Introduction

Nanotechnology, a groundbreaking boon in scientific progressions, has revolutionized different areas harmonizing technology and sustainability. With growing advancement, there is a basic need to utilize green innovations that accommodate development with ecological responsibility. Electrospinning is one such strategy that investigates nanotechnology applications, specifically the fabrication of biodegradable nanoscale filaments with alterable features. It works by utilizing electrical charges to mix biodegradable polymeric arrangements with organic substances into finely designed nanostructures, thus encouraging green innovation1. Hence, it leverages nanotechnology, chemistry, and biotechnological standards to address different concerns related to the ecosystem, discharging harmless end-products2. Besides, this strategy is beneficial as it possesses negligible equipment complexity, requires minimal samples and is confined inside a chamber.

Enzyme immobilization is a vital method to enhance the thermostability, catalytic productivity, reusability, and longevity of proteins3,4. Free/Unbound proteins have several impediments; they act in particular conditions and are prone to degradation5. These inadequacies can be viably addressed by utilizing the immobilization strategy6. Among various enzymes, carboxylesterase has become the enzyme of choice due to its ability to efficiently detoxify insecticides, such as organophosphate, pyrethroids, and carbamate, through different chemical reactions7. These cofactor-free enzymes specifically bind to substrates for performing transesterification, sequestration, hydrolysis, and esterification, thereby detoxifying harmful pollutants8. Carboxylesterases get chemically and physically incorporated into solid support during immobilization, and this prevents the denaturation of the enzyme caused by extreme conditions or proteolysis and enhances its potential as a valuable tool for real-world applications. Immobilization enhances enzyme stability and efficiency, reduces costs and improves process viability, thereby enhancing its bioremediation application. The essential objective for immobilization is the improvement of protein function and volume-specific protein stacking9. A few strategies of protein immobilization exist, counting retention on network, covalent interaction, entanglement, cross-linking and entrapment10,11. Within the realm of nanotechnology, the fabrication of nano-based tubes, particles, sheets, films and strands has picked up conspicuousness for protein immobilization12. Among these nanomaterials, nanofibers are the most favored choice due to their inalienable preferences, such as crosslinking capability, uniform scattering, high porosity, and capacity for self-assemblage13.

Chitosan, a semi-crystalline polysaccharide comprising of N-acetyl-d-glucosamine and d-glucosamine, is shaped through the deacetylation of chitin14,15. It is biodegradable, less harmful, and possesses antimicrobial properties with heightened biocompatibility16. The polycationic nature of chitosan encourages interaction with contrarily charged substances, such as proteins, through ionic cross-linking17. Chitosan has been selected as the immobilizing polymer among all synthetic and natural polymers because of its distinctive properties. Its distinctive features, such as gel-forming ability, mechanical strength, physiological inertness, high affinity for proteins, stability of the immobilized entity, facilitation of smooth biological interactions and abundant natural production, make it a suitable choice as an immobilization platform18. Moreover, chitosan is a noteworthy polymer due to its physicochemical properties, encompassing rigid crystallite formation in solid-state structure and fine microfibril formation in dissolved-state conformation1. However, its dissolvability in acidic fluid arrangements, agglomeration inclination, and electrostatic repulsion pose challenges for manufacturing chitosan strands14,19. To overcome these challenges in electrospinning, co-spinning with exceedingly electrospinnable biodegradable polymers like polyvinyl alcohol (PVA) is utilized20,21. These co-spinning polymers neutralize repulsions, permitting higher chain traps and the generation of smooth nanofibers17. PVA, known for its water dissolvability, thermostability, malleable quality, adaptability, and linear chain structure, is especially favored for electrospinning22,23. Various considerations have reported the broad utilization of PVA-chitosan nanofibers over differing applications, such as the medical field, filtration handle, wound recuperating, and biosensors24,25. The unique properties of chitosan and PVA make them great candidates for bioremediation applications.

In our earlier report, we broadly characterized the pesticide-resistant carboxylesterase protein Ha006a and detailed its bioremediation potential23. Building upon that, this investigation points to the progress of the enzyme's properties and broadening its application. The existing research acknowledges the immobilization of enzymes on various supports, there is a research gap regarding the immobilization of pesticide-resistant insect carboxylesterase on PVA-CS nanofibers and its subsequent characterization. The current study centers on optimizing and creating PVA-CS nanofibers utilizing changing proportions of polyvinyl alcohol and chitosan. The optimized polymer arrangement is utilized to immobilize Ha006a protein, making enzyme-based PVA-CS nanofibers. Electrospinning parameters, protein concentration, nanofiber morphology, and flow rate are explored in detail. The structure, crystallinity, thermal property, and other characteristics of the manufactured nanofibers have been examined. Strategies such as conductivity meter, viscometer, FESEM, TGA, XRD, and FTIR are utilized. Besides, the esterase property of the immobilized protein has been inspected, and its stability has been surveyed for one year.

