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Hyperactivation of crosslinked lipases in elastic hydroxyapatite microgel and their properties

Journal of Biological Engineering volume 18, Article number: 46 (2024) Cite this article

Abstract

Effective enzyme stabilization through immobilization is essential for the functional usage of enzymatic reactions. We propose a new method for synthesizing elastic hydroxyapatite microgel (E-HAp-M) materials and immobilizing lipase using this mesoporous mineral via the ship-in-a-bottle-neck strategy. The physicochemical parameters of E-HAp-M were thoroughly studied, revealing that E-HAp-M provides efficient space for enzyme immobilization. As a model enzyme, lipase (LP) was entrapped and then cross-linked enzyme structure, preventing leaching from mesopores, resulting in highly active and stable LP/E-HAp-M composites. By comparing LP activity under different temperature and pH conditions, it was observed that the cross-linked LP exhibited improved thermal stability and pH resistance compared to the free enzyme. In addition, they demonstrated a 156% increase in catalytic activity compared with free LP in hydrolysis reactions at room temperature. The immobilized LP maintained 45% of its initial activity after 10 cycles of recycling and remained stable for over 160 days. This report presents the first demonstration of a stabilized cross-linked LP in E-HAp-M, suggesting its potential application in enzyme-catalyzed processes within biocatalysis technology.

Introduction

Enzymes are sustainable biological catalysts that are efficient, robust, and derived from renewable resources [1]. Enzymatic processes proceed based on activity, specificity, and selectivity under mild conditions, closely resembling temperature, pressure, and physiological pH, including aqueous environments [2,3,4]. Enzymes offer greater economic viability than conventional organic synthesis methods, providing an energy-efficient and environmentally friendly synthesis pathway with reduced waste generation [5]. Therefore, enzymes are emerging as a crucial technology for producing green and sustainable chemicals in pharmaceuticals, food, and cosmetics. In clinical and biomedical applications, drug delivery systems (DDS) and tissue engineering further enhance the efficacy of therapeutic enzymes by improving their stability and activity. DDS enables controlled, targeted release, protecting enzymes and maintaining their effectiveness [6]. In tissue engineering, enzymes aid in tissue repair and integration by modifying biomaterials and promoting cell growth [7]. However, despite these advantages, enzyme applications in industry may face challenges such as reduced activity and difficulties in recycling, due to decreased enzyme stability from factors like low thermal stability, a narrow pH range, and structural modifications [8]. Therefore, improving enzyme stability is worthy of industrial attention [9]. Enzyme immobilization stabilizes enzymes during processes, enhancing their applications and uses by improving the physical and enzymatic stability of biocatalysts through tailored compositions that align with enzyme selectivity, stability, and kinetics, interacting with carriers via physical and chemical bonds [10,11,12,13]. Immobilized enzymes allow for efficient recovery and reuse, while their enhanced stability against denaturation or self-digestion under various conditions preserves the enzyme’s structure, resulting in high catalytic productivity [14,15,16,17,18,19]. Enzyme immobilization strengthens enzyme stability and enables reuse by stabilizing the enzyme structure and reinforcing the enzyme's dissociation resistance and mechanical strength through binding with a carrier [20, 21]. However, the process of enzyme immobilization can sometimes reduce enzyme activity, posing a significant challenge that needs to be addressed through the development of more efficient and practical methods [22, 23]. Recent advancements, such as cross-linked enzyme aggregates (CLEAs), have shown promise in providing high enzyme activity and spatial efficiency [24]. Nonetheless, issues like insufficient mechanical strength and difficulties in reuse remain unresolved [25].

