
==== Front
J Biol Eng
J Biol Eng
Journal of Biological Engineering
1754-1611
BioMed Central London

39223667
440
10.1186/s13036-024-00440-5
Research
Hyperactivation of crosslinked lipases in elastic hydroxyapatite microgel and their properties
Jeon Hyo Won 1
Lee Jun Seop 2
Lee Chan Hee 1
Kim Dain 1
Lee Hye Sun hslee@kicet.re.kr

2
Hwang Ee Taek ethwang@dau.ac.kr

1
1 https://ror.org/03qvtpc38 grid.255166.3 0000 0001 2218 7142 Department of Food Biotechnology, Dong-A University, Busan, Republic of Korea
2 https://ror.org/024t5tt95 grid.410900.c 0000 0004 0614 4603 Center for Convergence Bioceramic Materials, Korea, Institute of Ceramic Engineering & Technology, Cheongju-Si, Chungcheongbuk-Do Republic of Korea
2 9 2024
2 9 2024
2024
18 4624 6 2024
8 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
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.

Keywords

Enzyme immobilization
Enzyme stabilization
Elastic hydroxyapatite
Cross-linking
Lipase
issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcIntroduction

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 [2–4]. 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 [10–13]. 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 [14–19]. 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 [43–45]. 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 [53–55]. 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, 56–58]. 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 [59–62]. 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 [63–65].

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

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 [70–74]. 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 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)

Fig. 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)

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

	SBET	V	dad (average)	dde (average)	
(m2 g−1)	(cm3 g−1)	(nm)	(nm)	
Elastic HAp Microgel	105.38	0.68	32.89	28.44	
SBET is the BET surface area; V is the total pore volume

pore size, dad, and dde were calculated using the BJH (Barett–Joyner–Halenda) method

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

Sample	Enzyme loading	Enzyme loading efficiency	
wt %	%	
Immobilized LP	29.9	42.6	

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

Sample	Km	Vmax	Vmax Km−1	Ratio[a]	
	(mM)	(mM min⁻1)	(min⁻1)	(%)	
Free LP	0.82 ± 0.18	0.17 ± 0.04	0.21	100	
Immobilized LP	1.91 ± 0.30	0.54 ± 0.09	0.28	136	
[a]Ratio is taken from the relative Vmax Km−1 between free and immobilized LPs

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 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

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

	Specific activity (mM min−1 mg−1)	Specific activity (mM min−1 mg−1)	Ratio[a] (%)	
Free LP[b]	Immobilized LP[c]		
pH 6	0.0119	0.0141	1.18	
pH 7	0.0466	0.0745	1.60	
pH 8	0.1149	0.1789	1.56	
pH 9	0.1106	0.2462	2.23	
pH 10	0.0837	0.2495	2.98	
The ratio ([a] = [c]/[b]) was determined from the relative specific activity comparison between free and immobilized LP

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 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

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

Sample	Retained
specific activity	Storage stability	Reusability	Reference	
Ratio[a] (%)	(Day, Stability[b])	(Number of uses, Stability[c])	
E-HAp-M	156%	80 days, 50%	10 times, 45%	This work	
SiO2	60%	30 days, 76%	10 times, 34%	[93]	
Ceramic membranes (with Fuller’s earth clay and rice husk ash)	40%	Non specified	5 times, 40%	[94]	
Nanoflower based on Cu3(PO4)2	80.7%	28 days, 80%	8 times, 70%	[95]	
Trisilanolcyclohexyl POSS + lipase/ZrO2	70%	Non specified	3 times, 65%	[96]	
Trisilanolcyclohexyl POSS + lipase/ZrO2-NHx	60%	Non specified	3 times, 65%	[97]	
CLIL (cross-linked immobilized lipase)-SiO2	87.8%	10 days, 60%	5 times, 53%	[98]	
Epoxy-activated chitosan-coated silver oxide nanoparticles	67.5%	90 days, 50%	10 times, 50%	[99]	
Epoxy-activated Ag2O/Chitosan nanocomposites	50%	90 days, 50%	10 times, 50%	[100]	
Methyltrimethoxysilane (MTMS)-modified ceramic membranes	64%	Non specified	7 times, 64%	[101]	
Natural polyphenol-modified magnetic nanoparticles	75.3%	Non specified	8 times, 75.3%	[102]	
[a]Retained specific activity was determined specific activity comparison between free and immobilized LP from the relative

[b], [c]The stability of the immobilized LP was compared by considering the initial activity value as 100% of the first day and first uses of immobilized LP and evaluating the relative activity value as a percentage

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.

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).

Authors’ contributions

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

Funding

No funding was obtained for this study.

