanofibers are shown in Fig. 3 . As can be seen from the XRD patterns, PCL/CHI nanofibers have a semi-crystalline structure. The sharp peak observed around 2θ ≈ 21–22° confirms the (110) crystalline plane of PCL, while the broad shoulder in the range of 10–20° indicates the amorphous contribution of chitosan. The partial broadening of the peaks and the decrease in intensity in PCL/CHI@XyL fibers after Xylanase immobilization indicate that the crystallinity decreased and the amorphous structure increased due to enzyme coating and cross-linking with glutaraldehyde. This confirms that the nanofiber morphology was preserved during immobilization, but the enzyme modified the fiber surface [ 15 – 17 ]. Figure 3 . XRD patterns of PCL/CHI and PCL/CHI@XyL nanofibers Figure 4 presents comparative SEM images and histograms of nanofiber diameters of PCL/CHI and PCL/CHI@XyL nanofibers. As shown in Fig. 4 A, the PCL/CHI exhibits a smooth, homogeneous, and finer-diameter morphology, with fibers clearly separated from one another, low surface roughness, and a narrow average diameter distribution. This structure demonstrates that the electrospinning process is stable and controllable, and that the high flexibility of PCL and the hydrophilic properties of CHI are harmoniously combined in the form of fibers. Furthermore, an examination of the nanofiber diameter histogram reveals that the nanofiber diameter is distributed between 90 and 140 nm (Fig. 4 B). On the other hand, an examination of Fig. 4 C reveals that these nanofibers thicken significantly due to the modification with glutaraldehyde, stick together in some areas, and exhibit irregularities in their morphology. This change in morphology is thought to be due to glutaraldehyde cross-linking between the CHIs in the nanofiber structure and the restriction of polymer mobility following XyL immobilization. These changes in the nanofiber structure were confirmed by histogram distribution plots (Fig. 4 D). The histogram distribution plots showed that the nanofiber diameter distributions were concentrated in the range of approximately 200–300 nm. Consequently, the nanofibers were successfully synthesized, and the expected morphological changes following immobilization were clearly observed. Figure 4 . The SEM images and Nanofiber diameter distribution histograms of nanofibers (A, B) PCL/CHI nanofibers and (C, D) PCL/CHI@XyL nanofibers The elemental composition of the nanofibers was investigated using EDX analysis, and characteristic peaks belonging to carbon (C), oxygen (O), and nitrogen (N) were detected in the spectra (Fig. 5 ). Carbon and oxygen originate from the structural components of PCL and chitosan, while nitrogen originates from the amine groups of chitosan. The absence of metal or inorganic impurity peaks in the spectra confirms the purity of the produced nanofibers. This result is consistent with literature data, which indicates that only organic components are detected in EDX analysis of PCL/CHI nanofibers [ 18 ]. Figure 5 . EDX spectra of (A) PCL/CHI and (B) PCL/CHI@XyL nanofibers 3.2. Biochemical parameters 3.2.1. Optimum PH Figure 6 shows the optimum pH values ​​for free XyL and PCL/CHI@XyL nanofibers. Figure 6 clearly shows that the pH-activity profiles differ in terms of both location (optimum pH) and shape (width of the curve and tail behavior). As can be seen from the figure, the optimum pH value for free XyL was determined as 6, while the optimum pH value for PC/CHI@XyL was defined as 5. This change in the optimum pH value indicates that the immobilization process adapts the enzyme microframework to more acidic conditions [ 19 ]. The parameters that cause this change during the immobilization process can be listed as the alteration of substrate/product diffusion due to the arrangement of the water layer (hydration shell) on the solid carrier support, the acidic microclimate created by chitosan with -NH 3+ groups, or the rearrangement of pKa values ​​near the enzyme's active site due to electrostatic interactions in PCL/CHI nanofibers [ 20 ]. Furthermore, a review of the graph reveals that the immobilized enzyme retains a significant portion of its activity compared to the free enzyme at pH 8–9. This immobilization process not only enhances its catalytic activity under optimal conditions but also broadens the pH window, demonstrating a higher catalytic activity than the free enzyme even under alkaline pH conditions. Figure 6 . The effect of pH on the activity of free XyL and PCL/CHI@XyL 3.2.2. Optimum Temperature and Activation Energy Figure 7 below presents the findings regarding the optimum temperatures of free Xylanase and PCL/CHI@XyL. When the temperature-activity profiles of both enzyme forms are examined, it is evident that the optimum temperature for both is determined to be 50°C. However, as the temperature increases above the optimum temperature, significant decreases in the activity of the free enzyme are observed, while PCL/CHI@XyL maintains its activity significantly compared to the free enzyme, even at higher temperatures. This is because the immobilization process increases the enzyme's thermal resistance. While a significant loss of activity is observed in both enzymes between 70 and 80°C, the immobilized enzyme maintains its activity to a greater extent compared to the free form. These results indicate that immobilization provides structural rigidity to the enzyme, limiting conformational changes that may occur at high temperatures, thus providing a protective effect against thermal denaturation [ 21 ]. Similarly, Scharma et al. synthesized chitosan gel beads and immobilized xylenese on them [ 22 ]. Experiments to determine the optimum temperature determined the optimum temperature for both enzyme forms as 50°C. Furthermore, experiments conducted in parallel with our study showed that the immobilized enzyme maintained its activity more significantly at increased temperatures compared to the free enzyme. Based on these findings, it is possible to say that the results are consistent with the literature. Figure 7 . The effect of temperature on the activity of free XyL and PCL/CHI@XyL The Arrhenius curves of free XyL and PCL/CHI@XyL are presented in Fig. 8 . Activation energies (E a ) were calculated for both enzyme forms using Arrhenius curves, and the E a value for free XyL was determined to be 21.46 kJ/mol (Fig. 8 A), and for PCL/CHI@XyL was determined to be 1.17 kJ/mol (Fig. 8 B). This decrease in activation energy observed after immobilization indicates that immobilization reduces the energy barrier during the enzyme's catalytic process and facilitates substrate access to the active site. Furthermore, the lower activation energy of the immobilized enzyme suggests that the PCL/CHI support surface provides a suitable microenvironment for the enzyme, increasing catalytic efficiency. The higher E a observed in the free form suggests that the enzyme can be more easily denatured at high temperatures due to its structural flexibility, requiring more energy for reaction initiation. Similarly, it has been reported in the literature that the activation energies of hydrolytic enzymes immobilized on chitosan-based or functionalized supports decrease compared to their free forms, thus improving their catalytic activity [ 23 ]. Therefore, these results demonstrate that immobilization not only increases enzyme stability but also provides more advantageous catalytic performance in terms of kinetic parameters. Figure 8 . Arrhenius plots to calculate E a for the A) Free XyL and (B) PCL/CHI@XyL 3.2.3. Determination of Kinetic Parameters (K m and V max ) The Lineweaver–Burk plots shown in Fig. 9 and the resulting kinetic parameters calculated based on these