Most immobilization techniques require a carrier, making the characteristics of the carrier pivotal to enzyme immobilization [26]. The carrier must exhibit excellent stability and possess a porous structure to support various compounds effectively [27, 28]. Additionally, the carrier material should provide efficient immobilization space and be easily modifiable to promote enzyme immobilization [29]. Enzymes can be immobilized on solid carriers through various chemical and physical methods, including physical adsorption, chemical covalent bonding, entrapment or encapsulation within materials, and crosslinking [30, 31]. Adsorption relies on physical interactions between the support and the enzyme, such as van der Waals bonds, ionic interactions, and hydrogen bonds [32, 33]. While these bonds are relatively weak, they preserve the enzyme's original structure [34]. Successful adsorption depends on establishing affinity between the enzyme and carrier under specific conditions [35]. Entrapment involves confining an enzyme within a lattice network of carriers, improving enzyme stability and minimizing leaching and denaturation [36, 37]. Encapsulation captures enzymes within a spherical space, preserving their integrity and allowing a broader range of enzymes to be included [38, 39]. Both entrapment and encapsulation involve controlling the enzyme’s environment but differ in their structural confinement methods [40]. Cross-linking immobilization is an irreversible method that forms intermolecular cross-links between enzyme molecules using a crosslinking agent [41]. This method creates enzyme aggregates or crystals and utilizes dual-functional reagents to prepare carrier-free large particles [42]. While cross-linking offers enhanced stability and minimizes enzyme leakage from porous carriers, it may reduce bioactivity due to diffusion limitations caused by strong chemical bonds [43,44,45]. Crosslinking enzymes within mesoporous materials offers enhanced stability, high enzyme loading, and improved catalytic efficiency [43]. This method benefits from the unique "ship-in-a-bottle" approach, where enzymes are entrapped within the confined pores of the mesoporous material, providing robust protection and effective enzyme utilization [31, 46]. However, this process involves complex material synthesis, which can be expensive. In contrast, physical adsorption is simpler and cheaper but less stable, while chemical bonding offers strong attachment but is more complex and expensive [47]. Gel and polymer matrices provide stability and versatility but can be challenging to prepare and may limit enzyme activity [48]. Overall, while mesoporous materials, including those employing the ship-in-a-bottle approach, offer significant benefits, they come with challenges that must be weighed against other immobilization methods.

Various materials, both inorganic and organic, have been used as carriers for enzyme immobilization [49]. Among them, the inorganic materials used for enzyme immobilization exhibit high stability under reaction conditions such as high pressure and temperature and possess indefinite properties [50, 51]. For example, silica, inorganic oxides, mineral materials, and carbon-based materials have been adopted and applied [52]. However, they are limited in the creation of various geometric shapes and are sensitive to friction during stirring [52]. Hydroxyapatite (HAp) is a natural mineral used as a crucial component in the bones and teeth of humans and animals [53,54,55]. It is a non-toxic bioceramic material with a calcium phosphate (CaP) composition that is preferred for various applications in medical, environmental, and industrial fields [54, 56,57,58]. In addition, HAp possesses characteristics such as high porosity and ion exchange capacity, leading to its widespread use in various important fields, including its potential application as an auxiliary material in the design of gas sensors, fuel cells, and chromatographic separation of proteins [59,60,61,62]. Comprising calcium, phosphorus, oxygen, and hydrogen, HAp occurs naturally and can be easily chemically synthesized. HAp has primarily been used as a matrix for protein adsorption, facilitated by the charged side groups of proteins interacting with the phosphate and calcium groups of HAp [63]. Consequently, it has garnered significant attention from researchers. The calcium ions present in HAp can also chelate with the carboxylic acid groups present on the amino acids of enzymes, resulting in high resistance to various reaction conditions and forming very stable interactions for immobilization [63,64,65].

In this study, to practically enhance the application of enzyme-based processes, hydroxyapatite (HAp) was used as a carrier to immobilize enzymes in a model investigation. We explored the potential application of a new elastic HAp microgel (E-HAp-M) as an enzyme-stabilizing carrier by immobilizing lipase (LP) from Pseudomonas fluorescens into the pores of HAp materials. E-HAp-M can be synthesized as a well-formulated mesoporous micro-sized material. First, the prepared E-HAp-M was fully characterized by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), Fourier-transform infrared spectroscopy (FT-IR). X-ray diffraction (XRD), and particle size analysis (PSA). Additionally, Brunauer − Emmett − Teller (BET) analysis was employed to determine the surface area and pore structure. LP immobilization was performed in a two-step process based on the well-known ship-in-a-bottle approach, preventing enzyme denaturation and leakage [66]. Immobilized LP presents the prospect of enzyme immobilization by examining storage stability, recycling and pH stability, and temperature resistance. The crosslinked LP in E-HAp-M remained preserved for 160 days, with its catalytic activity remaining stable even after 10 hydrolysis cycles. This study highlights the applications of a stable and recyclable cross-linked enzyme in E-HAp-M, demonstrating its potential for practical enzyme-based processes.

