Immobilization of TtAlaDH on magnetic nanoparticles: effects on stability and reuse

Abstract

Abstract

L-Alanine dehydrogenase (L-AlaDH) is a microbial enzyme that catalyzes the reversible oxidative deamination of L-Alanine to pyruvate. This enzyme catalyzes the reversible conversion of L-Alanine to pyruvate, while concurrently maintaining redox homeostasis through the NAD + /NADH coenzyme system. Despite the critical role of the L-AlaDH enzyme in biotechnological amino acid production, its implementation is hampered by poor operational stability, specifically difficulties in maintaining activity at high temperatures and ensuring adequate reusability. In this study, the recombinant plasmid encoding TtAlaDH from Thermus thermophilus was transformed into E. coli BL21 (DE3) for heterologous expression and was successfully immobilized on the surface of functionalized magnetic nanoparticles (F-NH₂ and FS-NH₂) (immobilized TtAlaDHs). The chemical characterization of magnetic nanoparticles and immobilized TtAlaDHs was validated using FTIR, SEM, EDS, TGA, and XRD investigations. This immobilization conferred significant advantages, including enhanced activity at elevated temperatures, improved long-term storage stability (at 4 °C) and reusability. Specifically, the TtAlaDH enzyme immobilized on the surface of the F-NH₂ magnetic nanoparticles demonstrated superior thermal stability, retaining approximately 85% of its initial activity at the elevated temperature of 85 °C. Furthermore, the F-NH₂-E magnetic nanoparticle system (F-NH₂-E) maintained 50% of the initial TtAlaDH activity following ten (10) repeated catalytic cycles. Crucially, immobilized TtAlaDHs demonstrated excellent storage stability, maintaining nearly 100% of its activity at 4 °C. Consequently, the demonstrated superior thermal and operational stability of immobilized TtAlaDHs, coupled with its magnetic separability, establishes this biocatalyst as highly promising for continuous flow biocatalysis. This enhancement provides a clear path toward the economic viability and scalability of L-Alanine production in industrial biotechnological applications.

Key points

• F-NH₂-E retains 85% activity at 85 °C

• 50% activity after 10 cycles with F-NH₂-E

• Near 100% storage stability at 4 °C

Introduction

As one of the 20 canonical amino acids, L-Alanine is among the smallest chiral biological compounds (Liu et al. 2022; Han et al. 2023a). It serves as both a fundamental component of cellular proteins and a critical building block of peptidoglycan. The production of L-Alanine represents an evolutionary ancestral metabolic pathway. D-Alanine is exclusively essential for bacterial cell wall biosynthesis, where it forms one of the three indispensable amino acids in the peptidoglycan repeating unit. D-Alanine is primarily obtained through racemization of L-Alanine, catalyzed by the enzyme alanine racemase (Galkin et al. 1999; Dave and Kadeppagari 2019; Sidiq et al. 2021; Han et al. 2023b). Concurrently, within the bacterial cytoplasm, L-Alanine plays a pivotal role in protein synthesis and is abundantly incorporated into numerous functionally significant proteins (Dave and Kadeppagari 2019). L-Alanine has been widely utilized in chemical, food, pharmaceutical, and veterinary applications, with global annual demand reaching approximately 50,000 tons. In the medical field, L-Alanine plays significant roles in diabetes research and treatment, serving as both a low-calorie sweetener and fat substitute. Furthermore, it finds application in drug delivery systems and is incorporated into various cleaning products (Liu et al. 2022; Han et al. 2023b; Nie et al. 2024) L-Alanine biosynthesis occurs via two principal enzymatic routes: (i) L-aspartate decarboxylation catalyzed by aspartate decarboxylase and (ii) the NAD(H)-dependent reductive amination of pyruvate mediated by L-Alanine dehydrogenase (L-AlaDH; EC 1.4.1.1) (Dave and Kadeppagari 2019). As a member of the amino acid dehydrogenase family of oxidoreductases, L-AlaDH performs the reversible interconversion of L-Alanine and pyruvate. This reversibility allows L-AlaDH to participate in both nitrogen assimilation (pyruvate → L-Alanine) and energy generation (L-Alanine → pyruvate) (Demir et al. 2024).