plots (Table 1) comparatively demonstrate the catalytic behaviors of free XyL and PCL/CHI@XyL. The calculated K m value for free XyL was determined as 13.01 ± 2.23 mg/mL, while this value was determined as 4.80 ± 0.12 mg/mL for PCL/CHI@XyL. The significant decrease in the K m value after immobilization indicates that the affinity of the enzyme for its substrate increased. This can be explained by the fact that the hydrophilic and functional group-enriched microenvironment created by the PCL/CHI support surface facilitates the directing of substrate molecules to the active site. In particular, the amino groups of chitosan are thought to increase substrate retention through electrostatic interactions or hydrogen bonds, thereby increasing the enzyme's "effective substrate concentration" [ 24 ]. Thus, the immobilized enzyme can exhibit high activity even at lower substrate concentrations. In contrast, the maximum rate parameter (V max ) was calculated as 0.019 ± 0.005 µmol/min for the free enzyme and 0.010 ± 0.006 µmol/min for the immobilized enzyme. This decrease in V max reveals that immobilization partially limits the catalytic cycle rate. This is thought to be due to the substrate creating a diffusion barrier within the nanofiber as it reaches the active site or to diffusion limitations experienced during the removal of the formed product from the reaction medium, slowing down the catalytic cycle [ 25 , 26 ]. Furthermore, the high correlation coefficients obtained for both Lineweaver–Burk plots (R²=0.959 and R²=0.991) indicate that the results are reliable and that the experimental data used fit the linear kinetic model. This confirms that the inferences made from K m and V max values ​​are statistically strong. Figure 9 . Comparative Lineweaver–Burk plots of A) Free XyL and B) PCL/CHI@XyL Tablo 1. Serbest XyL ve PCL/CHI@XyL ait kinetik parametreler Enzyme K m (mg/mL) V max (µmol/min.mL) R 2 Free XyL 13.011 ± 5.781 0.019 ± 0.005 0.959 PCL/CHI@XyL 4.806 ± 0,121 0.010 ± 0.006 0.991 3.2.4. Effect of Metal Ions on Enzyme Activity Figure 10 compares the activities of free XyL and PCL/CHI@XyL in the presence of different metal ions (10 mM). Here, the control group represents the enzyme activities without any metal ions, and this value is accepted as the initial activity (%). From this point on, the presence of metal ions produces quite different effects on the free and immobilized enzymes. Monovalent ions (Na⁺ and K⁺) caused a slight increase in activity compared to the control in both enzyme forms. However, the increase was more pronounced in the immobilized enzyme, suggesting that ionic strength may facilitate the enzyme-substrate interaction. Remarkable differences were observed in the presence of divalent ions Ca²⁺ and Co²⁺. While the free enzyme exhibited only 1.1-1.2-fold activity, the activity of PCL/CHI@XyL increased approximately 1.9-2.10-fold. This suggests that divalent ions may generally have a cofactor-like effect in the active center of the enzyme and that these ions can bind more effectively to the enzyme surface thanks to the immobilization matrix (PCL/CHI) [ 27 ]. However, 3 + ions increased the activity of the immobilized enzyme approximately 1.8-fold, while the free enzyme increased it 1.3-fold. This can be attributed to the amino groups in the chitosan structure interacting with highly charged ions such as Al³⁺ to stabilize the enzyme's active conformation. One of the most striking results is the activity of both enzyme forms in the presence of Cu²⁺ ion. While the free enzyme retained only 20% of its activity compared to the control in the presence of this ion, the opposite was true in PCL/CHI@XyL, with an approximately 2.5-fold increase compared to the control. This opposing effect suggests that immobilization suppresses the inhibitory properties of the Cu²⁺ ion, protecting the enzyme from inactivation, and that Cu²⁺ may even facilitate the catalytic process when bound to the immobilized enzyme. This effect can be explained by the fact that the functional groups of the support material (e.g., amino groups of chitosan) chelate metal ions and regulate the ion concentration reaching the enzyme's active site [ 28 ]. Consequently, PCL/CHI@XyL nanofibers offer a much more advantageous biocatalyst alternative compared to free enzymes in industrial environments where metal ions are concentrated. Figure 10 . Effect of metal ions on enzyme activity (A) Free XyL and (B) PCL/CHI@XyL 3.2.5. Reusability of PCL/CHI@XyL Because free enzymes cannot be separated from the reaction medium and are easily affected by environmental conditions, they cannot be reused repeatedly. This restricts the industrial use of enzymes. To overcome this problem, the development of immobilization-based biocatalyst systems is widely used. The graph shown in Fig. 11 shows the reusability results of Xylanase immobilized on PCL/CHI nanofibers (PCL/CHI@XyL). The initial activity assumed to be 100% in the first cycle was retained at approximately 45% of its initial activity after the fifth cycle. This decrease in the immobilized enzyme is thought to be due to mechanisms such as partial inactivation, desorption from the support, or structural changes during the reuse process [ 29 ]. The results observed in the graph reveal that Xylanase immobilized on PCL/CHI nanofibers is a longer-lasting and economically viable biocatalyst candidate compared to its free form. Figure 11 . Reusability of PCL/CHI@XyL. 4. Conclusion Here, the immobilization of Xylanase enzyme onto PCL/CHI nanofibers obtained via electrospinning was successfully achieved. The structural and morphological properties of the resulting PCL/CHI@XyL were investigated through various characterizations, and their biochemical parameters were extensively studied. SEM, XRD, EDX, and FT-IR characterizations confirmed that the nanofibers maintained their integrity during the immobilization process and that covalent bonds were formed with the enzyme. In the biochemical evaluations, the optimum pH value was determined to be 6 for free XyL, while it decreased to 5 for PCL/CHI@XyL, indicating that the carrier matrix provided a more acidic microenvironment for the enzyme. Furthermore, although the optimum temperature was determined to be 50°C for both enzyme forms, the immobilized enzyme maintained its structural stability at higher temperatures and was more resistant to thermal denaturation. Kinetic parameters values ​​of the free and immobilized enzymes were also investigated, revealing that the immobilized enzyme has a lower Km value and a higher affinity for the substrate compared to the free enzyme. Furthermore, the significant decrease in activation energy indicates that immobilization enables catalytic reactions to occur under lower energy barriers. Tests conducted in the presence of metal ions revealed that the immobilized enzyme develops a protective resistance, particularly against inhibitory ions such as Cu²⁺, while some ions play a role in enhancing catalytic activity. Finally, reusability experiments revealed that the immobilized enzyme retained approximately 45% of its activity after five cycles, providing a significant economic advantage for industrial applications. In light of all these findings, PCL/CHI@XyL nanofibers have broad potential not only for Xylanase but also for the immobilization of various enzymes and their use in industrial bioprocesses. Therefore, this study provides an important contribution towards the development of sustainable, economical and high-performance biocatalysts using nanofiber-based support materials. Declarations Ethics Approval and Consent to Participate This study did not involve human participants or animals; therefore, ethics approval and consent