Methodology

Materials

All the chemicals, including PVA (cold water soluble, partially hydrolyzed, Molecular weight = ~ 160 kDa), CS (≥ 75% deacetylated, low molecular weight), and acetic acid (formula weight: 60.05 g/mol, purity > 99.5%) were bought from Sigma Aldrich and HiMedia. The columns for protein purification were obtained from GE Healthcare. From Millipore Corporation, the dialysis bag, syringe filters and 10 kDa protein concentrator were obtained.

Freshly purified Ha006a protein

A variety of chromatographic methods were used to purify the Ha006a protein (GenBank® accession number PP204289). For detailed information on the purification and characterization of Ha006a, refer to our previous work26. After purification, the Ha006a was dialyzed against 20 mM sodium phosphate buffer (pH 7.0), and 1 µM of the purified Ha006a was used for experimental procedures. The protein was then concentrated, filtered and kept at a constant temperature of 4 ℃ for sample preparation.

Preparation of nanofiber mixture

After adding 1.2 g of PVA to 10 mL of buffer with pH 5.0 sodium acetate composition, the mixture was stirred with a magnetic stirrer for a duration of 6–8 h to ensure its solubility at room temperature. Simultaneously, 0.2 g CS was added to another 10 mL buffer, along with 10% acetic acid to enhance solubility, and subjected to a magnetic stirrer for homogeneous mixing. Consequently, stock solutions of 12% PVA (w/v) and 2% CS (w/v) were concocted for the experiment. These stock solutions were combined in varying ratios PVA-CS (v/v) as specified in Table 1. Each sample PVA-CS solution mixture was kept on a magnetic stirrer for homogeneity for two hours at room temperature. All the sample PVA-CS polymer mixtures were subjected to electrospinning. The electrospun sample nanofibers were optimized based on factors such as fiber texture, flow rate, fiber morphology and most importantly, the conductivity of the nanofiber. The optimized PVA-CS mixture was consistently utilized throughout the subsequent experiments.

Table 1 Optimization of the ratio of PVA-CS polymer mixture for electrospinning nanofibers. PVA was dissolved in buffer and CS was dissolved in acidic aqueous solution.
Full size table

Immobilization of Ha006a protein

The electrospinning process maintained a steady application of 21 kV voltage, maintaining a gap of 12 cm for consistent production of fibers. The optimized electrospinning parameters are depicted in Table 2. Various concentrations of the carboxylesterase enzyme Ha006a were integrated into polymer solutions for electrospinning, housed in a 10 mL syringe with a 0.8 mm diameter needle. Optimization of the electrospun fibers was achieved by adjusting the flow rate and Ha006a concentration ranging from 0.1 to 0.7 mL/h and 0.5–3.0%, respectively. The detailed procedure for carrying out the electrospinning process is illustrated in Fig. 1. Additionally, nanofibers serving as control were produced by electrospinning the PVA-CS composite without the addition of enzyme and precisely maintaining the optimized parameters.

Table 2 Electrospinning parameters.
Full size table
Figure 1
Figure 1
Full size image

Detailed representation of the steps involved in the fabrication of control (PVA-CS) and Ha006a-PVA-CS nanostructures employing the electrospinning method.

Rheological properties and conductivity assessment of electrospinning solution

The rheological characteristic of the polymer solutions employed in the nanofiber fabrication was investigated. The viscosity of the control polymer solution and the Ha006a-PVA-CS polymer suspension was determined using a Rheometer (Rheoplus/32 Service V3.62 21000071-33086). The experiment involved varying the shear rate in the range of 0.1–1000 s−1, and all measurements were carried out at 25 ± 2 °C. The graphical representation was documented, illustrating the correlation between the viscosity of the polymer solutions to be electrospun and the shear rate. Additionally, another property, the conductivity of the PVA-CS polymer mixture and the Ha006a-PVA-CS polymer suspension was gauged at 25 ± 2 °C using a conductivity meter (DDSJ-308A, INESA & Scientific Instruments Co).