The increasing demand for sustainable production has resulted in a corresponding rise for enzymes (Almulaiky and Al-Harbi 2025). The high cost of enzymes, their restricted applicability, limited stability, and operational constraints within specific temperature and pH ranges present significant challenges (Zhou et al. 2022). Enzyme immobilization is necessary to address these issues (El-Shishtawy et al. 2025).

Magnetic nanomaterials enhance enzyme immobilization due to their extensive specific surface areas (Oraby et al. 2025) . The paramagnetic characteristics of Fe₃O₄ nanoparticles (F) enable the separation and recovery of immobilized enzymes through the application of an external magnetic field (Hadadi and Habibi 2025). Magnetic nanomaterial supports facilitate enzyme-based biocatalysis, minimizing operational costs. The surface of F is often modified with a coating of biocompatible, inert silica in biotechnological applications. This modification serves to enhance both functionality and biocompatibility, largely due to silica’s inherent high specific surface area. The main advantage of the silica-coated Fe₃O₄ (FS) surface exists in its resistance to highly acidic solutions and its straightforward conjugation with diverse functional groups. Studies indicate that FS nanoparticles exhibit biocompatibility in toxicity assessments (Sadeghi et al. 2023). The superparamagnetic properties of Fe₃O₄ are preferred in numerous applications, particularly in magnetic separation (You et al. 2021). Fe₃O₄ magnetic nanoparticles (MNPs) can be shown to be a more practical separation method via magnetic field interaction, for enzymes compared to conventional separation methods such as filtration and centrifugation (Guo et al. 2021). MNPs catalyze the oxidation of substrates in the presence of H₂O₂ and within a certain pH range, generating colorimetric reactions. This results from the peroxidase-like decomposition of H₂O₂, producing toxic hydroxyl radicals, disrupting bacterial cell walls, oxidizing cytoplasmic components, and causing cell death (Singh et al. 2018). Immobilizing enzymes onto Fe₃O₄ nanoparticles increases their effectiveness in chemical, biochemical, and industrial processes. Furthermore, immobilizing the enzyme with MNP increases its thermal and pH stability, enables rapid recovery, and enables repeated use of enzymes in different applications.

Existing literature indicates that investigations into the immobilization of the TtAlaDH enzyme are relatively rare. This study presents novel insights into the immobilization and optimization of the free TtAlaDH enzyme on F-NH₂ and FS-NH₂. The objective is to synthesize and characterize F-NH₂ and FS-NH₂ as supports for the free TtAlaDH enzyme, aiming to develop a biocatalytic system that exhibits improved activity and stability for biotechnological applications of free TtAlaDH. The magnetic characteristics of F-NH₂ and FS-NH₂ enable the easy recovery and reutilization of the immobilized enzyme, thereby addressing a major issue in this field. Examining the influence of magnetic forces and surface interactions on enzyme immobilization enhances comprehension of the nanoscale interactions between materials and enzymes.

Material and methodology

Material

Ferrous chloride hexahydrate (FeCl₃·6H₂O, 99%), ferric sulfate heptahydrate (FeSO₄·7H₂O, 99%), hexadecyltrimethylammonium bromide (CTAB, ≥ 98%), tetraethyl orthosilicate (TEOS, ≥ 99.0%, (GC)), and (3-aminopropyl) triethoxysilane (APTES) were purchased from Sigma-Aldrich (USA). Unless otherwise specified, all chemicals used in the experiments were purchased from Sigma-Aldrich (MO, USA). E. coli BL21 (DE3) cells for enzyme expression were obtained from Invitrogen (MA, USA). An Escherichia coli BL21(DE3) strain harboring the L-AlaDH from Thermus thermophilus (ATCC 27634) coding gene (ald) UniProt accession number Q5SLS7, cloned into a pET28(a) + vector with a C-terminal, non-cleavable 6*His-tag, was obtained from GERC (Gebze University Enzyme Recognition Center, Kocaeli).

Expression and purification of free TtAlaDH

Free TtAlaDH enzymes were expressed in 200 mL of auto-induction medium supplemented with 50 µg/mL kanamycin and incubated overnight at 30 °C with shaking at 200 rpm. Cells were harvested by centrifugation at 4000 rpm for 15 min, and the resulting pellets were resuspended in Buffer A (20 mM sodium phosphate or Tris-HCl, 500 mM NaCl, 30 mM imidazole, pH 7.4, supplemented with 1.0 mg/mL lysozyme). The suspension was incubated on ice for 45 min to facilitate cell wall degradation.