to participate were not required. Consent for Publication As the corresponding author of this study, I declare that I have read and approved the final version of the article titled “Electrospun Polycaprolactone-Chitosan Nanofiber Scaffolds for Covalent Immobilization of Xylanase: Structural Characterization and Enzyme Performance”, confirm that the work is original, unpublished, and free from legal or institutional restrictions, and give full approval for its submission to the journal, its publication in Catalysis Letters, and the transfer of all rights requested by the journal. Competing Interest The author declares that there are no known conflicts of interest or personal relationships that could influence the work reported in this article. Authors contributions The sole author conducted all research, analysis, and writing for this manuscript. Declaration of generative AI and AI-assisted technologies in the writing process The writers utilized ChatGPT 5 to enhance the text's coherence and clarity, while also refining the language and scientific terminology employed in this study. The writers took full responsibility for the publication's content after utilizing this tool/service, reviewing and editing it as necessary. Availability of Data and Materials The data and materials supporting the findings of this study are available from the corresponding author upon reasonable request. Acknowledgments I would like to thank Prof. Dr. Burhan Ateş, Assoc. Prof. Dr. Ahmet Ulu, Kübra Karadaş Gedik, and Gamze Dik for their valuable support in this study. 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Biotech 3:1–9. https://doi.org/10.1007/S13205-012-0071-7/FIGURES/1 Andrews AT, Williams RJH, Brownsell VL et al (2006) β-CN-5P and β-CN-4P components of bovine milk proteose–peptone: Large-scale preparation and influence on the growth of cariogenic microorganisms. Food Chem 96:234–241. https://doi.org/10.1016/J.FOODCHEM.2005.02.039 Karaca Açarı İ, Dik G, Bakar B et al (2023) Immobilization of α-Amylase onto Quantum Dots Prepared from Hypericum perforatum L. Flowers and Hypericum capitatum Seeds: Its Physicochemical and Biochemical Characterization. Top Catal 66:563–576. https://doi.org/10.1007/S11244-022-01699-Y/FIGURES/10 Wahba MI, Ismail SA, Hassan AA et al (2024) Xylanase immobilization using activated carrier of gellan gum-agar beads: Improved stability and catalytic activity for the production of antioxidant and anti-proliferative xylooligosaccharides. Biocatal Agric Biotechnol 56:103013. https://doi.org/10.1016/J.BCAB.2023.103013 Khan MR (2021) Immobilized enzymes: a comprehensive review. Bulletin of the National Research Centre 2021 45:1 45:1–13. https://doi.org/10.1186/S42269-021-00649-0 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 01 Nov, 2025 Reviews received at journal 31 Oct, 2025 Reviews received at journal 23 Oct, 2025 Reviewers agreed at journal 12 Oct, 2025 Reviewers agreed at journal 12 Oct, 2025 Reviewers agreed at journal 10 Oct, 2025 Reviewers invited by journal 10 Oct, 2025 Editor assigned by journal 06 Oct, 2025 Submission checks completed at journal 06 Oct, 2025 First submitted to journal 03 Oct, 2025 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. 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2","display":"","copyAsset":false,"role":"figure","size":7667,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectra of PCL/CHI and PCL/CHI@XyL nanofibers\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7775653/v1/1bc849377bc1b3561c2a333d.png"},{"id":94405162,"identity":"b95554c6-fb0b-4bcf-9d2d-3d1796ef3e8d","added_by":"auto","created_at":"2025-10-27 14:01:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9945,"visible":true,"origin":"","legend":"\u003cp\u003eXRD patterns of PCL/CHI and PCL/CHI@XyL nanofibers\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7775653/v1/7a1638e8efbd48bbfd3f57fc.png"},{"id":94405099,"identity":"db060946-e0b9-4d22-8ef3-b9dbbb80463f","added_by":"auto","created_at":"2025-10-27 14:01:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":111908,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM images and Nanofiber diameter distribution histograms of nanofibers (A, B) PCL/CHI nanofibers and (C, D) PCL/CHI@XyL nanofibers\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7775653/v1/05f41575ae33da6cbedc4450.png"},{"id":94404423,"identity":"cac206e3-dd11-47c1-9a8d-3960b4a92791","added_by":"auto","created_at":"2025-10-27 14:01:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11461,"visible":true,"origin":"","legend":"\u003cp\u003eEDX spectra of (A) PCL/CHI and (B) PCL/CHI@XyL 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plots of A) Free XyL and B) PCL/CHI@XyL\u003c/p\u003e","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7775653/v1/6dbb19b56da45fcfc1d9cf0d.png"},{"id":94403557,"identity":"4cef9f18-f794-44c8-8242-84fe94f87f0b","added_by":"auto","created_at":"2025-10-27 14:00:27","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":5583,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of metal ions on enzyme activity (A) Free XyL and (B) PCL/CHI@XyL\u003c/p\u003e","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7775653/v1/a57c9159584e10b1fc1a3b3c.png"},{"id":94404154,"identity":"6d5034db-0da4-4772-b3f8-f0b65bf79d6b","added_by":"auto","created_at":"2025-10-27 14:00:46","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":8349,"visible":true,"origin":"","legend":"\u003cp\u003eReusability of PCL/CHI@XyL.\u003c/p\u003e","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-7775653/v1/455a7d3c1fd85629b93cb07a.png"},{"id":94460320,"identity":"db0bf725-eb7c-4dac-8b1b-c3918d0c3044","added_by":"auto","created_at":"2025-10-27 14:55:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1096987,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7775653/v1/e13e6fc8-79d1-499c-8634-c79d088b98e4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Electrospun Polycaprolactone–Chitosan Nanofiber Scaffolds for Covalent Immobilization of Xylanase: Structural Characterization and Enzyme Performance","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe Xylanase (endo-1,4-β-Xylanase; EC 3.2.1.8) are enzymes classified primarily in glycoside hydrolase (GH) families GH10 and GH11 that hydrolyze the β-1,4 glycosidic bonds of xylan, the major component of plant cell wall hemicellulose, to form xylo-oligosaccharides and xylose; this classification is associated with differences in catalytic architecture and substrate specificity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Xylan is a complex polymer that interacts with cellulose and lignin, exhibiting branching and substituent diversity in the arabinoxylan structure, particularly in cereal straws and woody tissues. This structural heterogeneity contributes to the recalcitrance of biomass and determines the sensitivity of Xylanase activity to processing conditions [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The industrial applications, Xylanase play an important role in reducing chemical consumption and environmental burden by increasing the efficiency of biological pre-bleaching and bleaching agents in the paper/pulp industry; in improving pulp rheology, juice clarification[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and optimizing fiber functionality in food technology; in increasing digestive efficiency by degrading non-starch polysaccharides in feed additives; and in converting lignocellulosic residues to sugars in biorefinery/biofuel lines [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, like other enzymes, their protein structure can be easily affected by environmental conditions. Furthermore, if used in free form, they cannot be reused, making their applications extremely costly for industrial use. Therefore, researchers place significant emphasis on enzyme immobilization strategies.