Materials and methods

Chemicals

Ammonium phosphate ((NH4)2HPO4), Calcium nitrate tetrahydrate (Ca(NO3)2∙4H2O), Cetyltrimethylammonium Bromide(CTAB), Amano Lipase from Pseudomonas fluorescens, sodium phosphate monobasic, sodium phosphate dibasic, glutaraldehyde (GA), p-Nitrophenyl acetate, and acetonitrile were purchased from Sigma-Aldrich. and 1 M Tris–HCl (pH 8.0) was purchased from Bioneer. Finally, the BCA protein assay kit was purchased from Takara.

Synthesis of the elastic hydroxyapatite microgel (E-HAp-M)

Hydroxyapatite was produced using a precipitation method using Calcium nitrate tetrahydrate Ca(NO3)2∙4H2O (Sigma-Aldrich) and Ammonium Phosphate Dibasic (NH4)2HPO4 (Merck) as starting materials. A 0.6 M ammonium phosphate suspension was prepared by dissolving it in distilled water, and an appropriate amount of calcium nitrate tetrahydrate was dissolved to prepare a 1 M solution. Additionally, an appropriate amount of CTAB was dissolved in distilled water to prepare a 1 wt% solution.

The following reaction provides an explanation: 10Ca(NO3)2·4H2O + 6(NH4)2HPO4 + 8NH4OH → Ca10(PO4)6(OH)2 + 20NH4NO3 + 20H2O.

Subsequently, the ammonium phosphate solution was mixed with the CTAB solution to obtain a final CTAB/HAp weight ratio of 0.5% (w/w), based on the theoretical yield of hydroxyapatite. The pH of the mixed solution was adjusted to 10.0 using aqueous ammonia, a base solution. A solution of calcium nitrate tetrahydrate was slowly added dropwise to the CTAB/phosphate solution under constant stirring to maintain a pH of 10.0. The reaction mixture was then stirred at room temperature and atmospheric pressure for 12 h. The reaction mixture was centrifuged at 4500 rpm for 10 min to separate the precipitate. Following centrifugation, the supernatant was discarded, and the precipitate was resuspended in 50% ethanol. This centrifugation-washing cycle was repeated a total of six times to purify the precipitate. Subsequently, the obtained precipitate was aged in an oven at 70 °C for 12 h. Finally, to remove residual moisture and surfactant, the aged precipitate was freeze-dried at -70 °C.

Characterization of E-HAp-M

The crystallographic phases, functional groups, surface morphology, elemental composition, and particle size distribution of the synthesized hydroxyapatite microgel were characterized using various techniques. X-ray diffraction (XRD) was performed using a Rigaku miniflex600 (Japan) diffractometer in reflection mode with Cu Kα radiation (λ = 1.5405 Å). Data were collected in the 2θ range of 3° to 90° with a scanning speed of 0.02°/min. FT-IR analysis was conducted using a PerkinElmer Frontier (USA) spectrometer. FT-IR spectra were acquired over the 400–4,000 cm⁻1 region using the KBr pellet technique. The resolution of the spectrometer was 4 cm⁻1. Field Emission Scanning Electron Microscopy (FE-SEM) with energy dispersive X-ray spectroscopy (EDAX) (TESCAN-MIRAIII, Czech Republic) was employed to characterize the surface morphology, microstructural features, and elemental composition of the microgel. The average particle size of the microgel samples was determined by separate particle size analysis (HORIBA, Partica LA-960, Japan). N₂ adsorption/desorption isotherms (micromeritics Tristar ll PLUS, USA) were measured at 77 K to determine the specific surface area and pore size distribution of the samples using the BET and Barrett-Joyner-Halenda (BJH) models, respectively. The measurements were conducted with a known mass of the degassed solid sample in powder form (after degassing at 100 °C).