Cell lysis was performed by sonication, and the lysate was clarified by centrifugation at 10,000 rpm for 60 min. The supernatant was filtered through a 0.45-µm membrane and loaded onto a pre-equilibrated HisTrap affinity column (GE Healthcare) using Buffer A. After washing the column with 5 mL of Buffer A, bound proteins were eluted using a stepwise imidazole gradient (100, 200, 400, and 500 mM). Elution fractions were analyzed by SDS-PAGE, and those containing purified TtAlaDH were pooled. Protein concentration was quantified using the BCA Protein Assay Kit according to the manufacturer’s protocol (Demir et al. 2024, 2025).

Synthesis of nanoparticles and immobilization of TtAlaDH

Magnetic nanoparticles were synthesized following the methodologies established in our prior research (Kaptan Usul et al. 2022, 2024a). Initially, the Fe₃O₄ nanoparticles were dispersed in ethanol. The resulting mixture was sonicated for ten minutes to ensure uniform suspension. A solution containing distilled water, ammonium hydroxide, and TEOS was then gradually added to the nanoparticle suspension via a syringe under a nitrogen atmosphere. This reaction was allowed to proceed with stirring for 5 h. The resulting brown nanoparticles were isolated using an external magnet and subsequently washed repeatedly with distilled water. The modified nanoparticles (FS) underwent drying at room temperature for a duration of 1 day (Kaptan Usul et al. 2024b).

The surfaces of F and FS nanoparticles underwent modification using APTES. F and FS nanoparticles were subjected to ultrasonic dispersion for 10 min in a mixture of isopropyl alcohol and water, maintaining a volume ratio of 1:1. Subsequently, APTES (8 mmol) was introduced dropwise and mixed for a duration of 3 h at 60 °C under a nitrogen atmosphere. The mixture was cooled to ambient temperature and isolated from the solution utilizing a strong external magnet. The samples (F-NH₂ and FS-NH₂) underwent three washes with ethanol and deionized water, followed by drying at 60 °C for 24 h (Rajashekarappa et al. 2024; Hu et al. 2025).

F-NH₂ and FS-NH₂ nanoparticles (1 mg) were dispersed in a 50 mM Tris-HCl solution at pH 8.5 and incubated at 4 °C for 24 h with stirring at 600 rpm following the addition of the free TtAlaDH enzyme. After incubation, the immobilized TtAlaDHs were extracted using an external magnet, and unbound enzymes were eliminated through washing. The immobilization percentage was calculated based on the variation in enzyme activity in the solution prior to and following immobilization (Kaptan Usul et al. 2022, 2024a).

$$Xi=((X0-XS)/X0)\times 100$$

where X₀, added enzyme; X_s, free enzyme; and X_i, % immobilized enzyme.

Characterization of nanoparticles and immobilized enzymes

The FTIR absorption spectra of F-NH₂, F-NH₂-E, FS-NH₂, and FS-NH₂-E were obtained using a Perkin Elmer Spectrum 100 instrument. The morphology and structure of the prepared azole functional nanoparticles and immobilized enzyme were investigated using a scanning electron microscope (SEM), an elemental analyzer (EDX), X-ray diffraction (XRD), and thermogravimetric analysis (TGA) (Kaptan Usul et al. 2022, 2024a).

Determination effect of pH and temperature on free TtAlaDH and immobilized TtAlaDHs enzyme activity

Purified TtAlaDH enzyme activity was measured at pH 7–10: 50 mM Tris-HCl for pH levels 7–9, 50 mM sodium carbonate pH levels 9.5–10.0. For reductive amination of pyruvate, the reaction was monitored by following the consumption of NADH at 340 nm. To determine the activity of the purified TtAlaDH enzymes, multiple measurements were conducted using 10 mM pyruvate. These tests were performed under optimal pH and buffer conditions with 0.5 M NH₄Cl. The reaction was initiated by adding 0.5 mM NADH and pure TtAlaDH enzyme. Activity was calculated as units/mg using ε = 6220 M⁻¹ cm⁻¹ and by monitoring the decrease in absorbance based on NADH depletion over 10 min at 340 nm. One unit is defined as the amount of enzyme required for the formation of 1 μmol of NAD⁺ in 1 min (Demir et al. 2024). The thermal stability of both free and immobilized enzymes was assessed in a 50 mM Tris-HC buffer at pH 8.5 across a temperature range of 25 to 85 °C.