Availability of data and materials

Not applicable.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. Sheldon RA van Pelt S Enzyme immobilisation in biocatalysis: why, what and how Chem Soc Rev 2013 42 15 6223 6235 10.1039/C3CS60075K 23532151
Sheldon RA, van Pelt S. Enzyme immobilisation in biocatalysis: why, what and how. Chem Soc Rev. 2013;42(15):6223–35.23532151 10.1039/C3CS60075K
2. Jesionowski T Zdarta J Krajewska B Enzyme immobilization by adsorption: a review Adsorption 2014 20 801 821 10.1007/s10450-014-9623-y
Jesionowski T, Zdarta J, Krajewska B. Enzyme immobilization by adsorption: a review. Adsorption. 2014;20:801–21.10.1007/s10450-014-9623-y
3. Rios NS Mendez-Sanchez C Arana-Peña S Rueda N Ortiz C Gonçalves LR Fernandez-Lafuente R Immobilization of lipase from Pseudomonas fluorescens on glyoxyl-octyl-agarose beads: Improved stability and reusability Biochim Biophys Acta Proteins Proteom 2019 1867 9 741 747 10.1016/j.bbapap.2019.06.005 31202001
Rios NS, Mendez-Sanchez C, Arana-Peña S, Rueda N, Ortiz C, Gonçalves LR, Fernandez-Lafuente R. Immobilization of lipase from Pseudomonas fluorescens on glyoxyl-octyl-agarose beads: Improved stability and reusability. Biochim Biophys Acta Proteins Proteom. 2019;1867(9):741–7.31202001 10.1016/j.bbapap.2019.06.005
4. Spasojevic M Prodanovic O Pantic N Popovic N Balaz AM Prodanovic R The enzyme immobilization: carriers and immobilization methods Journal of Engineering & Processing Management 2019 11 2 89 105
Spasojevic M, Prodanovic O, Pantic N, Popovic N, Balaz AM, Prodanovic R. The enzyme immobilization: carriers and immobilization methods. Journal of Engineering & Processing Management. 2019;11(2):89–105.
5. Almulaiky YQ Khalil N El-Shishtawy RM Altalhi T Algamal Y Aldhahri M Al-Harbi SA Allehyani ES Bilal M Mohammed MM Hydroxyapatite-decorated ZrO2 for α-amylase immobilization: Toward the enhancement of enzyme stability and reusability Int J Biol Macromol 2021 167 299 308 10.1016/j.ijbiomac.2020.11.150 33275970
Almulaiky YQ, Khalil N, El-Shishtawy RM, Altalhi T, Algamal Y, Aldhahri M, Al-Harbi SA, Allehyani ES, Bilal M, Mohammed MM. Hydroxyapatite-decorated ZrO2 for α-amylase immobilization: Toward the enhancement of enzyme stability and reusability. Int J Biol Macromol. 2021;167:299–308.33275970 10.1016/j.ijbiomac.2020.11.150
6. Vargason AM, Anselmo AC, Mitragotri S. The evolution of commercial drug delivery technologies. Nat Biomed Eng. 2021;5:951–67.
7. Tandon S Sharma A Singh S Sharma S Sarma SJ Therapeutic enzymes: Discoveries, production and applications Journal of Drug Delivery Science and Technology 2021 63 102455 10.1016/j.jddst.2021.102455
Tandon S, Sharma A, Singh S, Sharma S, Sarma SJ. Therapeutic enzymes: Discoveries, production and applications. Journal of Drug Delivery Science and Technology. 2021;63: 102455.10.1016/j.jddst.2021.102455
8. Nguyen HH Kim M An overview of techniques in enzyme immobilization Applied Science and Convergence Technology 2017 26 6 157 163 10.5757/ASCT.2017.26.6.157
Nguyen HH, Kim M. An overview of techniques in enzyme immobilization. Applied Science and Convergence Technology. 2017;26(6):157–63.10.5757/ASCT.2017.26.6.157
9. Maghraby YR El-Shabasy RM Ibrahim AH Azzazy HME-S: Enzyme immobilization technologies and industrial applications ACS Omega 2023 8 6 5184 5196 10.1021/acsomega.2c07560 36816672
Maghraby YR, El-Shabasy RM, Ibrahim AH. Azzazy HME-S: Enzyme immobilization technologies and industrial applications. ACS Omega. 2023;8(6):5184–96.36816672 10.1021/acsomega.2c07560
10. Rios NS Morais EG dos Santos GW Andrade Neto DM dos Santos JCS Bohn F Correa MA Fechine PBA Fernandez-Lafuente R Gonçalves LRB Further stabilization of lipase from Pseudomonas fluorescens immobilized on octyl coated nanoparticles via chemical modification with bifunctional agents Int J Biol Macromol 2019 141 313 324 10.1016/j.ijbiomac.2019.09.003 31491511
Rios NS, Morais EG, dos Santos GW, Andrade Neto DM, dos Santos JCS, Bohn F, Correa MA, Fechine PBA, Fernandez-Lafuente R, Gonçalves LRB. Further stabilization of lipase from Pseudomonas fluorescens immobilized on octyl coated nanoparticles via chemical modification with bifunctional agents. Int J Biol Macromol. 2019;141:313–24.31491511 10.1016/j.ijbiomac.2019.09.003