Lipase immobilization in E-HAp-M

Immobilization of the enzyme in E-HAp-M was performed via a two-step process, as shown in Fig. 1. First, LP (5 mg mL−1, pH 7.0) and E-HAp-M (5 mg mL−1, pH 7.0) solutions were added to a glass vial and vigorously vortexed for 10 s. The mixture was then incubated at 200 rpm for 2 h at 25 ℃ to allow adsorption into the E-HAp-M pores. Once the adsorption process was complete, centrifugation was performed to collect the immobilized enzyme sample. To cross-link the adsorbed enzyme, 2 mL of a 0.5% glutaraldehyde solution (diluted with 100 mM PB buffer, pH 7.0) was added to the enzyme and hydroxyapatite mixture. The solution was left undisturbed for 1 h and then incubated at 200 rpm for an additional 1.5 h at 25 ℃. Subsequently, centrifugation was applied to collect the immobilized LP, and the supernatant was discarded. To remove unreacted glutaraldehyde, the sample was washed five times with 100 mM PB buffer (pH 7.0). In addition to capping the aldehyde groups, 2 mL of 100 mM Tris–HCl buffer (pH 8.0) was added to the sample and incubated at 200 rpm for 30 min at 25 ℃. Subsequently, centrifugation was performed to collect the immobilized enzyme sample, and the supernatant was discarded. The sample was washed five times using the same procedure as before. Finally, the sample was stored in 20 mM phosphate buffer (pH 8.0) at 4 ℃ until the next use. The mass of immobilized lipase (LP) and its efficiency in E-HAp-M were determined using a BCA protein assay kit. The immobilization efficiency was assessed by measuring the concentration of LP before and after the immobilization process. Specifically, the concentration of unimmobilized LP was measured during the process to calculate the percentage of lipase successfully immobilized in the E-HAp-M material relative to the initial amount used.

Fig. 1
figure 1

Schematic illustrations of the immobilization of LP in E-HAp-M

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Immobilized lipase activity and stability measurements

After immobilization, the activity recovery (retained activity) of the immobilized LP was also evaluated. This assessment measures the percentage of the lipase’s original specific activity that was retained after immobilization in the E-HAp-M material, by comparing it to the specific activity of an equivalent mass of free LP. The activity of the immobilized LP in E-HAp-M was assessed by monitoring the absorbance change during the hydrolysis of 0.5 mM p-nitrophenyl acetate into p-nitrophenol and acetate. The activity was calculated based on the initial activity, which was measured by absorbance per reaction time and is proportional to the concentration of p-nitrophenol. To measure the activity of immobilized LP, 30 μL of immobilized LP was mixed with 1,455 μL of 20 mM phosphate buffer (pH 8.0), and 15 μL of 50 mM p-nitrophenyl acetate was added. The mixture was shaken at 200 rpm at 25 ℃.

To investigate the pH effect on immobilizing LP, the activity was measured in the range of 6.0–10.0. In addition, a temperature profile was established by measuring the activity in the range of 25 ℃ to 65 ℃. Stability during long-term storage and recycling use was also evaluated using the same method as described above. The experimental results were compared using relative activity values based on the initial room conditions by storage at room temperature. All experiments were performed three times to calculate the standard deviation, which is represented by error bars in the figure.

Result and discussion

Characterization of E-HAp-M

Contrary to other methods for synthesizing hydroxyapatite (HAp), our process was conducted at room temperature, eliminating the need for high temperatures or pressures and thereby conserving energy [54, 67, 68]. Additionally, we minimized the use of organic solvents, which not only reduces energy consumption but also significantly lowers wastewater treatment costs. The synthesis of E-HAp-M involved three types of reagents, most of which were used at low concentrations, with distilled water serving as the solvent. The reagents constituted only 1/10 of the total solvent volume, significantly reducing resource consumption and waste generation. This approach facilitates the easy large-scale production of E-HAp-M. Figure 2A shows the FE-SEM image of the synthesized Elastic HAp microgel. Each sample was presented at magnifications of 10 kx and 30 kx. This micrograph illustrates the morphology of HAp microgel with sizes ranging from approximately 2 to 5 µm. The HAp particles exhibited pores, including intra- and inter-particle pores, underscoring the substantial adsorption capacity of porous materials. EDS analysis of the E-HAp-M specimen (Fig. 2B) revealed that it was primarily composed of calcium (Ca), phosphorus (P), and oxygen (O) elements. The Ca/P ratio obtained from EDS analysis was 1.89, which falls within the synthetic HA Ca/P ratio range of 1.25–2.10 [69]. PSA analysis (Fig. 2C) revealed an average diameter of 3.03 μm for E-HAp-M, with a distribution ranging from 4.02 to 5.09 μm at a cumulative value of 90%. Notably, these PSA results showed larger diameters compared with the FE-SEM micrographs. This most likely occurred because of particle aggregation during dispersion in water. In Fig. 3A, major peaks were observed at 2θ angles of 25.7°, 31.7°, 39.5°, 46.4°, and 49.3°. This pattern exhibited good correlation with the XRD pattern of pure hydroxyapatite (JCPDS-09–0432). Notably, there was a significant peak at 31.7°, corresponding to HA [70,71,72,73,74]. The observed strong diffraction peaks at 2θ values can be attributed to the hydroxyapatite structure. These peaks corresponded to the hkl values of 002, 102, 210, 211, 112, 300, and 202, respectively, as reported in previous studies [69]. The FTIR spectrum is shown in Fig. 3B. All samples showed characteristic bands of the PO43− part at 569.9 cm−1 (P-O antisymmetric bending), 969 cm−1 (P-O symmetric stretching), and 1044 cm−1 (P-O antisymmetric stretching). These peaks indicated hydroxyapatite (HA). The E-HAp-M spectrum also showed a broad band at 3456 cm−1, which indicates the presence of H2O molecules. Additional peaks at 1643.9 cm−1 and 628.3 cm−1, indicating O–H groups. Weak bands at 1435 cm−1 and 869 cm−1 were also observed, which may be related to CO32− groups in the sample. Therefore, FTIR spectroscopy confirmed the presence of all these functional groups in E-HAp-M. Figure 3C shows the nitrogen adsorption/desorption isotherms. The isotherm exhibited a type IV profile with an H1 hysteresis loop, which is characteristic of mesoporous materials according to the International Union of Pure and Applied Chemistry (IUPAC) classification [74]. A larger pore volume of 0.68 cm3 g⁻1 was observed for E-HAp-M (Table 1). The BET surface area of E-HaP-M was calculated to be 105.38 m2 g⁻1. In a previous study, Safi et al. reported a surface area of 85 m2 g⁻1 and a pore volume of 0.4227 cm3 g⁻1 for synthesized mesoporous HAp [75]. The results of the present study showed that the synthesized E-HAp-M has a larger surface area and pore volume compared with previous reports, which described mesoporous HAp as a good adsorbent [61, 62]. Both BET and BJH analyses confirmed the porous structure observed in the FE-SEM micrographs.