Reusability and stability

There was an investigation into whether the immobilized TtAlaDH enzyme could be reused. The activity assessment of the immobilized enzyme was conducted ten (10) times. Following each procedure, the immobilized enzyme was extracted from the mixture using an external magnet and subsequently washed multiple times with the suitable buffer solution. The specific activity of the initial cycle was designated as 100%. The storage stability of free TtAlaDH and immobilized TtAlaDHs was assessed every 10 days through incubation at 4 °C (Kaptan Usul et al. 2022, 2024a).

Results

Characterization of nanoparticles and immobilized enzymes

APTES is an organosilane that readily bonds to surfaces containing an amino group in its main chain, providing a suitable platform for biological and chemical functionalization. F and FS nanoparticles functionalized with APTES enable enzymes to bind covalently or physically to the surface of the nanoparticles. This ensures the stability of the TtAlaDH enzyme immobilized on the support surface. Studies on the immobilization of the TtAlaDH enzyme are quite limited in the literature. In this study, to evaluate the immobilization efficiency, the % immobilization rate was calculated after binding the TtAlaDH enzyme to F-NH₂ and FS-NH₂ surfaces. This enzyme was immobilized at a rate of 83.2% on the F-NH₂ surface, while immobilization at a rate of 74.5% occurred on the FS-NH₂ surface. This decrease in immobilization is attributed to the coating of Fe₃O₄ nanoparticles with a porous silica shell on their surface (Zhou et al. 2022). Figure 1 shows the FT-IR spectra of F-NH₂ and FS-NH₂ nanoparticles prior to and after immobilization.

Fig. 1.

FTIR spectra of F-NH₂, F-NH₂-E, FS-NH₂, and FS-NH₂-E

In Fig. 1, the peak resulting from the stretching vibrations of Fe-O bonds in F nanoparticles was detected within the 530–580 cm⁻¹ wavenumber region (Mohammed et al. 2022; Sadeghi et al. 2023; Oraby et al. 2025). A peak resulting from the asymmetric Si-O-Si stretching vibration has been observed in the 1000–1100 cm⁻¹ range of FS nanoparticles (Hu et al. 2025). The strain vibrations from the ethyl groups in the TEOS structure vary from 2850 to 3000 cm⁻¹. The peaks detected between 1513 and 1629 cm⁻¹ in APTES-functionalized nanoparticles arise from NH₂ group deformation modes, whereas the peaks between 1390 and 1337 cm⁻¹ are from C-N stretching vibrations (Mohammed et al. 2022; Hu et al. 2025). The absence of the peak in the 1500–1650 cm⁻¹ range upon immobilization of the TtAlaDH enzyme on the F-NH₂ nanoparticle surface corroborates the immobilization process. Moreover, in the FS-NH₂ nanoparticle, following the covalent attachment of TtAlaDH to the surface, the peak near 3000 cm⁻¹ vanished, while a peak emerged in the 900–1000 cm⁻¹ range. The peaks identified in the 900–1000 cm⁻¹ range stem from the fingerprint region characteristic of the TtAlaDH enzyme. Peaks at 1600–1700 cm⁻¹ are detected, attributed to C=O vibrations from the secondary structure of proteins (Baysal et al. 2007). Scanning electron microscopy (SEM) was employed to analyze the morphology and surface characteristics of immobilized TtAlaDHs utilizing produced Fe₃O₄-NH₂ and Fe₃O₄-SiO₂-NH₂ materials (Fig. 2).

Fig. 2.