Characterization of fabricated nanofibers

The synthesized nanofibers underwent comprehensive characterization to elucidate their chemical composition, structural features, morphology, thermal behavior, and various other parameters. In order to ensure authenticity and obtain valuable insights into the nanofibers, both prior to and following the enzyme immobilization approach, a series of instrumental analyses were conducted. Notable techniques employed are discussed.

Apreo field emission scanning electron microscope (FESEM)

The structural characterization involved a comprehensive examination of the diameter and morphological attributes of the fabricated nanofibers. This scrutiny was conducted utilizing an Apreo Field Emission Scanning Electron Microscope (FESEM) with a low vacuum of the Quanta 200F Model, manufactured by FEI, Netherlands. To optimize and enrich the conductivity of the nanofibers, specimens measuring 10 × 10 mm were subjected to gold coating, a process facilitated by the Baltech SC005 sputter coater, Switzerland. Subsequently, high-resolution imaging of PVA-CS and Ha006a-PVA-CS nanofibers was captured at various resolutions in 5000–10,000 magnification employing the FESEM. Quantitative analysis of nanofiber diameters was accomplished through the calculation of 100 randomly selected calibrated diameter sizes from FESEM images, executed using ImageJ software27.

Fourier Transform-Infrared Spectroscopy (FTIR)

The assessment of the functional groups, chemistry, and composition of the nanofibers was executed using Perkin Elmer's Fourier Transform Infrared (FTIR) spectroscopy. A systematic process was employed wherein the nanofibers were effectively crushed and blended with potassium bromide (KBr) to form KBr pellets. Subsequently, the resulting infrared spectrum was captured from the wavenumber span of 4000 cm−1 to 400 cm−1, utilizing a scanning resolution of 4 cm−1.

X-ray Diffraction (XRD)

The Thin Film X-ray Diffraction (XRD) instrument, Rigaku, was utilized to examine the structural characteristics of nanofibers both before and after enzyme immobilization. Assignment of peaks, peak shifts and intensities were observed to assess the composition of the manufactured nanofibers. The study employed a 2-theta angle from 2° to 60°, with a fixed 0.02° step size. The scan speed was maintained at 2.7°/min, and a spacer of 0–3 mm was consistently applied throughout the 20-min total scan duration.

Thermo Gravimetric Analyzer (TGA)

The thermal analysis of the control and enzyme-incorporated fibers was conducted using Thermo Gravimetric Analyzer (TGA) (Model:—SII 6300 EXSTAR). The nanofibers were 10 mg in weight, and before the onset of the thermal analysis, the fibers were placed under vacuum for a day. The thermal stability was examined by heating samples up to 500 °C, maintaining a scanning speed of 10 °C/min in nitrogen environment.

Enzyme activity

Esterase activity of unbound and bound Ha006a protein was conducted following the methodology outlined in the previous research26. The generation of the product, α-naphthol at 450 nm was assessed spectrophotometrically. The reaction mixture of pH 7.0 with sodium phosphate buffer, a conjugate dye, Fast Blue B salt (3 mM) and substrate, α-naphthyl acetate (α-NA) (10–160 µM), was used to evaluate the enzyme response26,28. The intensity of the purple color development is directly proportional to the substrate hydrolysis rate. The diazonium group reacts with the phenolic group of α-naphthol, producing a conjugated azo compound, which gets detected spectrophotometrically at 450 nm. Around 4 µL of diluted Ha006a (20 nM) or 4 mg of enzyme-immobilized nanofiber disc was incubated in the buffer for 10 min. Subsequently, the readings were recorded by introducing substrate solution (having dye), and color change was monitored spectrophotometrically for 10 min. Respective blanks were maintained for both the free Ha006a and immobilized protein reaction mixture. The coefficient of the product (9.25 mM−1 cm−1) was considered for the calculation of kinetic parameters (Km, Vmax) as reported by researchers26,29,30. Three readings were recorded for each substrate concentration, and the analysis was conducted by performing respective blank subtraction.

Evaluating temperature range of unbound and immobilized Ha006a

The best temperature range for immobilized and free Ha006a enzyme was analyzed, the activity was evaluated between 5 and 70 °C with a 5 min incubation time. The instructions provided in “Enzyme activity” were rigorously adhered to by the enzyme assay processes. In order to evaluate the thermostability in both conditions, the incubation period was extended to thirty minutes. The activity was plotted as relative percentage; 100% was considered to be the maximum activity.