Fig. 2
figure 2

A Field Emission Scanning Electron Microscopy (FESEM) images of elastic hydroxyapatite microgel (E-HAp-M) at various magnifications (10kx, 30 kx), B Elemental composition of elastic hydroxyapatite microgel (E-HAp-M) determined by energy dispersive spectrometry (EDS) analysis, and C particle size analysis (PSA) particle size distributions of elastic hydroxyapatite microgel (E-HAp-M)

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Fig. 3
figure 3

A X-ray Diffraction (XRD) patterns of elastic hydroxyapatite microgel (E-HAp-M), B Fourier Transform Infrared (FTIR) spectra from 400 to 4000 cm−1 for elastic hydroxyapatite microgel (E-HAp-M), and C N2 adsorption–desorption isotherms measured at 77°k for elastic hydroxyapatite microgel (E-HAp-M)

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Table 1 Summary of the characteristics of the E-HAp-M, including its physical properties, as determined by BET analysis
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Characteristics of the immobilized lipase in E-HAp-M

The immobilization of LP was performed through a simple two-step process: LP adsorption and crosslinking, as illustrated in Fig. 1. The mesoporosity and pore volume of E-HAp-M provided a conducive environment for LP enzyme adsorption. When 5 mg of LP and 5 mg of E-HAp-M were incubated in 2 mL of 20 mM sodium phosphate buffer (pH 8.0) at room temperature with shaking at 200 rpm, approximately 2.1 mg was loaded. This corresponds to a loading efficiency of 42.6%, indicating the completion of LP adsorption (Table 2). To prevent enzyme leaching from E-HAp-M, the adsorbed LP was cross-linked through glutaraldehyde (GA) treatment. This process resulted in a combined effect of adsorption and crosslinking, with the enzymes forming a hybrid structure within the E-HAp-M bottleneck pores. The crosslinked LP created multi-point covalent linkages, enhancing resistance against denaturation and leaching. After immobilization, LP loadings were determined to be 29.9 wt% (Table 2). Measurement of the initial activities indicated that the LP enzymes were effectively retained within the mesopores of E-HAp-M, demonstrating the success of glutaraldehyde (GA) treatment using the ship-in-a-bottle neck approach.