\u003c/p\u003e\u003cp\u003eEnzyme immobilization is the binding of an enzyme to a solid carrier support through physical interactions or chemical bonds. Any nano or microcarrier can be used as a solid support for immobilization [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among these, nanoparticles, metal organic frameworks, and nanofibers are the main ones. For example, Dik et al. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] synthesized starch nanoparticles and immobilized xylenease onto these nanoparticles. They observed a significant improvement in the enzyme's catalytic performance, with the immobilized enzyme retaining approximately 62.8% of its initial activity even after seven cycles of use. On the other hand, in a study conducted by Bakar et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], Xylanase enzyme was immobilized on ZIF-67 and manganese-doped ZIF-67 MOFs. Both material groups exhibited higher environmental resistance and higher reuse potential compared to the free enzyme. As can be seen, there are ongoing studies, particularly these, aimed at improving Xylanase immobilization. Nanofibers obtained by electrospinning offer suitable structures for high-capacity enzyme loading due to their high surface area-to-volume ratio, adjustable porosity, and ease of functionalization [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Poly(ε-caprolactone) (PCL), one of the materials widely used in electrospinning, stands out with its biodegradability, mechanical strength, and biocompatibility [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, PCL's hydrophobic structure and limited functional groups are insufficient for enzyme binding. Therefore, PCL is often combined with natural polymers such as chitosan (CHI). Chitosan, a valuable component derived from chitin, improves the biochemical properties of the support material through its reactive amino groups, hydrophilic structure, and antimicrobial properties, while also providing suitable surfaces for covalent binding [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHerein, PCL/CHI nanofibers were first synthesized via electrospinning (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For this synthesis, 10% PCL and 0.5% CHI were prepared with 10 mL of acetic acid: formic acid (6:4) and stirred for 4 hours. Following the stirring period, electrospinning parameters were investigated, and experiments determined that 22 kV, 1 mL/h flow rate, and 18 cm spinning were the optimum electrospinning conditions. These nanofibers were then cut at a specific surface area and treated with glutaraldehyde. The glutaraldehyde solution was removed from the fibers, and they were washed once with distilled water to remove impurities. Following this process, 150 IU of Xylanase enzyme solution was added to each fiber and stirred overnight at +\u0026thinsp;4\u0026deg;C. Following this process, the supernatants were separated and stored at +\u0026thinsp;4\u0026deg;C for subsequent experiments. They were then washed once with distilled water to remove impurities. The structural and morphological properties of the resulting PCL/CHI and PCL/CHI@XyL nanofibers were then investigated using various analysis techniques, including scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDX), X-ray diffraction analysis (XRD), and Fourier Transform Infrared Spectroscopy (FT-IR). Then, the optimum pH value for Free XyL and the optimum temperature values ​​for both enzyme forms were investigated. Additionally, activation energies (E\u003csub\u003ea\u003c/sub\u003e) were calculated based on Arrhenius curves, and a significant decrease in E\u003csub\u003ea\u003c/sub\u003e value was recorded after the immobilization process. Also, the reusability of PCL/CHI@XyL was investigated, and it was observed that it retained approximately 45% of its initial activity even after 5 usage cycles. On the other hand, the effect of metal ions on the activity of both enzyme forms was investigated, and it was determined that the immobilized form exhibited quite good catalytic performance. Consequently, a promising nanocarrier platform for the immobilization of various enzymes was successfully obtained.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Schematic diagram of PCL/CHI Nanofibers fabrication and enzyme immobilization.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Material\u003c/h2\u003e\u003cp\u003eAll materials used in this study were of analytical grade and were used without additional purification unless otherwise stated. Xylanase enzyme from Trichoderma viride (Purity: 100,000 U/g) was purchased from Bostonchem, and trichloroacetic acid (TCA, \u0026ge; 99.0%) was purchased from Sigma-Aldrich (St. Louis, MO). Polycaprolactone (PCL, 99%) and chitosan were also supplied by Sigma-Aldrich. Potassium sodium tartrate tetrahydrate (99%), sodium hydroxide (NaOH, \u0026ge; 98%), and sodium dihydrogen phosphate (NaH₂PO₄\u0026middot;H₂O, \u0026ge; 98%) were purchased from Merck. Substrate solutions were prepared fresh on each experimental day and used on the same day.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Fabrication of PCL/CHI Nanofibers via Electrospinning\u003c/h2\u003e\u003cp\u003ePreparation of nanofibers was carried out as described in the literature by Balcıoğlu [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. First, 10% PCL and 0.5% CHI were prepared in 6 mL of formic acid and 4 mL of cetic acid. This solution was thoroughly mixed for 4 hours to ensure homogeneity. Electrospinning was then performed using a voltage of 22 kV, a distance of 18 cm, and a flow rate of 1 mL/h. The resulting nanofibers were deposited on an aluminum foil on a collector plate. They were then stored at room temperature for subsequent analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Covalent Immobilization of XyL onto PCL/CHI Nanofibers\u003c/h2\u003e\u003cp\u003eThe resulting nanofibers were cut onto a collector plate to measure 1 cm x 1 cm. A 0.5% glutaraldehyde solution was prepared in pH 7.4 PBS buffer, and 1 mL was added to each cut nanofiber. The glutaraldehyde-added nanofibers were incubated on a shaker at 40\u0026deg;C for 1.5 hours. Following the incubation process, the glutaraldehyde solution was removed from the fibers, and they were washed once with distilled water to remove potential impurities. Then, 1 mL of a 150 IU enzyme solution prepared in distilled water was added to each nanofiber and allowed to stir overnight at +\u0026thinsp;4\u0026deg;C. Following this mixing process, the supernatants were removed from the nanofibers and stored at +\u0026thinsp;4\u0026deg;C for use in later experiments. The nanofibers were then washed once with distilled water to remove any remaining impurities and unbound enzymes and allowed to dry.