Evaluating the pH range of unbound and immobilized Ha006a

Various buffer conditions were incorporated in the study to explore the optimal pH for esterase activity pre- and post-immobilization. The reaction mixture consisted of alpha naphthyl acetate, Fast Blue B salt and 20 nM Ha006a in different pH buffers. Respective blanks were maintained for each condition. Enzyme response was monitored and represented in the form of percentage, with the highest activity set at 100%.

Stability of unbound and bound Ha006a

The enzyme response was assessed at 60-day intervals throughout the course of one year. Spectrophotometric analysis was employed to evaluate the catalytic efficiency of the enzyme over the 12-month period. Consistent enzyme mixtures were utilized, spanning from month 0 to month 12, with corresponding blanks maintained for each reaction mixture. The activity was plotted in the form of percentage, declaring the maximum response as 100%.

Statistical analysis

A one-way ANOVA analysis was performed to examine the distribution of nanofiber size diameters (https://astatsa.com/OneWay_Anova_with_TukeyHSD/). The statistical significance of an input parameter, as indicated by the p-value, was comparatively assessed, with values of ≤ 0.05 considered statistically significant. Furthermore, an unpaired t-test was employed to compare and evaluate the means of the optimized control and enzyme-immobilized electrospun nanofibers. The objective was to evaluate whether there was a significant difference in the mean nanofiber diameter between the two samples.

Results and discussion

Fiber morphology, conductivity and rheological properties

The concentration of polymer immensely contributes to shaping the texture and morphology of fibers, influencing parameters such as surface tension, viscosity and conductivity. This connection is evident in the obtained FESEM images for different combinations of PVA-CS nanofibers, wherein the analysis of fiber diameter was conducted (Fig. 2). All the nanofibers exhibited fine texture and uniform distribution, except for the PVA100 mixture, which displayed some bead-like structure within its fiber composition. To enhance the overall quality of the nanofibers, chitosan, serving as an ionic additive, was introduced into the polymer solution. Previous studies have emphasized the role of chitosan as a thickening agent, effectively enhancing solution viscosity without compromising functionality31. The incorporation of the cationic polyelectrolyte, chitosan, acting as a thickener, resulted in the production of non-beaded and uniform fibers. It is well-established that polyvinyl alcohol (PVA) strongly interacts with chitosan through hydrogen bonding31. To enhance tensile strength, a higher concentration of PVA is preferable for fabricating uniform nanofibers, and in this context, the PVA concentration was maintained higher throughout the optimization process. The breadth of the sample nanofibers was 218–550 nm, depicting the smoothest and best morphology in the range of 218–287 nm. Additionally, this sample exhibited an optimal fiber morphology with no observable beads. The augmentation of chitosan concentration significantly improved fiber morphology, aligning with findings from previous research1,32. With the gradual elevation of CS, the homogeneous mixing of polymer solution leading to a decrease in bead density and fiber diameter was observed (Fig. 3A). The chemistry behind this can be explained as CS is a cationic polymer; at pH 5, the amino group (–NH2) is protonated, attributing to repulsive forces and hence expanding the CS to link with the co-spinning partner14. The –OH group of PVA thereby reacts with the protonated amino group of CS, generating intermolecular hydrogen bonds. With increasing CS concentration, the existence of both intra- and intermolecular bonds between PVA-CS increased and hence, the decrease in diameter was observed owing to compact interaction. The comprehensive information regarding the properties and attributes of each sample nanofiber is provided in Table 3.

Figure 2
Figure 2
Full size image

FESEM images (10,000× magnification) and fiber diameter histograms of fabricated nanofibers. (A) PVA100 FESEM image, (B) PVA100 histogram plot, (C) PVA96 FESEM image, (D) PVA96 histogram plot, (E) PVA94 FESEM image, (F) PVA94 histogram plot, (G) PVA92 FESEM image, (H) PVA92 histogram plot, (I) PVA90 FESEM image and (J) PVA90 histogram plot. For each fabricated nanofiber sample, a total of 100 readings of calibrated fiber diameter were considered using ImageJ software. The histogram plot depicting the mean value of each sample is represented using Origin software.

Figure 3
Figure 3
Full size image

The representation of fiber diameter and the conductivity of each sample nanofiber. (A) The average fiber diameter with standard deviation in the form of error bars is depicted for all nanofiber samples. The statistical analysis was performed to compare the diameter of PVA100 with four other samples. The p-value observed in all the cases was < 0.01 and hence displayed on the graph. (B) The relationship between solution conductivity and average fiber diameter for PVA-CS nanofiber samples is depicted.