SEM analysis of F-NH₂, F-NH₂-E, FS-NH₂, and FS-NH₂-E

Figure 2 illustrates that, in contrast to prior studies, nanoparticles treated with amino groups demonstrated enhanced aggregation and complex surface characteristics resulting from surface functionalization with APTES (Kaptan Usul et al. 2022, 2024a). EDX analysis was performed to investigate the elemental alterations in functionalized F-NH₂ and FS-NH₂ nanoparticles before and after the immobilization of the TtAlaDH enzyme. The presence of nitrogen atoms in the structure following the functionalization of F and FS nanoparticles with APTES confirms that functionalization has occurred (Table 1).

Table 1.

Mass percentage distribution of atoms obtained from EDX analysis

Materials	Fe	O	C	N	Si
F	71.6	28.4	-	-	-
FS	20.3	38.8	12.9	-
FS-NH2	19.1	42.5	12.4	4.7	21.3
FS-NH2-E	61.4	24.6	7.7	3.3	3.0
F-NH2	60.9	24.1	8.2	3.7	3.1
F-NH2-E	71.2	15.7	7.2	4.1	1.8

*Fe, iron; O, oxygen; C, carbon; N, nitrogen; Si, silisium

Table 1 demonstrates that carbon and sulfur atoms inside the APTES structure are detected in the structure following the functionalization of the F surface (Tarhan et al. 2023). Moreover, the attachment of the free TtAlaDH enzyme to the support materials demonstrates effective immobilization, as indicated by alterations in the atomic mass of the structure (Chen et al. 2010; Losito et al. 2021). X-ray diffraction (XRD) patterns were utilized to explain the crystal structure of F and FS magnetic nanoparticles (Fig. 3).

Fig. 3.

XRD graph of F (Fe₃O₄), and FS (Fe₃O₄-SiO₂)

In Fig. 3, the unique peaks at 220°, 311°, 400°, 422°, 440°, and 511° for F and FS nanoparticles, in comparison to the literature, demonstrated magnetic characteristics. Consequently, it was proven that the synthesized carrier retained its magnetic characteristics (Chen et al. 2010; Kaptan Usul et al. 2024a). The thermogravimetric analysis results for the F-NH₂ and FS-NH₂ nanoparticles are presented in Fig. 4.

Fig. 4.

TG analysis of F-NH₂ (Fe₃O₄-NH₂), and FS-NH₂ (Fe₃O₄-SiO₂-NH₂)

Figure 4 illustrates the thermal characteristics of F-NH₂ and FS-NH₂ nanoparticles. F-NH₂ nanoparticles demonstrate a weight loss of around 13.7% between 25 and 900 °C, whereas FS-NH₂ nanoparticles exhibit a weight loss of 21.9%. The mass decomposition disparity between F-NH₂ and FS-NH₂ nanoparticles (8.2%) is attributed to the TEOS coating on the Fe₃O₄ surface and the variation in the NH₂ binding ratio within the structure. Furthermore, the results indicate that magnetic nanoparticles exhibit resistance to elevated temperatures (Tarhan et al. 2023; Kaptan Usul et al. 2024a).

pH and temperature effects

Enzyme activity was determined for both free and immobilized TtAlaDHs at pH levels ranging from 7.5 to 10 (Fig. 5). The findings demonstrated that immobilized TtAlaDHs displayed greater stability and activity within this pH range relative to their free form. This indicates possible benefits for industrial applications with fluctuating pH values.

Fig. 5.

pH activity profiles for free TtAlaDH, F-NH₂-E, and FS-NH₂-E

Figure 5 illustrates that the unbound enzyme, F-NH₂-E, and FS-NH₂-E attained peak relative activity at pH 8.5. While the attachment of the enzyme to a support surface did not alter its maximum relative activity at pH 8.5, there were reductions in the activity of the immobilized enzyme compared to that of the free enzyme at various pH levels. Steric hindrance or restricted accessibility of the enzyme’s active site upon binding to the support may result in this reduction in activity (Oraby et al. 2025). Subsequent research could investigate optimizing ways to improve the efficacy of immobilized enzymes across diverse pH values. Furthermore, investigating the impact of temperature and ionic strength on enzyme stability may provide additional insights for enhancing immobilization strategies. By comprehending these factors, researchers could create more efficient uses for immobilized enzymes in industrial processes (Mohammed et al. 2022; Behshad et al. 2024). The relative activities of free TtAlaDH and immobilized TtAlaDHs in the 25–85 °C range are shown in Fig. 6. The relative activity profiles of the free TtAlaDH enzyme and immobilized TtAlaDHs across the temperature range of 25–85 °C are presented in Fig. 6.