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Determination of enzyme activity\u003c/h2\u003e\u003cp\u003eXyL activity was determined according to the method reported in the literature [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Here, it was determined by measuring the amount of reducing sugars (xylose equivalents) produced from xylan hydrolysis using the DNS method. First, 1% xylan solution was prepared with a pH of 5 in a sodium acetate solution. Then, 1 mL of the prepared xylan solution was added to free Xylanase (0.4 U) and 1 cm X 1 cm PCL/CHI@XyL nanofibers and incubated at 50\u0026deg;C for 30 min. After the incubation period, 100 \u0026micro;L of 1.5 M TCA was added to the free enzymes. Both free XyL and PCL/CHI@XyL were then centrifuged, and after centrifugation, 500 \u0026micro;L of the supernatant was taken to determine the activity of each sample, and 500 \u0026micro;L was added and left in boiling water for 5 min. At the end of 5 min, the samples were taken to room temperature, and absorbance measurements of the released reducing sugars at 540 nm were performed on an Eon Microplate Reader controlled by Gen5 2.0 Data Analysis Software (BioTek Instruments, Inc., Winooski, VT, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Determination of Optimal Temperature and Activation Energy\u003c/h2\u003e\u003cp\u003eTo investigate the temperature effect on enzyme activity, a substrate solution prepared at the optimum pH for the immobilized enzyme was used. 1 mL of substrate solution was added to each PCL/CHI/@XyL sample and incubated for 30 minutes at different temperature conditions (30, 40, 50, 60, 70, and 80\u0026deg;C). Absorbance values ​​were measured at the end of the incubation period. The highest absorbance value obtained was accepted as 100% activity, and relative activities at other temperatures were calculated according to this value. The activation energies of the PCL/CHI/@XyL samples were determined using the Arrhenius equation given below (Eq.\u0026nbsp;1). In this calculation, the activation energy was calculated by plotting the logarithm of the relative activity against 1000/T (T: temperature in Kelvin).\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Slope=\\frac{Ea}{R}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Determination of Kinetic Parameters (K\u003csub\u003em\u003c/sub\u003e and V\u003csub\u003emax\u003c/sub\u003e)\u003c/h2\u003e\u003cp\u003eHere, to determine the kinetic parameters of PCL/CHI/@XyL samples, the enzyme was incubated with different substrate concentrations under optimal conditions. After incubation, absorbance values ​​were measured, and K\u003csub\u003em\u003c/sub\u003e and V\u003csub\u003emax\u003c/sub\u003e were calculated using a Lineweaver-Burk plot. These values ​​were compared with data for free enzyme reported in the literature.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Effect of Metal Ions on Enzyme Activity\u003c/h2\u003e\u003cp\u003eA series of experiments was conducted to evaluate the effects of metal ions and organic solvents on the activity of the immobilized enzyme. First, metal ion solutions (Al\u0026sup3;⁺, Cu\u0026sup2;⁺, Ca\u0026sup2;⁺, Co\u0026sup2;⁺, Na⁺, Mg\u0026sup2;⁺, K⁺) were prepared at a concentration of 5 mM. 200 \u0026micro;L of each solution was added to the nanofiber samples and incubated overnight. Then, substrate solution adjusted to pH 5 was added to the samples and incubated at 50\u0026deg;C for 30 min. After incubation, the absorbance of the samples was measured as previously described. The absorbance of the PCL/CHI@XyL sample that was not exposed to the metal solution was considered 100%, and the results of the other samples were expressed as relative activity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Reusability of PCL/CHI@XyL\u003c/h2\u003e\u003cp\u003eReusability is a key parameter that limits the use of free enzymes in industrial settings. For this purpose, the reusability of PCL/CHI/@Xyl was measured for five cycles under optimal conditions. After each measurement, the PCL/CHI@XyL nanofiber was filtered and washed with distilled water. Fresh substrate was then added to the immobilized enzyme to restart the reaction. The initial enzyme activity was considered 100%, and this process was repeated for five consecutive replicates.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Characterization techniques\u003c/h2\u003e\u003cp\u003eCharacterization studies were conducted to compare the structural and morphological properties of free nanofibers and enzyme-immobilized nanofibers. FTIR spectra were obtained with a Perkin-Elmer spectrophotometer in the wavelength range of 400\u0026ndash;4000 cm⁻\u0026sup1;. X-ray diffraction (XRD) analyses were performed using a Rigaku RadB-DMAX II powder XRD instrument (USA) with a 2θ angle between 10\u0026deg; and 80\u0026deg;. The surface morphology of the samples was examined using a scanning electron microscope (SEM) using an EVO 40-LEO instrument (LEO Ltd., Cambridge, UK). Elemental analyses were performed using a 125 eV resolution EDX detector manufactured by Bruker AXS (Berlin, Germany).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Characterization\u003c/h2\u003e\u003cp\u003eThe FT-IR spectra given in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e show the intermolecular interactions of PCL/CHI nanofibers and PCL/CHI@XyL nanofibers. In PCL/CHI nanofibers, the bands at 1725 cm⁻\u0026sup1; (C\u0026thinsp;=\u0026thinsp;O) and 2950 cm⁻\u0026sup1; (CH₂) confirm the presence of the PCL structure, while the band around 3450 cm⁻\u0026sup1; corresponds to the \u0026ndash;OH and \u0026ndash;NH groups of chitosan. The significant broadening of the 3450 cm⁻\u0026sup1; band after Xylanase immobilization indicates increased hydrogen bonding due to the hydroxyl and amine groups of the enzyme. In addition, the shift observed around 1590\u0026ndash;1620 cm⁻\u0026sup1; confirms the Schiff base (C\u0026thinsp;=\u0026thinsp;N) bonds formed through glutaraldehyde and the covalent binding of the enzyme to the nanofiber surface. Overall, these analyses demonstrate that the PCL/CHI nanofibers retain their structural integrity, successfully immobilize Xylanase, and achieve covalent binding via glutaraldehyde. The resulting composite structure has the potential to increase the enzyme's stability and reusability under industrial conditions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. FT-IR spectra of PCL/CHI and PCL/CHI@XyL nanofibers\u003c/p\u003e\u003cp\u003eThe XRD patterns of PCL/CHI and PCL/CHI@XyL nanofibers are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. As can be seen from the XRD patterns, PCL/CHI nanofibers have a semi-crystalline structure. The sharp peak observed around 2θ\u0026thinsp;\u0026asymp;\u0026thinsp;21\u0026ndash;22\u0026deg; confirms the (110) crystalline plane of PCL, while the broad shoulder in the range of 10\u0026ndash;20\u0026deg; indicates the amorphous contribution of chitosan. The partial broadening of the peaks and the decrease in intensity in PCL/CHI@XyL fibers after Xylanase immobilization indicate that the crystallinity decreased and the amorphous structure increased due to enzyme coating and cross-linking with glutaraldehyde. This confirms that the nanofiber morphology was preserved during immobilization, but the enzyme modified the fiber surface [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. XRD patterns of PCL/CHI and PCL/CHI@XyL nanofibers\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e presents comparative SEM images and histograms of nanofiber diameters of PCL/CHI and PCL/CHI@XyL nanofibers. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, the PCL/CHI exhibits a smooth, homogeneous, and finer-diameter morphology, with fibers clearly separated from one another, low surface roughness, and a narrow average diameter distribution. This structure demonstrates that the electrospinning process is stable and controllable, and that the high flexibility of PCL and the hydrophilic properties of CHI are harmoniously combined in the form of fibers. Furthermore, an examination of the nanofiber diameter histogram reveals that the nanofiber diameter is distributed between 90 and 140 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). On the other hand, an examination of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC reveals that these nanofibers thicken significantly due to the modification with glutaraldehyde, stick together in some areas, and exhibit irregularities in their morphology. This change in morphology is thought to be due to glutaraldehyde cross-linking between the CHIs in the nanofiber structure and the restriction of polymer mobility following XyL immobilization. These changes in the nanofiber structure were confirmed by histogram distribution plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The histogram distribution plots showed that the nanofiber diameter distributions were concentrated in the range of approximately 200\u0026ndash;300 nm. Consequently, the nanofibers were successfully synthesized, and the expected morphological changes following immobilization were clearly observed.