Table 3 Properties of PVA-CS fabricated samples.
Full size table

The investigation of fiber diameter size involved One-way ANOVA analysis. The ANOVA results signified that when comparing PVA100 with other fabricated nanofibers containing CS, the p-value was significantly lower than the test value (α = 0.05) (Table 4). These results provide confirmation that the diameter of the nanofibers is associated with CS, with statistical analysis highlighting a significant difference.

Table 4 One-way ANOVA with post-hoc Tukey HSD test for comparing the PVA100 fabricated nanofibers with CS-containing nanofibers (PVA96, PVA94, PVA92 and PVA90).
Full size table

Previous studies have reported that an elevated PVA concentration leads to an increase in conductivity31. Other studies have also established a direct correlation between PVA concentration and solution conductivity33,34. Therefore, in this investigation, a higher PVA concentration (10.8–12%) was maintained in comparison to CS. On the other hand, with the increasing concentration of CS from PVA100 to PVA90, a slight elevation in acetic acid concentration occurred, as acetic acid is essential for CS solubility. This increase in CS concentration resulted in a corresponding rise in conductivity, aligning with previous findings34. Consequently, it can be asserted that the presence of acetate ions and protons in the solution significantly contributed to the heightened conductivity. Figure 3B illustrates a graph displaying an ascending trend in conductivity.

Rheological parameters, such as viscosity, play a pivotal role in determining the morphology of nanofibers. Generally, there is a documented trend indicating that the average diameter of electrospun nanofibers tends to increase with the rise in viscosity. Our study aligns with this observation, as evidenced by the plotted flow curves illustrating viscosity as a function of shear rate for Ha006a-PVA-CS and PVA-CS nanofibers (Fig. 4). It was observed that the viscosity escalated from 4790 to 15,600 mPa.s following the incorporation of Ha006a into the polymer solution. The flow curves distinctly affirm the shear rate dependence in both scenarios, exhibiting heightened viscosity in the case of enzyme-immobilized nanofibers. As the shear rate increased, both polymer mixtures showed shear-thinning property, wherein viscosity declined with the ascending shear rate. This phenomenon could be attributed to the disruption of crosslinked networks at high shear rates, while the polymer solutions demonstrated elevated viscosity at low shear rates35.

Figure 4
Figure 4
Full size image

Viscosity vs. shear rate plot of control (PVA-CS) and enzyme immobilized (Ha006a-PVA-CS) polymer solution employed in the electrospinning method.

Optimization of electrospinning parameters

The quality assessment of enzyme-immobilized nanofiber involves the optimization of various factors, including polymer rheological property, fiber morphology, flow rate, and enzyme concentration36. The electrospun fibers were cut in uniform disc shapes with 0.4 cm diameter and placed in the 96 well plate containing assay solution. The enzyme activity was investigated following the protocol mentioned in “Enzyme activity”. For each assay condition, a respective blank was maintained. The enzyme response of the immobilized Ha006a was analyzed at a flow rate spanning from 0.1 to 0.7 mL/h. The esterase activity was very low in the range of 0.1–0.3 mL/h, possibly attributed to prolonged exposure to high voltage impacting catalytic activity. Moreover, beads were seen in the spun fibers when the rate was set to greater than 0.6 mL/h. The highest esterase activity was obtained at 0.5 mL/h flow rate (Fig. 5A), and this activity level was sustained during the investigation.

Figure 5
Figure 5
Full size image

Optimization of electrospinning parameters to achieve the highest enzyme activity. (A) Flow rate optimization graph. (B) Enzyme concentration optimization graph. The highest activity is depicted as 100% in both the plots.

The esterase activity was elevated when enzyme concentration was progressively changed from 0.5 to 3% in the nanofiber mixture. Notably, the esterase activity exhibited over 50% activity within 1.0–3.0% enzyme concentration range. At an enzyme concentration of 2.0%, the highest catalytic activity was noted (Fig. 5B). Due to the limited interacting surface of the polymer, a decrease in esterase activity was seen as the enzyme concentration rose above the saturation point37. A number of studies have postulated that factors like steric hindrance, enzyme aggregation or absence of electron transferring media may be the reason behind low catalytic activity at high enzyme concentration38,39. Consequently, the 2.0% enzyme concentration was identified as the optimum level for fabricating Ha006a-PVA-CS nanofibers.