11. Verma ML Kumar S Das A Randhawa JS Chamundeeswari M Chitin and chitosan-based support materials for enzyme immobilization and biotechnological applications Environ Chem Lett 2020 18 2 315 323 10.1007/s10311-019-00942-5
Verma ML, Kumar S, Das A, Randhawa JS, Chamundeeswari M. Chitin and chitosan-based support materials for enzyme immobilization and biotechnological applications. Environ Chem Lett. 2020;18(2):315–23.10.1007/s10311-019-00942-5
12. de Oliveira UMF Lima de Matos LJB, de Souza MCM, Pinheiro BB, dos Santos JCS, Gonçalves LRB: Effect of the Presence of Surfactants and Immobilization Conditions on Catalysts’ Properties of Rhizomucor miehei Lipase onto Chitosan Appl Biochem Biotechnol 2018 184 4 1263 1285 10.1007/s12010-017-2622-1 29019010
de Oliveira UMF. Lima de Matos LJB, de Souza MCM, Pinheiro BB, dos Santos JCS, Gonçalves LRB: Effect of the Presence of Surfactants and Immobilization Conditions on Catalysts’ Properties of Rhizomucor miehei Lipase onto Chitosan. Appl Biochem Biotechnol. 2018;184(4):1263–85.29019010 10.1007/s12010-017-2622-1
13. de Menezes FL Freire TM de Castro Monteiro RR Antunes RA Melo RLF Freire RM dos Santos JCS Fechine PBA L-cysteine-coated magnetite nanoparticles as a platform for enzymes immobilization: Amplifying biocatalytic activity of Candida antarctica Lipase A Mater Res Bull 2024 177 112882 10.1016/j.materresbull.2024.112882
de Menezes FL, Freire TM, de Castro Monteiro RR, Antunes RA, Melo RLF, Freire RM, dos Santos JCS, Fechine PBA. L-cysteine-coated magnetite nanoparticles as a platform for enzymes immobilization: Amplifying biocatalytic activity of Candida antarctica Lipase A. Mater Res Bull. 2024;177: 112882.10.1016/j.materresbull.2024.112882
14. Kumar A Dhar K Kanwar SS Arora PK Lipase catalysis in organic solvents: advantages and applications Biological procedures online 2016 18 1 11 10.1186/s12575-016-0033-2 26740800
Kumar A, Dhar K, Kanwar SS, Arora PK. Lipase catalysis in organic solvents: advantages and applications. Biological procedures online. 2016;18:1–11.26740800 10.1186/s12575-016-0033-2
15. Khan MR Immobilized enzymes: a comprehensive review Bulletin of the National Research Centre 2021 45 1 1 13 10.1186/s42269-021-00649-0
Khan MR. Immobilized enzymes: a comprehensive review. Bulletin of the National Research Centre. 2021;45(1):1–13.10.1186/s42269-021-00649-0
16. Riaz R Ashraf M Hussain N Baqar Z Bilal M Iqbal HMN Redesigning Robust Biocatalysts by Engineering Enzyme Microenvironment and Enzyme Immobilization Catal Lett 2023 153 6 1587 1601 10.1007/s10562-022-04137-6
Riaz R, Ashraf M, Hussain N, Baqar Z, Bilal M, Iqbal HMN. Redesigning Robust Biocatalysts by Engineering Enzyme Microenvironment and Enzyme Immobilization. Catal Lett. 2023;153(6):1587–601.10.1007/s10562-022-04137-6
17. Li W Bilal M Singh AK Sher F Ashraf SS Franco M Américo-Pinheiro JHP Iqbal HMN Broadening the Scope of Biocatalysis Engineering by Tailoring Enzyme Microenvironment: A Review Catal Lett 2023 153 5 1227 1239 10.1007/s10562-022-04065-5
Li W, Bilal M, Singh AK, Sher F, Ashraf SS, Franco M, Américo-Pinheiro JHP, Iqbal HMN. Broadening the Scope of Biocatalysis Engineering by Tailoring Enzyme Microenvironment: A Review. Catal Lett. 2023;153(5):1227–39.10.1007/s10562-022-04065-5
18. Garcia-Galan C dos Santos JCS Barbosa O Torres R Pereira EB Corberan VC Gonçalves LRB Fernandez-Lafuente R Tuning of Lecitase features via solid-phase chemical modification: Effect of the immobilization protocol Process Biochem 2014 49 4 604 616 10.1016/j.procbio.2014.01.028
Garcia-Galan C, dos Santos JCS, Barbosa O, Torres R, Pereira EB, Corberan VC, Gonçalves LRB, Fernandez-Lafuente R. Tuning of Lecitase features via solid-phase chemical modification: Effect of the immobilization protocol. Process Biochem. 2014;49(4):604–16.10.1016/j.procbio.2014.01.028
19. Cui J Ren S Lin T Feng Y Jia S Shielding effects of Fe3+-tannic acid nanocoatings for immobilized enzyme on magnetic Fe3O4@silica core shell nanosphere Chem Eng J 2018 343 629 637 10.1016/j.cej.2018.03.002
Cui J, Ren S, Lin T, Feng Y, Jia S. Shielding effects of Fe3+-tannic acid nanocoatings for immobilized enzyme on magnetic Fe3O4@silica core shell nanosphere. Chem Eng J. 2018;343:629–37.10.1016/j.cej.2018.03.002