Table 2 Summary of LP loading and LP loading efficiency during immobilization in E-HAp-M. This includes data on the initial amount of LP used, the amount successfully immobilized, and the corresponding loading efficiency, calculated as the percentage of LP retained in the E-HAp-M
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The kinetic parameters of the free enzyme and immobilized enzyme in E-HAp-M were investigated at different substrate concentrations ranging from 0.1 to 4.5 mM, and the values were obtained for Km and Vmax using a generally double reciprocal Lineweaver Burk plot. The values for Km and Vmax of the immobilized enzyme were measured to be 1.91 ± 0.30 mM and 0.54 ± 0.09 mM min−1, respectively. To compare the efficiency of each enzyme, their Vmax Km−1 values were measured, as shown in Table 3. The Km value defines the affinity of the enzyme for the substrate, with a smaller Km value indicating decreased affinity of the enzyme for the substrate. Changes in enzyme affinity for the substrate may be caused by alterations in the enzyme structure during the immobilization process or by reduced access of the substrate to the active site of the immobilized enzyme, as immobilization restricts access to the enzyme active site [76]. Most immobilization decrease Vmax values[36]. However, because the cross-linking immobilization method induces kinetic changes influenced by mass transfer, certain structural changes in enzymes, and microenvironmental changes in an inorganic circumstance, [66] the morphology of the E-HAp-M could, on the other hand, impose diffusion constraints and influence enzyme–substrate interactions, resulting in a higher Vmax than the free LP. This could be attributed to an increase in substrate concentration, leading to the acceleration of substrate diffusion toward the cross-linked LP in E-HAp-M, which may eventually reach a threshold level. Once the immobilized enzyme achieves maximum velocity only after reaching complete substrate saturation, the Vmax value will be higher than that of the free enzyme in the media solution, as summarized in Table 3 [76].

Table 3 Summary of enzyme kinetics for free and immobilized LP in E-HAp-M. The enzyme kinetics were measured at room temperature and pH 8.0
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Properties of the immobilized lipase in response to changes in temperature and pH

To characterize the LP within the E-HAp-M structure, we investigated the LP-catalyzed hydrolysis of p-nitrophenyl acetate by comparing its activity before and after immobilization. The optimum pH of LP from Pseudomonas fluorescens has been reported to be 8.0, and the temperature was 55 °C. The specific activity of immobilized LP measured to be 0.1789 mM−1 min−1 mg−1, as shown in Fig. 4a, whereas free LP exhibited specific activities of 0.1149 mM−1 min−1 mg−1. This demonstrates that the immobilized LP in E-HAp-M retains approximately 156% of the specific activity of free LP. Considering that most crosslinked enzymes displayed < 10% of specific activity retention [77], 156% catalytic activity represents a significant hyperactivation of specific activity. This effect was achieved by preserving lipase activity, preventing denaturation and autolysis, and providing a favorable microenvironment through E-HAp-M. The structure of E-HAp-M facilitated substrate access and promoted the open form of Amano lipase from Pseudomonas fluorescens. The lipase's lid domain, an alpha-helical structure of 20–30 amino acids, was crucial for regulating active site access and influencing substrate interactions [78]. However, activation was not solely dependent on the lid's structure. It resulted from the combined effects of the lid's properties, carrier hydrophobicity, inorganic surface interactions, and environmental factors such as pH, temperature, and calcium ions [79]. Specifically, calcium ions in E-HAp-M enhanced lipase activity by stabilizing the enzyme's structure and facilitating hyperactivation through improved interfacial interactions [80]. Furthermore, E-HAp-M offered advantages such as a high specific surface area and large pore size, which facilitated substrate transport [81]. The elastic properties of the hydroxyapatite improved substrate accessibility to enzyme active sites and increased the diffusion rate, collectively leading to enhanced reaction rates [82].

Fig. 4
figure 4

Specific activity of (A) free LP and immobilized LP in E-HAp-M under different temperature conditions at pH 8.0, and B Effect of temperature on LP activity in comparison with immobilized LP and free LP. The results were standardized, with the optimum value of each set assigned the value of 100% activity at pH 8.0 and 55 °C

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To investigate the behavior of immobilized LP, the stability of free LP and crosslinked LP was tested under different temperature ranges (Fig. 4b). Temperature resistance was measured under the same conditions from 25 ℃ to 65 ℃ for both free and immobilized lipases. Relative values were compared by setting 55 ℃, the optimal temperature for free LP, as 100%. At 65 ℃, free LP exhibited an activity of 100%, whereas immobilized LP showed an activity of 98%, similar to the optimal temperature of 55 ℃. However, at temperatures lower than the optimal temperature, free LP showed activity that dropped by up to 25%, whereas immobilized LP demonstrated activity better than the optimal temperature. Improved thermal stability of the immobilized LP has been previously reported, and this behavior of the immobilized LP can be attributed to the following factors: Glutaraldehyde cross-linker stabilizes and rigidifies the structure of the substrate [83]. Therefore, as the temperature increases, the structural changes in the substrate decrease, and the temperature stability over a wide range increases [84].