Characterization of electrospun fibers

Characterization by FESEM

The FESEM images of control nanofibers and Ha006a-PVA-CS fibers are presented in Fig. 6. The nanofibers displayed homogenous uniformity in texture with a diameter range of 170–225 nm. The fibers were smooth and bead-free in morphology. The enzyme-immobilized nanofibers depicted homogenous texture and proper mixing with the polymer solution. The average diameter of the control and Ha006a-PVA-CS fibers was 170.13 ± 44.22 nm and 225.28 ± 66.56 nm, respectively. With the introduction of the enzyme, the diameter of the nanofibers increased.

Figure 6
Figure 6
Full size image

FESEM images (5000–10,000× magnification) and fiber diameter histograms of PVA90 optimized electrospun nanofibers. (A) FESEM image (5000× magnification) of PVA-CS control nanofiber with the embedded image showing an image of (10,000× magnification) of the same sample. (B) Histogram plot of PVA-CS nanofiber diameter depicting the mean and standard deviation values. (C) FESEM image (5000 × magnification) of Ha006a-PVA-CS control nanofiber with the embedded image showing an image of (10,000× magnification) of the same sample. (D) The histogram plot of Ha006a-PVA-CS nanofiber diameter depicts the mean and standard deviation values. The values were obtained by considering 100 readings of calibrated fiber diameter using ImageJ software. The histogram plot depicting the mean value of each sample is represented using Origin software.

The nanofiber diameter of Ha006a-PVA-CS exhibited a significant difference compared to the control group (PVA-CS) (t = 6.988, df = 198, p < 0.0001, two-tailed). Moreover, the F-test indicated a significant difference in variances between the reaction and control groups (F = 2.264, (DFn) = 99, (Dfd) = 99, p < 0.0001). This statistical analysis collectively establishes evidence for a notable divergence in both means and variances between the enzyme-embedded and control nanofibers.

Characterization by TGA

The electrospun nanofibers showed an initial weight loss at 50–150 °C, which is attributed to the moisture vaporization. The major weight loss for Ha006a-PVA-CS was noted between 250 and 330 °C, which could be associated with PVA sidechain decomposition40. Whereas the PVA-CS nanofibers exhibited major weight loss between 240 and 310 °C. The last weight loss of Ha006a-PVA-CS and PVA-CS started at 350 °C and 320 °C, respectively and can be attributed to the degradation of polymeric chains of chitosan41. Thus, it was observed that Ha006a-PVA-CS nanofibers exhibited higher thermal stability as compared to the control nanofibers (Fig. 7).

Figure 7
Figure 7
Full size image

TGA plot of control and Ha006a-PVA-CS nanofibers.

Characterization by XRD

The X-ray diffraction plot of the control and Ha006a-PVA-CS fibers is depicted in Fig. 8. The control fiber revealed XRD peaks at 2θ = 7.18°, 19.46°, 22.2°, 38.48° and 44.7°. Whereas, Ha006a-PVA-CS fiber showed XRD peaks at 2θ = 19.46°, 22.2°, 30.0°, 38.48° and 44.7°. The diffraction impression around 2θ = 7.18 was observed for PVA-CS nanofibers, but it disappeared with the addition of the enzyme. The reason behind it may be the strong interaction of Ha006a with PVA-CS resulted in weaker impression at 7.18° as the literature depicts that such peaks signified pure PVA fibers34. A prominent peak at ~ 19.5° was observed, which is reported to represent the PVA content in the solution42,43. A small peak at 22.2 indicates crystalline peak of CS which signifies the presence of intra- and inter-molecular hydrogen bonds44. A shoulder peak at 2θ = 30° was observed in Ha006a-PVA-CS fiber; this peak signified a strong interaction between nanofiber and enzyme45. The peaks at 2θ from 35° to 55° represent the amorphous content of chitosan44. Moreover, the semi-crystalline property of polyvinyl alcohol is denoted by a peak at 2θ = 38.48°; this is attributed to intramolecular and intermolecular hydrogen bonds. According to the XRD peak results, uniform mixing of the Ha006a into the PVA-CS fiber content was observed as identical peaks were observed in the control and enzyme-immobilized nanofibers.

Figure 8
Figure 8
Full size image

XRD graph of control and Ha006a-PVA-CS fabricated fibers.