20. Cui J Zhao Y Liu R Zhong C Jia S Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance Sci Rep 2016 6 1 27928 10.1038/srep27928 27297609
Cui J, Zhao Y, Liu R, Zhong C, Jia S. Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance. Sci Rep. 2016;6(1):27928.27297609 10.1038/srep27928
21. Feng Y Du Y Kuang G Zhong L Hu H Jia S Cui J Hierarchical micro- and mesoporous ZIF-8 with core–shell superstructures using colloidal metal sulfates as soft templates for enzyme immobilization J Colloid Interface Sci 2022 610 709 718 10.1016/j.jcis.2021.11.123 34863543
Feng Y, Du Y, Kuang G, Zhong L, Hu H, Jia S, Cui J. Hierarchical micro- and mesoporous ZIF-8 with core–shell superstructures using colloidal metal sulfates as soft templates for enzyme immobilization. J Colloid Interface Sci. 2022;610:709–18.34863543 10.1016/j.jcis.2021.11.123
22. Rueda N dos Santos JCS Ortiz C Torres R Barbosa O Rodrigues RC Berenguer-Murcia Á Fernandez-Lafuente R Chemical Modification in the Design of Immobilized Enzyme Biocatalysts: Drawbacks and Opportunities Chem Rec 2016 16 3 1436 1455 10.1002/tcr.201600007 27166751
Rueda N, dos Santos JCS, Ortiz C, Torres R, Barbosa O, Rodrigues RC, Berenguer-Murcia Á, Fernandez-Lafuente R. Chemical Modification in the Design of Immobilized Enzyme Biocatalysts: Drawbacks and Opportunities. Chem Rec. 2016;16(3):1436–55.27166751 10.1002/tcr.201600007
23. Shen X Du Y Du Z Tang X Li P Cheng J Yan R Cui J Construction of enzyme@glutathione hybrid metal-organic frameworks: glutathione-boosted microenvironment fine-tuning of biomimetic immobilization for improving catalytic performance Materials Today Chemistry 2023 27 101326 10.1016/j.mtchem.2022.101326
Shen X, Du Y, Du Z, Tang X, Li P, Cheng J, Yan R, Cui J. Construction of enzyme@glutathione hybrid metal-organic frameworks: glutathione-boosted microenvironment fine-tuning of biomimetic immobilization for improving catalytic performance. Materials Today Chemistry. 2023;27: 101326.10.1016/j.mtchem.2022.101326
24. Chen N Chang B Shi N Yan W Lu F Liu F Cross-linked enzyme aggregates immobilization: preparation, characterization, and applications Crit Rev Biotechnol 2023 43 3 369 383 10.1080/07388551.2022.2038073 35430938
Chen N, Chang B, Shi N, Yan W, Lu F, Liu F. Cross-linked enzyme aggregates immobilization: preparation, characterization, and applications. Crit Rev Biotechnol. 2023;43(3):369–83.35430938 10.1080/07388551.2022.2038073
25. Cui J Cui L Jia S Su Z Zhang S Hybrid Cross-Linked Lipase Aggregates with Magnetic Nanoparticles: A Robust and Recyclable Biocatalysis for the Epoxidation of Oleic Acid J Agric Food Chem 2016 64 38 7179 7187 10.1021/acs.jafc.6b01939 27595982
Cui J, Cui L, Jia S, Su Z, Zhang S. Hybrid Cross-Linked Lipase Aggregates with Magnetic Nanoparticles: A Robust and Recyclable Biocatalysis for the Epoxidation of Oleic Acid. J Agric Food Chem. 2016;64(38):7179–87.27595982 10.1021/acs.jafc.6b01939
26. Thangaraj B Solomon PR Immobilization of lipases–a review Part II: carrier materials ChemBioEng Reviews 2019 6 5 167 194
Thangaraj B, Solomon PR. Immobilization of lipases–a review. Part II: carrier materials ChemBioEng Reviews. 2019;6(5):167–94.
27. Chauhan V Kaushal D Dhiman VK Kanwar SS Singh D Dhiman VK Pandey H An insight in developing carrier-free immobilized enzymes Frontiers in Bioengineering and Biotechnology 2022 10 794411 10.3389/fbioe.2022.794411 35309979
Chauhan V, Kaushal D, Dhiman VK, Kanwar SS, Singh D, Dhiman VK, Pandey H. An insight in developing carrier-free immobilized enzymes. Frontiers in Bioengineering and Biotechnology. 2022;10: 794411.35309979 10.3389/fbioe.2022.794411
28. Xue P Hu CM Yan XH Fang GL Shen HF Enhancement of activity and reusability of lipase immobilized on magnetic mesoporous silica for the resolution of racemic secondary alcohols J Chin Chem Soc 2019 66 4 427 433 10.1002/jccs.201800193
Xue P, Hu CM, Yan XH, Fang GL, Shen HF. Enhancement of activity and reusability of lipase immobilized on magnetic mesoporous silica for the resolution of racemic secondary alcohols. J Chin Chem Soc. 2019;66(4):427–33.10.1002/jccs.201800193
29. Mohamad NR Marzuki NHC Buang NA Huyop F Wahab RA An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes Biotechnol Biotechnol Equip 2015 29 2 205 220 10.1080/13102818.2015.1008192 26019635