The effect of pH on enzyme activity was tested on both free and immobilized LP. The pH profile was measured under the same conditions, ranging from pH 6.0 to 10.0, for both free and immobilized LP. The specific activity of free and immobilized LP was investigated at room temperature. As shown in Table 4, under different pH conditions, immobilized LP retained a significantly higher specific activity than free LP. The specific activity of free LP decreased under acidic conditions but slightly decreased at high pH. These results indicate a shift in enzyme activity preference toward higher pH values. After immobilization at pH 6.0, both free and immobilized LP exhibited similar specific activity. In contrast, crosslinked LP (highest at pH 10.0) demonstrated improved specific activity compared with free LP, and between pH 7.0 and 9.0, much higher values of specific activity were obtained compared with free LP. This suggests that immobilization confers enhanced thermal stability through crosslinking. Thus, the immobilized LP demonstrated an improved pH resistance range compared with its free counterpart. Therefore, we can conclude that immobilized LP in E-HAp-M via crosslinking presents increased resistance to pH. However, different pH shift characteristics were observed and supported by other previous studies, indicating that various immobilization methods induce outcomes that are largely enzyme-dependent [85, 86].

Table 4 Comparison of the specific activities of free lipase and immobilized lipase in E-HAp-M under different pH conditions at room temperature. This table illustrates the specific activities of free and immobilized lipase in the E-HAp-M matrix across a range of pH conditions at room temperature. It shows how enzyme activity varies with pH for both free and immobilized LP
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Storage and recycling stability of immobilized lipase in E-HAp-M

Storage stability is a key consideration for evaluating the properties of immobilized enzymes. Prevention of LP leaching from E-HAp-M was investigated to enhance enzyme stability. The stability of the immobilized LP was confirmed by measuring its hydrolysis activity by storing it at room temperature over a long period of time. The stability of the immobilized enzyme was compared by considering the initial activity value as 100%, and the relative activity value was evaluated. Free LP showed 50% activity after 48 h and its activity decreased to less than 41% within 72 h, but immobilized LP maintained more than 85% of its initial activity within a week and maintained 50% of its initial activity after 80 days of storage, and even 34% of relative activity was preserved within 160 days of measurement, as shown in Fig. 5a. It is assumed that the high stability of cross-linked LP was maintained because cross-linking prevents the release of the enzyme entrapped in the HAp pore and inhibits denaturation of the enzyme structure by inducing chemical bonding [66]. Based on these results, LP stably immobilized inside E-HAp-M showed great potential for industrial applications. Immobilized LP are crucial in various fields: they catalyze biodiesel production, enhance flavors in the food industry, synthesize pharmaceuticals, optimize cosmetics, treat fabrics in textiles, improve paper quality, boost detergent efficacy, and support environmental efforts such as waste treatment, oil spill cleanup, and bioremediation [87, 88]. Hyperactivated versions of LP offer even greater efficiency and cost-effectiveness, making them particularly valuable in large-scale biodiesel production and industrial waste treatment. However, before these processes can be fully industrialized, effective scaling-up is essential. Scaling up immobilized LP processes involves several challenges, such as optimizing immobilization techniques, ensuring enzyme stability, and addressing mass transfer limitations [89]. Strategies to address these challenges include standardizing immobilization methods and using robust support materials like E-HAp-M, which is both cost-effective and simple to produce [90]. Additionally, improvements in reactor design and agitation systems are necessary to tackle mass transfer issues, while managing operational costs and maintaining quality control are crucial [91]. Compliance with regulatory standards and integrating sustainable practices are important for ensuring long-term stability and environmental compliance [92].