Characterization by FTIR spectroscopy

FTIR spectra of four samples: polyvinyl alcohol, chitosan, PVA-CS fiber, and the Ha006a-PVA-CS are shown in Fig. 9. The FTIR spectra with specific assignment of the functional group are depicted in Table 5. A prominent band in the area 3310–3319 cm−1 corresponding to N–H/O–H stretching, intermolecular and intramolecular hydrogen bonds were observed in spectra of all the samples46. The shift in the band was observed, which further validated the interaction of the polymer solution with the enzyme. The absorption bands around 2909–2943 and 2870 cm−1, assigned by earlier researchers to denote C-H symmetric and asymmetric stretching, respectively were observed in the samples37,47. In the case of CS, both the peaks were observed, but all other three samples depicted symmetric stretching. The C=O carbonyl stretching from the acetate group was observed around 1720–1731 cm−1 for PVA containing nanofibers48,49. The C=O stretching was observed for CS at around 1642 cm−1 and this peak was absent in all three other samples46. A band at 1562–1586 cm−1 denoting the N–H bending was observed in the spectra containing CS in their polymer solution50. Other bending and symmetrical deformations were validated by the occurrence of peaks in the range of 1422–1428 and 1372–1375 cm−1, respectively46. The C-H deformation or C-N stretching in CS is depicted by a peak around 1325 cm−1, and this was observed in all the samples46. The C–H wagging mode appeared around 1239 cm−1 for PVA and in fabricated fibers49,51. The wagging behavior occurs after the acetylation process, signifying the conformational change of PVA on interaction with enzyme and CS. Bands in 1066 and 1027 cm−1 correspond to C–O stretching and the samples showed peaks around this range37,48,49. The C–O–C bridge of PVA-containing fibers was signified at 1023 cm−1 position37,46. The band around 840 cm−1 signified C–C stretching48.

Figure 9
Figure 9
Full size image

FTIR spectra of PVA, CS, control, and Ha006a-PVA-CS electrospun fibers.

Table 5 FTIR absorption bands of PVA, CS, PVA-CS and Ha006a-PVA-CS samples.
Full size table

The FTIR data indicated that the peaks of stretching, bending, wagging, and other conformations were shifted to upper frequency after the addition of Ha006a enzyme to the PVA-CS mixture. Some other bands of stretching and hydrogen bonds in CS got sifted to a lower frequency in the case of Ha006a-PVA-CS fibers. No shift in frequencies was observed for C=O carbonyl stretching from the acetate group, CH2 bending, CH3 symmetrical deformation, and C–O–C stretching in control and Ha006a-PVA-CS nanofibers. These shifts in bands further validate the strong interaction profile of the polymer solution with the enzyme.

Determination of optimum temperature

To gain insights into the temperature relation to the esterase activity of Ha006a, the relative response of the unbound and bound enzyme was evaluated from 5 to 70 °C (Fig. 10A). The unbound enzyme showed enhanced esterase activity at temperatures up to 40 °C and had negligible activity at 50–70 °C. The most favorable temperature of the unbound Ha006a was calculated as 35 °C. Whereas the bound enzyme showed an optimum esterase response of 40 °C. The bound enzyme showed > 50% enzyme response from 20 to 45 °C condition. The highest response was observed in the range of 30–40 °C. Thus, it was observed that there was an increase in enzyme stability by 5 °C after immobilization. The reason behind the enhanced temperature stability can be attributed to the covalent interaction between the enzyme and the nanofiber matrix. This interaction stabilized the enzyme, thereby making it stable and preventing it from denaturation. Similar justifications supporting the elevated stability of the immobilized enzyme have been documented by other researchers52.

Figure 10
Figure 10
Full size image

Comparative analysis of the esterase activity of unbound and immobilized enzyme, Ha006a. Graph to determine (A) the optimum temperature, (B) the thermostability, (C) the optimum pH of Ha006a. In each case, three readings were taken, and the plotted values represent averages, with error bars indicating standard deviation. The esterase enzyme activity was analyzed by (D) the Michaelis–Menten graph of the immobilized enzyme, (E) the Lineweaver–Burk Plot of the immobilized enzyme, and (F) the stability of both unbound and immobilized enzyme.

Thermostability of unbound and bound/immobilized enzyme

Thermostability of the unbound and bound Ha006a was evaluated by performing enzyme activity from 5 to 70 °C and subjecting the enzyme to these temperatures for a longer duration (Fig. 10B). In lower temperatures, 5–15 °C, the free enzyme showed low catalytic activity. It was noted that the unbound enzyme exhibited esterase activity till 40 °C, and it almost lost its activity after 50 °C. Whereas the bound enzyme exhibited an esterase response up to 45 °C. The bound enzyme revealed a good catalytic response at a lower temperature (10–35 °C) and exhibited the highest activity at 40 °C. The enzyme was active till 55 °C, depicting the temperature resistance of the immobilized enzyme. Furthermore, it was noted that the immobilized enzyme exhibited elevated enzyme activity throughout all temperatures (10–65 °C) as compared to the free enzyme. Previous studies have explored the rationale behind the increased thermostability of the enzymes, focusing on factors such as interactions involving hydrogen bonds and covalent bonds. This understanding contributes to their ability to develop thermal resistance, enabling them to endure elevated temperatures53.