Mohamad NR, Marzuki NHC, Buang NA, Huyop F, Wahab RA. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnol Biotechnol Equip. 2015;29(2):205–20.26019635 10.1080/13102818.2015.1008192
30. Hwang ET Lee B Zhang M Jun S-H Shim J Lee J Kim J Gu MB Immobilization and stabilization of subtilisin Carlsberg in magnetically-separable mesoporous silica for transesterification in an organic solvent Green Chem 2012 14 7 1884 1887 10.1039/c2gc35559k
Hwang ET, Lee B, Zhang M, Jun S-H, Shim J, Lee J, Kim J, Gu MB. Immobilization and stabilization of subtilisin Carlsberg in magnetically-separable mesoporous silica for transesterification in an organic solvent. Green Chem. 2012;14(7):1884–7.10.1039/c2gc35559k
31. Lee CH Lee HS Lee JW Kim J Lee JH Jin ES Hwang ET Evaluating enzyme stabilizations in calcium carbonate: Comparing in situ and crosslinking mediated immobilization Int J Biol Macromol 2021 175 341 350 10.1016/j.ijbiomac.2021.02.028 33556395
Lee CH, Lee HS, Lee JW, Kim J, Lee JH, Jin ES, Hwang ET. Evaluating enzyme stabilizations in calcium carbonate: Comparing in situ and crosslinking mediated immobilization. Int J Biol Macromol. 2021;175:341–50.33556395 10.1016/j.ijbiomac.2021.02.028
32. Garcia-Galan C Berenguer-Murcia Á Fernandez-Lafuente R Rodrigues RC Potential of different enzyme immobilization strategies to improve enzyme performance Adv Synth Catal 2011 353 16 2885 2904 10.1002/adsc.201100534
Garcia-Galan C, Berenguer-Murcia Á, Fernandez-Lafuente R, Rodrigues RC. Potential of different enzyme immobilization strategies to improve enzyme performance. Adv Synth Catal. 2011;353(16):2885–904.10.1002/adsc.201100534
33. Liu D-M Dong C Recent advances in nano-carrier immobilized enzymes and their applications Process Biochem 2020 92 464 475 10.1016/j.procbio.2020.02.005
Liu D-M, Dong C. Recent advances in nano-carrier immobilized enzymes and their applications. Process Biochem. 2020;92:464–75.10.1016/j.procbio.2020.02.005
34. Zucca P Sanjust E Inorganic materials as supports for covalent enzyme immobilization: methods and mechanisms Molecules 2014 19 9 14139 14194 10.3390/molecules190914139 25207718
Zucca P, Sanjust E. Inorganic materials as supports for covalent enzyme immobilization: methods and mechanisms. Molecules. 2014;19(9):14139–94.25207718 10.3390/molecules190914139
35. Zhang M Jun S-H Wee Y Kim HS Hwang ET Shim J Hwang SY Lee J Kim J Activation of crosslinked lipases in mesoporous silica via lid opening for recyclable biodiesel production Int J Biol Macromol 2022 222 2368 2374 10.1016/j.ijbiomac.2022.10.023 36216105
Zhang M, Jun S-H, Wee Y, Kim HS, Hwang ET, Shim J, Hwang SY, Lee J, Kim J. Activation of crosslinked lipases in mesoporous silica via lid opening for recyclable biodiesel production. Int J Biol Macromol. 2022;222:2368–74.36216105 10.1016/j.ijbiomac.2022.10.023
36. Ashkan Z Hemmati R Homaei A Dinari A Jamlidoost M Tashakor A Immobilization of enzymes on nanoinorganic support materials: An update Int J Biol Macromol 2021 168 708 721 10.1016/j.ijbiomac.2020.11.127 33232698
Ashkan Z, Hemmati R, Homaei A, Dinari A, Jamlidoost M, Tashakor A. Immobilization of enzymes on nanoinorganic support materials: An update. Int J Biol Macromol. 2021;168:708–21.33232698 10.1016/j.ijbiomac.2020.11.127
37. Villeneuve P Muderhwa JM Graille J Haas MJ Customizing lipases for biocatalysis: a survey of chemical, physical and molecular biological approaches J Mol Catal B Enzym 2000 9 4–6 113 148 10.1016/S1381-1177(99)00107-1
Villeneuve P, Muderhwa JM, Graille J, Haas MJ. Customizing lipases for biocatalysis: a survey of chemical, physical and molecular biological approaches. J Mol Catal B Enzym. 2000;9(4–6):113–48.10.1016/S1381-1177(99)00107-1
38. Liu S Bilal M Rizwan K Gul I Rasheed T Iqbal HM Smart chemistry of enzyme immobilization using various support matrices–a review Int J Biol Macromol 2021 190 396 408 10.1016/j.ijbiomac.2021.09.006 34506857
Liu S, Bilal M, Rizwan K, Gul I, Rasheed T, Iqbal HM. Smart chemistry of enzyme immobilization using various support matrices–a review. Int J Biol Macromol. 2021;190:396–408.34506857 10.1016/j.ijbiomac.2021.09.006
39. Zdarta J Meyer AS Jesionowski T Pinelo M A general overview of support materials for enzyme immobilization: characteristics, properties, practical utility Catalysts 2018 8 2 92 10.3390/catal8020092