Fig. 6.

Effect of temperature on catalytic activity

According to Fig. 6, the optimum temperature for the free TtAlaDH enzyme is 55 °C. The unbound enzyme maintains 50% of its activity at temperatures up to 85 °C (Fig. 6). At temperatures below 55 °C, the reduction in activity is under 30%. The support material affects the activity profile of immobilized TtAlaDHs between 25 and 85 °C. F-NH₂-E, serving as a support system, elevates the optimal operating temperature to 65 °C and improves thermal stability, restricting activity loss to under 20% within the range of 65–85 °C. Conversely, FS-NH₂-E serves as an efficient support for low-temperature applications, reducing the optimal temperature to 35 °C.

Reusability and stability

Enzyme immobilization provides benefits including cost-effectiveness and savings from enzyme reusability, continuous production and process regulation, enhanced product purity and quality, and reduced synthesis and purification expenses. Following 10 successive reactions, F-NH₂-E maintained 38.1% of its activity, whereas FS-NH₂-E preserved 16.8% of its activity (Fig. 7).

Fig. 7.

Reusability of F-NH₂-E, and FS-NH₂-E

In Fig. 7, the application of TEOS to the surface of Fe₃O₄ nanoparticles rendered them less advantageous for repeated use in comparison to Fe₃O₄ alone. The substantial expense of enzymes underscores the potential advantages of this immobilization technique for applications necessitating prolonged repetitive enzymatic activity (Cui et al. 2024; Hadadi and Habibi 2025). The stability of the free TtAlaDH enzyme, F-NH₂-E, and FS-NH₂-E systems stored at 4 °C is presented in Fig. 8.

Fig. 8.

Storage stability of free TtAlaDH, F-NH₂-E, and FS-NH₂-E

Figure 8 illustrates the storage stability of the free TtAlaDH, F-NH₂-E, and FS-NH₂-E which exhibits remarkable stability, maintaining an average of 97.3% of its activity after 40 days of storage at 4 °C in a 50 mM Tris-HCl solution at pH 8.5 (Fig. 8). After 40 days, the average stability of FS-NH₂-E was determined to be 98.6%, whereas that of F-NH₂-E was 98.7%. These data demonstrate that immobilization maintains enzyme activity, thereby prolonging shelf life.

Discussion

An examination of existing literature indicates a limited number of investigations focused on L-AlaDH enzyme immobilization (Table 2).

Table 2.

Investigation of the immobilization of the TtAlaDH enzyme

Source	Materials	Notes	References
Bacillus cereus	CNBr-activated Sepharose 4B	FE: pH 9.0–9.5 IE: pH 9.0–9.5	Mureşan et al. (1983)
Bacillus subtilis	Ag-CB	FE: pH 9.0 IE: pH 9.0	da Silva et al. (2017)
Bacillus subtilis	Ag-G	FE: pH 9.0 IE: pH 8.0	da Silva et al. (2017)
Bacillus stearothermophilus	CA–Zr alkoxide gel fibers	IE: six reaction cycles, activity ~ 60%	Nakane et al. (2010)
Thermus thermophilus	F-NH2	FE: pH 8.5 IE: pH 8.5 FE: 55 °C maximum activity IE: 65 °C maximum activity FE: storage stability (~ 97) IE: storage stability (~ 99) IE: six reaction cycles, activity ~ 70%	This study
Thermus thermophilus	FS-NH2	FE: pH 8.5 IE: pH 8.5 FE: 55 °C maximum activity IE: 35 °C maximum activity FE: storage stability (~ 97) IE: storage stability (~ 99) IE: six reaction cycles, activity ~ 30%	This study