Determination of optimum pH

The catalytic response of Ha006a was evaluated at pH conditions ranging from 4.0 to 11.0 (Fig. 10C). The catalytic response of the unbound Ha006a enzyme was best at pH 7.0, with good activity observed at pH 6.0–9.0. The immobilized enzyme showed optimal activity in the pH 7.0–10.0 range, with its maximum catalytic activity occurring at pH 8.0. For the unbound and immobilized condition, a noticeable change in the ideal pH for maximum enzymatic activity was thus observed. The enzyme showed improved stability and favorable activity in an alkaline environment. The strong connection between the enzyme and the nanofiber matrix was responsible for the notable change in stability at different pH levels54. This secondary interaction manifested through hydrogen bonds, impeded the enzyme mobility and played a crucial role in preserving the native conformation in diverse conditions55.

Enzyme activity

Kinetic parameters (Km and Vmax) of immobilized Ha006a were investigated employing varying substrate concentrations at optimized pH and temperature conditions and were compared with the reported value of free Ha006a26. A graphical representation of the Michaelis–Menten curve was generated by plotting the initial velocity achieved at substrate concentrations ranging from 10 to 160 µM (Fig. 10D). With the aid of the Lineweaver–Burk equation, the kinetic parameters of the immobilized Ha006a were calculated (Fig. 10E). The Km and Vmax values obtained were 12.27 ± 0.238 µM and 0.359 ± 0.006 µmoles/min, respectively. The immobilization of the enzyme prevented rapid substrate accessibility for its activity, contrasting with the free enzyme, leading to increased Km values. Additionally, the porous nature of the nanofibers and the steric hindrance induced by the polymer might be other contributing factors for the lower binding of the enzyme towards the substrate56.

Evaluating stability of bound and unbound Ha006a

The esterase response of the free and bound Ha006a enzyme was evaluated at an interval of every 60 days, and the relative activity is depicted in Fig. 10F. It was observed that the immobilized Ha006a was active for 12 months and it exhibited > 50% activity till a year when compared with day zero. Whereas, the free enzyme was observed to be active with activity > 50% up to 5 months. The detrimental activity was observed after 5 months as compared to day 0. Thus, the longevity of the enzyme activity increased drastically when it was immobilized on PVA-CS nanofibers. Several research investigations have demonstrated that the stability of enzymes can be improved through immobilization, and the present study aligns with those findings4.

Conclusion

In summary, we fabricated nanofibers using various mixture ratios of PVA/CS (v/v) polymers (100/0, 96/4, 94/6, 92/8, 90/10) and characterized the nanofiber samples by FESEM and conductivity meter. The outcomes underscored the significant influence of chitosan concentration on polymer solution conductivity, viscosity, fiber morphology, and diameter. Subsequently, the optimal PVA/CS (90/10) mixture was identified, and carboxylesterase enzyme Ha006a was incorporated at various concentrations (0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 wt. %). To improve stability, the Ha006a enzyme, which is renowned for its remarkable pesticide remediation abilities, was combined with PVA-CS polymer via the electrospinning technique. The control and Ha006a-PVA-CS fibers had average diameters of 170.13 ± 44.22 nm and 225.28 ± 66.56 nm, respectively. The structural, chemical, and thermal properties of the control (PVA-CS) and enzyme-immobilized (Ha006a-PVA-CS) nanofibers were thoroughly investigated. The diameter, homogeneity, and shape of the nanofibers were verified by FESEM and the uniform dispersion of the enzyme within the nanofibers was verified by X-ray diffraction. Thermal stability was revealed by TGA, and changes in chemical composition brought about by interactions between enzymes and polymers were tracked by FTIR spectra. The esterase activity of the unbound and bound Ha006a enzyme was systematically analyzed using diverse criteria and stability evaluations. This study offers improved results in pesticide remediation, ensuring enhanced enzyme stability and an economical approach by encapsulating carboxylesterase within nanofibers in a sustainable manner. Systematic analysis of esterase activity for both free and immobilized Ha006a enzyme encompassed various parameters and stability assessments. The meticulously designed and characterized nanofibers in this study enhance the authenticity and reproducibility, expanding the bioremediation potential of Ha006a post-immobilization.