Zdarta J, Meyer AS, Jesionowski T, Pinelo M. A general overview of support materials for enzyme immobilization: characteristics, properties, practical utility. Catalysts. 2018;8(2):92.10.3390/catal8020092
40. Cavalcante FT Cavalcante AL de Sousa IG Neto FS dos Santos JC Current status and future perspectives of supports and protocols for enzyme immobilization Catalysts 2021 11 10 1222 10.3390/catal11101222
Cavalcante FT, Cavalcante AL, de Sousa IG, Neto FS, dos Santos JC. Current status and future perspectives of supports and protocols for enzyme immobilization. Catalysts. 2021;11(10):1222.10.3390/catal11101222
41. Ismail AR Baek K-H Lipase immobilization with support materials, preparation techniques, and applications: Present and future aspects Int J Biol Macromol 2020 163 1624 1639 10.1016/j.ijbiomac.2020.09.021 32916199
Ismail AR, Baek K-H. Lipase immobilization with support materials, preparation techniques, and applications: Present and future aspects. Int J Biol Macromol. 2020;163:1624–39.32916199 10.1016/j.ijbiomac.2020.09.021
42. Mulinari J Oliveira JV Hotza D Lipase immobilization on ceramic supports: An overview on techniques and materials Biotechnol Adv 2020 42 107581 10.1016/j.biotechadv.2020.107581 32590050
Mulinari J, Oliveira JV, Hotza D. Lipase immobilization on ceramic supports: An overview on techniques and materials. Biotechnol Adv. 2020;42: 107581.32590050 10.1016/j.biotechadv.2020.107581
43. Govardhan CP Crosslinking of enzymes for improved stability and performance Curr Opin Biotechnol 1999 10 4 331 335 10.1016/S0958-1669(99)80060-3 10449320
Govardhan CP. Crosslinking of enzymes for improved stability and performance. Curr Opin Biotechnol. 1999;10(4):331–5.10449320 10.1016/S0958-1669(99)80060-3
44. Yang Y Yu H Zhou X Zhou Z Shallow porous microsphere carriers with core-shell structure based on glass beads cross-linking chitosan for immobilizing inulinase Molecular Catalysis 2020 486 110871 10.1016/j.mcat.2020.110871
Yang Y, Yu H, Zhou X, Zhou Z. Shallow porous microsphere carriers with core-shell structure based on glass beads cross-linking chitosan for immobilizing inulinase. Molecular Catalysis. 2020;486: 110871.10.1016/j.mcat.2020.110871
45. Kim MI Kim J Lee J Jia H Na HB Youn JK Kwak JH Dohnalkova A Grate JW Wang P Crosslinked enzyme aggregates in hierarchically-ordered mesoporous silica: A simple and effective method for enzyme stabilization Biotechnol Bioeng 2007 96 2 210 218 10.1002/bit.21107 16986168
Kim MI, Kim J, Lee J, Jia H, Na HB, Youn JK, Kwak JH, Dohnalkova A, Grate JW, Wang P. Crosslinked enzyme aggregates in hierarchically-ordered mesoporous silica: A simple and effective method for enzyme stabilization. Biotechnol Bioeng. 2007;96(2):210–8.16986168 10.1002/bit.21107
46. Kim J Lee J Na HB Kim BC Youn JK Kwak JH Moon K Lee E Kim J Park J A Magnetically Separable, Highly Stable Enzyme System Based on Nanocomposites of Enzymes and Magnetic Nanoparticles Shipped in Hierarchically Ordered, Mesocellular Mesoporous Silica Small 2005 1 12 1203 1207 17193420
Kim J, Lee J, Na HB, Kim BC, Youn JK, Kwak JH, Moon K, Lee E, Kim J, Park J, et al. A Magnetically Separable, Highly Stable Enzyme System Based on Nanocomposites of Enzymes and Magnetic Nanoparticles Shipped in Hierarchically Ordered, Mesocellular. Mesoporous Silica Small. 2005;1(12):1203–7.17193420
47. Hirsh SL Bilek MMM Nosworthy NJ Kondyurin A dos Remedios CG McKenzie DR A Comparison of Covalent Immobilization and Physical Adsorption of a Cellulase Enzyme Mixture Langmuir 2010 26 17 14380 14388 10.1021/la1019845 20704318
Hirsh SL, Bilek MMM, Nosworthy NJ, Kondyurin A, dos Remedios CG, McKenzie DR. A Comparison of Covalent Immobilization and Physical Adsorption of a Cellulase Enzyme Mixture. Langmuir. 2010;26(17):14380–8.20704318 10.1021/la1019845
48. Rodrigues RC Berenguer-Murcia Á Carballares D Morellon-Sterling R Fernandez-Lafuente R Stabilization of enzymes via immobilization: Multipoint covalent attachment and other stabilization strategies Biotechnol Adv 2021 52 107821 10.1016/j.biotechadv.2021.107821 34455028
Rodrigues RC, Berenguer-Murcia Á, Carballares D, Morellon-Sterling R, Fernandez-Lafuente R. Stabilization of enzymes via immobilization: Multipoint covalent attachment and other stabilization strategies. Biotechnol Adv. 2021;52: 107821.34455028 10.1016/j.biotechadv.2021.107821