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The SEM images and Nanofiber diameter distribution histograms of nanofibers (A, B) PCL/CHI nanofibers and (C, D) PCL/CHI@XyL nanofibers\u003c/p\u003e\u003cp\u003eThe elemental composition of the nanofibers was investigated using EDX analysis, and characteristic peaks belonging to carbon (C), oxygen (O), and nitrogen (N) were detected in the spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Carbon and oxygen originate from the structural components of PCL and chitosan, while nitrogen originates from the amine groups of chitosan. The absence of metal or inorganic impurity peaks in the spectra confirms the purity of the produced nanofibers. This result is consistent with literature data, which indicates that only organic components are detected in EDX analysis of PCL/CHI nanofibers [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. EDX spectra of (A) PCL/CHI and (B) PCL/CHI@XyL nanofibers\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Biochemical parameters\u003c/h2\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1. Optimum PH\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the optimum pH values ​​for free XyL and PCL/CHI@XyL nanofibers. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e clearly shows that the pH-activity profiles differ in terms of both location (optimum pH) and shape (width of the curve and tail behavior). As can be seen from the figure, the optimum pH value for free XyL was determined as 6, while the optimum pH value for PC/CHI@XyL was defined as 5. This change in the optimum pH value indicates that the immobilization process adapts the enzyme microframework to more acidic conditions [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The parameters that cause this change during the immobilization process can be listed as the alteration of substrate/product diffusion due to the arrangement of the water layer (hydration shell) on the solid carrier support, the acidic microclimate created by chitosan with -NH\u003csup\u003e3+\u003c/sup\u003e groups, or the rearrangement of pKa values ​​near the enzyme's active site due to electrostatic interactions in PCL/CHI nanofibers [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Furthermore, a review of the graph reveals that the immobilized enzyme retains a significant portion of its activity compared to the free enzyme at pH 8\u0026ndash;9. This immobilization process not only enhances its catalytic activity under optimal conditions but also broadens the pH window, demonstrating a higher catalytic activity than the free enzyme even under alkaline pH conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The effect of pH on the activity of free XyL and PCL/CHI@XyL\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2. Optimum Temperature and Activation Energy\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e below presents the findings regarding the optimum temperatures of free Xylanase and PCL/CHI@XyL. When the temperature-activity profiles of both enzyme forms are examined, it is evident that the optimum temperature for both is determined to be 50\u0026deg;C. However, as the temperature increases above the optimum temperature, significant decreases in the activity of the free enzyme are observed, while PCL/CHI@XyL maintains its activity significantly compared to the free enzyme, even at higher temperatures. This is because the immobilization process increases the enzyme's thermal resistance. While a significant loss of activity is observed in both enzymes between 70 and 80\u0026deg;C, the immobilized enzyme maintains its activity to a greater extent compared to the free form. These results indicate that immobilization provides structural rigidity to the enzyme, limiting conformational changes that may occur at high temperatures, thus providing a protective effect against thermal denaturation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Similarly, Scharma et al. synthesized chitosan gel beads and immobilized xylenese on them [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Experiments to determine the optimum temperature determined the optimum temperature for both enzyme forms as 50\u0026deg;C. Furthermore, experiments conducted in parallel with our study showed that the immobilized enzyme maintained its activity more significantly at increased temperatures compared to the free enzyme. Based on these findings, it is possible to say that the results are consistent with the literature.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. The effect of temperature on the activity of free XyL and PCL/CHI@XyL\u003c/p\u003e\u003cp\u003eThe Arrhenius curves of free XyL and PCL/CHI@XyL are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Activation energies (E\u003csub\u003ea\u003c/sub\u003e) were calculated for both enzyme forms using Arrhenius curves, and the E\u003csub\u003ea\u003c/sub\u003e value for free XyL was determined to be 21.46 kJ/mol (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA), and for PCL/CHI@XyL was determined to be 1.17 kJ/mol (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). This decrease in activation energy observed after immobilization indicates that immobilization reduces the energy barrier during the enzyme's catalytic process and facilitates substrate access to the active site. Furthermore, the lower activation energy of the immobilized enzyme suggests that the PCL/CHI support surface provides a suitable microenvironment for the enzyme, increasing catalytic efficiency. The higher E\u003csub\u003ea\u003c/sub\u003e observed in the free form suggests that the enzyme can be more easily denatured at high temperatures due to its structural flexibility, requiring more energy for reaction initiation. Similarly, it has been reported in the literature that the activation energies of hydrolytic enzymes immobilized on chitosan-based or functionalized supports decrease compared to their free forms, thus improving their catalytic activity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, these results demonstrate that immobilization not only increases enzyme stability but also provides more advantageous catalytic performance in terms of kinetic parameters.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Arrhenius plots to calculate E\u003csub\u003ea\u003c/sub\u003e for the A) Free XyL and (B) PCL/CHI@XyL\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3. Determination of Kinetic Parameters (K\u003csub\u003em\u003c/sub\u003e and V\u003csub\u003emax\u003c/sub\u003e)\u003c/h2\u003e\u003cp\u003eThe Lineweaver\u0026ndash;Burk plots shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e and the resulting kinetic parameters calculated based on these plots (Table\u0026nbsp;1) comparatively demonstrate the catalytic behaviors of free XyL and PCL/CHI@XyL. The calculated K\u003csub\u003em\u003c/sub\u003e value for free XyL was determined as 13.01\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23 mg/mL, while this value was determined as 4.