Fig. 5
figure 5

A Effect of long-term storage on enzyme activity in comparison between immobilized LP in E-HAp-M and free LP and (B) recycling stability of immobilized LP in E-HAp-M. The stability of the immobilized LP was compared by considering the initial activity value as 100% and evaluating the relative activity value as a percentage

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Recycling is one of the most important properties of immobilized enzymes for the economics of industrial applications. After measuring the activity of each sample for recycling of the immobilized enzyme, each sample was washed three times with 20 mM PB buffer (pH 8.0), stored in 20 mM Tris–HCl buffer (pH 8.0), and stored at room temperature. To measure the efficiency of recycling, relative activation values were compared by setting the initial activation value to 100%. The experiment was conducted under optimal conditions and performed 10 times. As a result, the relative activity of recycling the immobilized enzyme was maintained at approximately 45% of the initial activity value after 10 rounds of enzymatic use (Fig. 5b). This preservation can be mainly attributed to the immobilization of LP into the E-HAp-M pore by the crosslinker glutaraldehyde, thus forming a crosslinked enzyme structure in the E-HAp-M via the "ship-in-a-bottle" mechanism, leading to the strong LP structure [35, 52]. The stabilization of the enzyme could be attributed to the improvement in intrinsic enzyme stability, effectively conserving the active form of LP molecules and demonstrating high recycling stability. However, we observed a continuous decline in enzyme activity over time, illustrating the inherent challenges in reactivating crosslinked enzymes. As seen in the previous ship-in-a-bottle approach, where reactivation of the enzyme was not achievable, this trend further indicated that restoring full enzymatic function after crosslinking was also a challenge in our study. Given these findings, it may have been more practical to consider alternative approaches, such as utilizing newly synthesized enzymes or exploring different strategies for future study. As shown in Table 5, the storage stability and reusability of the hyperactivated immobilized LP exhibited commendable performance. Compared to other immobilization systems, the hyperactivated LP immobilized on E-HAp-M demonstrated a significant advantage in maintaining high stability over extended periods and through repeated use. This improved stability and reusability were important for practical applications, making it a cost-effective and sustainable option for continuous lipase-catalyzed enzymatic processes. The enhanced performance of the hyperactivated immobilized LP ensured consistent activity over time, which helped reduce the need for frequent replacements and lowered operational costs, crucial for industrial and long-term use.

Table 5 Comparison of the specific activity, storage stability, and reusability of immobilized lipase with inorganic carriers as reported in the literature
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Conclusions

The synthesis of E-HAp-M and its characterization, including PSA, XRD, SEM, EDS, BET surface area, pore size, and adsorption/desorption pore volumes, were demonstrated in this study. The crosslinked LP in this new E-HAp-M material was successfully assembled and exhibited advantages such as high reaction rates and an increase in Vmax. After immobilization through crosslinking methods, 42% of the LP was immobilized into the pores of E-HAp-M, and 156% of the specific activity was preserved at room temperature. The enzyme retained over 45% of its relative value even after 10 reuse cycles when stored for 160 days at 25 ℃, indicating high recycling and storage stabilities. Therefore, these new E-HAp-M materials may provide a novel enzyme-immobilizing carrier for investigating various mineral-based enzymatic reactions.

Availability of data and materials

Not applicable.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

This work was supported by a grant from the Fundamental R&D program and funded by the Korea Institute of Ceramic Engineering and Technology (KICET) and Ministry of Trade, Industry and Energy (MOTIE), Republic of Korea (NTIS: 1415187241), the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00242532), and the Biomaterials Specialized Graduate Program through the Korea Environmental Industry & Technology Institute (KEITI) funded by the Ministry of Environment (MOE).

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  1. Department of Food Biotechnology, Dong-A University, Busan, Republic of Korea

    Hyo Won Jeon, Chan Hee Lee, Dain Kim & Ee Taek Hwang

  2. Center for Convergence Bioceramic Materials, Korea, Institute of Ceramic Engineering & Technology, Cheongju-Si, Chungcheongbuk-Do, Republic of Korea

    Jun Seop Lee & Hye Sun Lee

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Hyo Won Jeon: Conceptualization, Methodology, Data curation, Writing- Original draft preparation; Jun Seop Lee: Resource, Investigation, Chan Hee Lee: Software; Dain Kim: Validation; Hye Sun Lee: Writing-Original draft preparation, Supervision; Ee Taek Hwang: Writing- Reviewing and Editing, Supervision

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Jeon, H.W., Lee, J.S., Lee, C.H. et al. Hyperactivation of crosslinked lipases in elastic hydroxyapatite microgel and their properties. J Biol Eng 18, 46 (2024). https://doi.org/10.1186/s13036-024-00440-5

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  • DOI: https://doi.org/10.1186/s13036-024-00440-5

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Journal of Biological Engineering

ISSN: 1754-1611