49. Yushkova ED Nazarova EA Matyuhina AV Noskova AO Shavronskaya DO Vinogradov VV Skvortsova NN Krivoshapkina EF Application of immobilized enzymes in food industry J Agric Food Chem 2019 67 42 11553 11567 10.1021/acs.jafc.9b04385 31553885
Yushkova ED, Nazarova EA, Matyuhina AV, Noskova AO, Shavronskaya DO, Vinogradov VV, Skvortsova NN, Krivoshapkina EF. Application of immobilized enzymes in food industry. J Agric Food Chem. 2019;67(42):11553–67.31553885 10.1021/acs.jafc.9b04385
50. Magnan E Catarino I Paolucci-Jeanjean D Preziosi-Belloy L Belleville M Immobilization of lipase on a ceramic membrane: activity and stability J Membr Sci 2004 241 1 161 166 10.1016/j.memsci.2004.05.023
Magnan E, Catarino I, Paolucci-Jeanjean D, Preziosi-Belloy L, Belleville M. Immobilization of lipase on a ceramic membrane: activity and stability. J Membr Sci. 2004;241(1):161–6.10.1016/j.memsci.2004.05.023
51. Kamori M Hori T Yamashita Y Hirose Y Naoshima Y Immobilization of lipase on a new inorganic ceramics support, toyonite, and the reactivity and enantioselectivity of the immobilized lipase J Mol Catal B Enzym 2000 9 4–6 269 274 10.1016/S1381-1177(99)00105-8
Kamori M, Hori T, Yamashita Y, Hirose Y, Naoshima Y. Immobilization of lipase on a new inorganic ceramics support, toyonite, and the reactivity and enantioselectivity of the immobilized lipase. J Mol Catal B Enzym. 2000;9(4–6):269–74.10.1016/S1381-1177(99)00105-8
52. Hwang ET Gu MB Enzyme stabilization by nano/microsized hybrid materials Eng Life Sci 2013 13 1 49 61 10.1002/elsc.201100225
Hwang ET, Gu MB. Enzyme stabilization by nano/microsized hybrid materials. Eng Life Sci. 2013;13(1):49–61.10.1002/elsc.201100225
53. Coutinho TC Tardioli PW Farinas CS Phytase immobilization on hydroxyapatite nanoparticles improves its properties for use in animal feed Appl Biochem Biotechnol 2020 190 1 270 292 10.1007/s12010-019-03116-9 31342308
Coutinho TC, Tardioli PW, Farinas CS. Phytase immobilization on hydroxyapatite nanoparticles improves its properties for use in animal feed. Appl Biochem Biotechnol. 2020;190(1):270–92.31342308 10.1007/s12010-019-03116-9
54. Fihri A Len C Varma RS Solhy A Hydroxyapatite: A review of syntheses, structure and applications in heterogeneous catalysis Coord Chem Rev 2017 347 48 76 10.1016/j.ccr.2017.06.009
Fihri A, Len C, Varma RS, Solhy A. Hydroxyapatite: A review of syntheses, structure and applications in heterogeneous catalysis. Coord Chem Rev. 2017;347:48–76.10.1016/j.ccr.2017.06.009
55. Emtiazi G Shapoorabadi F Mirbagheri M Chemical and Biological Synthesis of HydroxyApatite: Advantage and Application Int J Microbiol Curr Res 2019 1 1 20 22 10.18689/ijmr-1000103
Emtiazi G, Shapoorabadi F, Mirbagheri M. Chemical and Biological Synthesis of HydroxyApatite: Advantage and Application. Int J Microbiol Curr Res. 2019;1(1):20–2.10.18689/ijmr-1000103
56. DileepKumar VG Sridhar MS Aramwit P Krut'ko VK Musskaya ON Glazov IE Reddy N A review on the synthesis and properties of hydroxyapatite for biomedical applications 2022 33 2 229 261
DileepKumar VG, Sridhar MS, Aramwit P, Krut’ko VK, Musskaya ON, Glazov IE, Reddy N. A review on the synthesis and properties of hydroxyapatite for biomedical applications. 2022;33(2):229–61.
57. Bushra A Subhani A Islam N A comprehensive review on biological and environmental applications of chitosan-hydroxyapatite biocomposites Composites Part C: Open Access 2023 12 100402
Bushra A, Subhani A, Islam N. A comprehensive review on biological and environmental applications of chitosan-hydroxyapatite biocomposites. Composites Part C: Open Access. 2023;12: 100402.
58. Hong L Hengchang X de Groot K Tensile strength of the interface between hydroxyapatite and bone J Biomed Mater Res 1992 26 1 7 18 10.1002/jbm.820260103 1315777
Hong L, Hengchang X, de Groot K. Tensile strength of the interface between hydroxyapatite and bone. J Biomed Mater Res. 1992;26(1):7–18.1315777 10.1002/jbm.820260103
59. Wu X Song X Li D Liu J Zhang P Chen X Preparation of Mesoporous Nano-Hydroxyapatite Using a Surfactant Template Method for Protein Delivery J Bionic Eng 2012 9 2 224 233 10.1016/S1672-6529(11)60105-4