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 mg/mL for PCL/CHI@XyL. The significant decrease in the K\u003csub\u003em\u003c/sub\u003e value after immobilization indicates that the affinity of the enzyme for its substrate increased. This can be explained by the fact that the hydrophilic and functional group-enriched microenvironment created by the PCL/CHI support surface facilitates the directing of substrate molecules to the active site. In particular, the amino groups of chitosan are thought to increase substrate retention through electrostatic interactions or hydrogen bonds, thereby increasing the enzyme's \"effective substrate concentration\" [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Thus, the immobilized enzyme can exhibit high activity even at lower substrate concentrations. In contrast, the maximum rate parameter (V\u003csub\u003emax\u003c/sub\u003e) was calculated as 0.019\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005 \u0026micro;mol/min for the free enzyme and 0.010\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006 \u0026micro;mol/min for the immobilized enzyme. This decrease in V\u003csub\u003emax\u003c/sub\u003e reveals that immobilization partially limits the catalytic cycle rate. This is thought to be due to the substrate creating a diffusion barrier within the nanofiber as it reaches the active site or to diffusion limitations experienced during the removal of the formed product from the reaction medium, slowing down the catalytic cycle [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Furthermore, the high correlation coefficients obtained for both Lineweaver\u0026ndash;Burk plots (R\u0026sup2;=0.959 and R\u0026sup2;=0.991) indicate that the results are reliable and that the experimental data used fit the linear kinetic model. This confirms that the inferences made from K\u003csub\u003em\u003c/sub\u003e and V\u003csub\u003emax\u003c/sub\u003e values ​​are statistically strong.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. Comparative Lineweaver\u0026ndash;Burk plots of A) Free XyL and B) PCL/CHI@XyL\u003c/p\u003e\u003cp\u003e\u003cb\u003eTablo 1.\u003c/b\u003e Serbest XyL ve PCL/CHI@XyL ait kinetik parametreler\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnzyme\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003csub\u003em\u003c/sub\u003e (mg/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eV\u003csub\u003emax\u003c/sub\u003e (\u0026micro;mol/min.mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFree XyL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13.011\u0026thinsp;\u0026plusmn;\u0026thinsp;5.781\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.019\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.959\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePCL/CHI@XyL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e4.806\u0026thinsp;\u0026plusmn;\u0026thinsp;0,121\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.010\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.991\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.2.4. Effect of Metal Ions on Enzyme Activity\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e compares the activities of free XyL and PCL/CHI@XyL in the presence of different metal ions (10 mM). Here, the control group represents the enzyme activities without any metal ions, and this value is accepted as the initial activity (%). From this point on, the presence of metal ions produces quite different effects on the free and immobilized enzymes. Monovalent ions (Na⁺ and K⁺) caused a slight increase in activity compared to the control in both enzyme forms. However, the increase was more pronounced in the immobilized enzyme, suggesting that ionic strength may facilitate the enzyme-substrate interaction. Remarkable differences were observed in the presence of divalent ions Ca\u0026sup2;⁺ and Co\u0026sup2;⁺. While the free enzyme exhibited only 1.1-1.2-fold activity, the activity of PCL/CHI@XyL increased approximately 1.9-2.10-fold. This suggests that divalent ions may generally have a cofactor-like effect in the active center of the enzyme and that these ions can bind more effectively to the enzyme surface thanks to the immobilization matrix (PCL/CHI) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, 3\u003csup\u003e+\u003c/sup\u003e ions increased the activity of the immobilized enzyme approximately 1.8-fold, while the free enzyme increased it 1.3-fold. This can be attributed to the amino groups in the chitosan structure interacting with highly charged ions such as Al\u0026sup3;⁺ to stabilize the enzyme's active conformation. One of the most striking results is the activity of both enzyme forms in the presence of Cu\u0026sup2;⁺ ion. While the free enzyme retained only 20% of its activity compared to the control in the presence of this ion, the opposite was true in PCL/CHI@XyL, with an approximately 2.5-fold increase compared to the control. This opposing effect suggests that immobilization suppresses the inhibitory properties of the Cu\u0026sup2;⁺ ion, protecting the enzyme from inactivation, and that Cu\u0026sup2;⁺ may even facilitate the catalytic process when bound to the immobilized enzyme. This effect can be explained by the fact that the functional groups of the support material (e.g., amino groups of chitosan) chelate metal ions and regulate the ion concentration reaching the enzyme's active site [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Consequently, PCL/CHI@XyL nanofibers offer a much more advantageous biocatalyst alternative compared to free enzymes in industrial environments where metal ions are concentrated.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e. Effect of metal ions on enzyme activity (A) Free XyL and (B) PCL/CHI@XyL\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e3.2.5. Reusability of PCL/CHI@XyL\u003c/h2\u003e\u003cp\u003eBecause free enzymes cannot be separated from the reaction medium and are easily affected by environmental conditions, they cannot be reused repeatedly. This restricts the industrial use of enzymes. To overcome this problem, the development of immobilization-based biocatalyst systems is widely used. The graph shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e shows the reusability results of Xylanase immobilized on PCL/CHI nanofibers (PCL/CHI@XyL). The initial activity assumed to be 100% in the first cycle was retained at approximately 45% of its initial activity after the fifth cycle. This decrease in the immobilized enzyme is thought to be due to mechanisms such as partial inactivation, desorption from the support, or structural changes during the reuse process [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The results observed in the graph reveal that Xylanase immobilized on PCL/CHI nanofibers is a longer-lasting and economically viable biocatalyst candidate compared to its free form.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e. Reusability of PCL/CHI@XyL.