*FE, free enzyme; IE, immobilized enzyme

The absence of a shift in the optimal pH following free TtAlaDH enzyme immobilization suggests that the immobilization process does not affect the enzyme’s pH optimum. This outcome is typically observed when immobilization successfully preserves the enzyme’s tertiary structure and the ionization states of the active site amino acid residues, thus maintaining the original pH profile. Collectively, this indicates that the enzyme’s structural integrity and pH sensitivity were maintained throughout the immobilization procedure. For instance, da Silva et al. (2017) reported a shift in optimum pH following immobilization, attributing the change to the support material’s surface chemistry which can alter local surface charge and thus influence enzyme activity at different pH. Conversely, Mureşan et al. (1983) found no change in optimum pH after immobilization, suggesting that the enzyme’s structural and functional properties were fully retained. Consistent with the latter, the pH stability observed for free TtAlaDH after immobilization likely results from the preservation of its native conformation and active site microenvironment during the process. Furthermore, the type of support material is a critical factor influencing the activity profile of immobilized TtAlaDHs across the temperature range of 25 to 85 °C. Immobilization using the F-NH₂-E support successfully raised the enzyme’s optimal operating temperature to between 65 °C and significantly enhanced its thermal stability, limiting activity loss to less than 20% across the range of 65 to 85 °C. In stark contrast, the FS-NH₂-E support lowered the optimal temperature of TtAlaDH to 35 °C, making it more suitable for low-temperature applications. This latter support resulted in a substantial activity reduction (60%) across the elevated temperature range (35–85 °C). These findings demonstrate that selecting F-NH₂ for high-temperature or FS-NH₂ for low-temperature processes offers direct and practical benefits that align with the diverse operational requirements for TtAlaDH in biotechnological applications (Mazlan and Hanifah 2017; de Andrade Silva et al. 2022). Enzyme immobilization, by enabling repeated use, offers a substantial advantage in significantly reducing enzyme costs. Furthermore, utilizing magnetic nanoparticles as support materials provides a key benefit: their ease of separation from reaction mixtures, which surpasses that of many other support types. Specifically, immobilized TtAlaDHs successfully demonstrated that the enzyme maintained high activity and stability while exhibiting improved reusability. This performance indicates that these supports effectively preserve the enzyme’s functional properties throughout the immobilization process, making them a highly promising strategy for developing cost-effective and efficient biocatalyst applications. This level of reusability compares favorably to other literature; for instance, the study by Nakane et al. (2010) reported the retention of approximately 60% of enzyme activity after six reuses in a similar system. Consistent with the promising reusability observed in other systems, the immobilized TtAlaDH on the F-NH₂ support maintained over 60% of its initial activity after six operational cycles, thus demonstrating good operational stability. In sharp contrast, the enzyme immobilized on the FS-NH₂ support exhibited a significant decline in reusability, with activity plummeting to only 20–30% after six uses. This marked reduction in operational stability for FS-NH₂-E is likely attributable to the silica coating on the magnetic nanoparticles. The silica layer has been observed as a disadvantage for repeated enzyme use, plausibly due to its detrimental effects on enzyme-support interaction stability or restricted substrate accessibility within the support microenvironment (da Silva et al. 2017). Thus, while F-NH₂ supports effectively retain enzymatic activity during reuse, the silica-coated FS-NH₂ supports appear to compromise enzyme stability and operational performance in repeated cycles. Shifting focus to storage stability, the TtAlaDH enzyme exhibited high inherent stability, with its activity decreasing by less than 3% after 40 days of storage at 4 °C. Crucially, the immobilized enzymes on both the F-NH₂-E and FS-NH2-E supports demonstrated superior storage stability compared to the free enzyme, exhibiting activity reductions of only 1 to 2% over the same period (Fig. 8). Furthermore, the presence of the silica coating on the iron nanoparticle surface resulted in a markedly lower activity retention, specifically below 2%. Generally, the immobilization process enhances enzyme stability by limiting its conformational flexibility, which in turn protects the enzyme from various denaturation and degradation mechanisms (Sadeghi et al. 2023; Kaptan Usul et al. 2024a; Hadadi and Habibi 2025).

Author contribution

S.K.U. developed methods, designed and performed experiments, analyzed data and wrote the paper. Ğ.D. performed experiments, designed and contributed to writing the paper. A.A.C. developed methods, analyzed data and wrote the paper. B.B. designed the experiments and contributed to writing the paper.

Funding

This research received no external funding.

Data availability

All data are contained within the manuscript.

Declarations

Ethical approval

No procedures requiring ethical approval were conducted in this study.

Competing interests

The authors declare no competing interests.