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eHere, the immobilization of Xylanase enzyme onto PCL/CHI nanofibers obtained via electrospinning was successfully achieved. The structural and morphological properties of the resulting PCL/CHI@XyL were investigated through various characterizations, and their biochemical parameters were extensively studied. SEM, XRD, EDX, and FT-IR characterizations confirmed that the nanofibers maintained their integrity during the immobilization process and that covalent bonds were formed with the enzyme. In the biochemical evaluations, the optimum pH value was determined to be 6 for free XyL, while it decreased to 5 for PCL/CHI@XyL, indicating that the carrier matrix provided a more acidic microenvironment for the enzyme. Furthermore, although the optimum temperature was determined to be 50\u0026deg;C for both enzyme forms, the immobilized enzyme maintained its structural stability at higher temperatures and was more resistant to thermal denaturation. Kinetic parameters values ​​of the free and immobilized enzymes were also investigated, revealing that the immobilized enzyme has a lower Km value and a higher affinity for the substrate compared to the free enzyme. Furthermore, the significant decrease in activation energy indicates that immobilization enables catalytic reactions to occur under lower energy barriers. Tests conducted in the presence of metal ions revealed that the immobilized enzyme develops a protective resistance, particularly against inhibitory ions such as Cu\u0026sup2;⁺, while some ions play a role in enhancing catalytic activity. Finally, reusability experiments revealed that the immobilized enzyme retained approximately 45% of its activity after five cycles, providing a significant economic advantage for industrial applications. In light of all these findings, PCL/CHI@XyL nanofibers have broad potential not only for Xylanase but also for the immobilization of various enzymes and their use in industrial bioprocesses. Therefore, this study provides an important contribution towards the development of sustainable, economical and high-performance biocatalysts using nanofiber-based support materials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not involve human participants or animals; therefore, ethics approval and consent to participate were not required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs the corresponding author of this study, I declare that I have read and approved the final version of the article titled “Electrospun Polycaprolactone-Chitosan Nanofiber Scaffolds for Covalent Immobilization of Xylanase: Structural Characterization and Enzyme Performance”, confirm that the work is original, unpublished, and free from legal or institutional restrictions, and give full approval for its submission to the journal, its publication in Catalysis Letters, and the transfer of all rights requested by the journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declares that there are no known conflicts of interest or personal relationships that could influence the work reported in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sole author conducted all research, analysis, and writing for this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe writers utilized ChatGPT 5 to enhance the text's coherence and clarity, while also refining the language and scientific terminology employed in this study. The writers took full responsibility for the publication's content after utilizing this tool/service, reviewing and editing it as necessary.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and materials supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI would like to thank Prof. Dr. Burhan Ateş, Assoc. Prof. Dr. Ahmet Ulu, Kübra Karadaş Gedik, and Gamze Dik for their valuable support in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMendon\u0026ccedil;a M, Barroca M, Collins T (2023) Endo-1,4-β-Xylanase-containing glycoside hydrolase families: characteristics, singularities and similarities. 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Bulletin of the National Research Centre 2021 45:1 45:1\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/S42269-021-00649-0\u003c/span\u003e\u003cspan address=\"10.1186/S42269-021-00649-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"catalysis-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Catalysis Letters](https://link.springer.com/journal/10562)","snPcode":"10562","submissionUrl":"https://submission.springernature.com/new-submission/10562/3","title":"Catalysis Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Xylanases, Enzyme Immobilization, Electrospinning, Nanofibers, Biocatalysts","lastPublishedDoi":"10.21203/rs.3.rs-7775653/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7775653/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eXylanases are critical enzymes that release xylose and its derivatives through the hydrolysis of xylan, the primary component of hemicellulose, and are of great importance to many industrial fields. However, there are several factors that limit their use in free form. Enzyme immobilization stands out as an important strategy to overcome these limitations. In this study, PCL/CHI nanofibers were synthesized by electrospinning, and an innovative nanocarrier platform was developed for Xylanase immobilization. A solution containing 10% PCL and 0.5% CHI was converted into nanofibers by electrospinning under optimized conditions (22 kV, 1 mL/h, 18 cm) and then cross-linked with glutaraldehyde to make them suitable for enzyme immobilization. SEM, EDX, XRD, and FT-IR analyses confirmed the morphological and structural integrity of the nanofibers. When comparing free and immobilized xylenese, the optimum temperature was determined as 50\u0026deg;C for both forms, while the optimum pH was determined as 6 for the free form and 5 for the immobilized form. The decrease in activation energy from 21.46 kJ/mol to 1.17 kJ/mol in the immobilized form indicated that the reaction occurred with a lower energy barrier. Furthermore, the decrease in Km value revealed that immobilization enhanced enzyme-substrate interaction, while reusability tests showed that the immobilized enzyme retained 45% of its initial activity after five cycles. The fact that the immobilized form maintained its high catalytic performance in the presence of metal ions highlights the system's potential for adaptation to industrial conditions. In conclusion, this developed platform has been demonstrated to be a promising approach for sustainable and economical solutions in enzyme technologies.\u003c/p\u003e","manuscriptTitle":"Electrospun Polycaprolactone–Chitosan Nanofiber Scaffolds for Covalent Immobilization of Xylanase: Structural Characterization and Enzyme Performance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-26 08:40:32","doi":"10.21203/rs.3.rs-7775653/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-01T08:55:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-31T08:31:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T19:59:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"220149778677797810970444305159842166614","date":"2025-10-12T15:31:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"127033744191573627013951384485962435060","date":"2025-10-12T13:39:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52427234570266987972757594529901039946","date":"2025-10-10T13:04:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-10T11:19:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-06T13:50:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-06T13:49:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Catalysis Letters","date":"2025-10-03T18:14:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"catalysis-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Catalysis Letters](https://link.springer.com/journal/10562)","snPcode":"10562","submissionUrl":"https://submission.springernature.com/new-submission/10562/3","title":"Catalysis Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"acdb9850-f660-42ad-824a-e350503847b3","owner":[],"postedDate":"October 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-23T12:09:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-26 08:40:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7775653","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7775653","identity":"rs-7775653","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}