Wu X, Song X, Li D, Liu J, Zhang P, Chen X. Preparation of Mesoporous Nano-Hydroxyapatite Using a Surfactant Template Method for Protein Delivery. J Bionic Eng. 2012;9(2):224–33.10.1016/S1672-6529(11)60105-4
60. Odusote JK Danyuo Y Baruwa AD Azeez AA Synthesis and characterization of hydroxyapatite from bovine bone for production of dental implants Journal of Applied Biomaterials & Functional Materials 2019 17 2 2280800019836829 10.1177/2280800019836829 31041872
Odusote JK, Danyuo Y, Baruwa AD, Azeez AA. Synthesis and characterization of hydroxyapatite from bovine bone for production of dental implants. Journal of Applied Biomaterials & Functional Materials. 2019;17(2):2280800019836829.31041872 10.1177/2280800019836829
61. Cengiz B Gokce Y Yildiz N Aktas Z Calimli A Synthesis and characterization of hydroxyapatite nanoparticles Colloids Surf, A 2008 322 1 29 33 10.1016/j.colsurfa.2008.02.011
Cengiz B, Gokce Y, Yildiz N, Aktas Z, Calimli A. Synthesis and characterization of hydroxyapatite nanoparticles. Colloids Surf, A. 2008;322(1):29–33.10.1016/j.colsurfa.2008.02.011
62. Shylaja S Prashanthi Y Nageswara Rao T Synthesis and evaluating the effects of nano hydroxyapatite on germination, growth and yield of cluster beans Materials Today: Proceedings 2022 64 917 921
Shylaja S, Prashanthi Y, Nageswara Rao T. Synthesis and evaluating the effects of nano hydroxyapatite on germination, growth and yield of cluster beans. Materials Today: Proceedings. 2022;64:917–21.
63. Coutinho TC Tardioli PW Farinas CS Hydroxyapatite nanoparticles modified with metal ions for xylanase immobilization Int J Biol Macromol 2020 150 344 353 10.1016/j.ijbiomac.2020.02.058 32045612
Coutinho TC, Tardioli PW, Farinas CS. Hydroxyapatite nanoparticles modified with metal ions for xylanase immobilization. Int J Biol Macromol. 2020;150:344–53.32045612 10.1016/j.ijbiomac.2020.02.058
64. Coutinho TC Rojas MJ Tardioli PW Paris EC Farinas CS Nanoimmobilization of β-glucosidase onto hydroxyapatite Int J Biol Macromol 2018 119 1042 1051 10.1016/j.ijbiomac.2018.08.042 30098358
Coutinho TC, Rojas MJ, Tardioli PW, Paris EC, Farinas CS. Nanoimmobilization of β-glucosidase onto hydroxyapatite. Int J Biol Macromol. 2018;119:1042–51.30098358 10.1016/j.ijbiomac.2018.08.042
65. Yewle JN Wei Y Puleo DA Daunert S Bachas LG Oriented Immobilization of Proteins on Hydroxyapatite Surface Using Bifunctional Bisphosphonates as Linkers Biomacromol 2012 13 6 1742 1749 10.1021/bm201865r
Yewle JN, Wei Y, Puleo DA, Daunert S, Bachas LG. Oriented Immobilization of Proteins on Hydroxyapatite Surface Using Bifunctional Bisphosphonates as Linkers. Biomacromol. 2012;13(6):1742–9.10.1021/bm201865r
66. Lee CH Jin ES Lee JH Hwang ET Immobilization and Stabilization of Enzyme in Biomineralized Calcium Carbonate Microspheres Frontiers in bioengineering and biotechnology 2020 8 553591 10.3389/fbioe.2020.553591 33163476
Lee CH, Jin ES, Lee JH, Hwang ET. Immobilization and Stabilization of Enzyme in Biomineralized Calcium Carbonate Microspheres. Frontiers in bioengineering and biotechnology. 2020;8: 553591.33163476 10.3389/fbioe.2020.553591
67. Obada DO Osseni SA Sina H Oyedeji AN Salami KA Okafor E Csaki S Abolade SA Akande A Dauda M Hydroxyapatite materials-synthesis routes, mechanical behavior, theoretical insights, and artificial intelligence models: a review J Aust Ceram Soc 2023 59 3 565 596 10.1007/s41779-023-00854-2
Obada DO, Osseni SA, Sina H, Oyedeji AN, Salami KA, Okafor E, Csaki S, Abolade SA, Akande A, Dauda M, et al. Hydroxyapatite materials-synthesis routes, mechanical behavior, theoretical insights, and artificial intelligence models: a review. J Aust Ceram Soc. 2023;59(3):565–96.10.1007/s41779-023-00854-2
68. Najmi H Singh RP A review on synthesis, characterisation, and applications of hydroxyapatite nanoparticles Advances in manufacturing and materials: 2024 2024 Singapore Springer Nature Singapore 703 720
Najmi H, Singh RP. A review on synthesis, characterisation, and applications of hydroxyapatite nanoparticles. In: Advances in manufacturing and materials: 2024. Singapore: Springer Nature Singapore; 2024. p. 703–20.
69. Chaikina MV Bulina NV Vinokurova OB Gerasimov KB Prosanov IY Kompankov NB Lapina OB Papulovskiy ES Ishchenko AV Makarova SV Possibilities of Mechanochemical Synthesis of Apatites with Different Ca/P Ratios Ceramics 2022 5 3 404 422 10.3390/ceramics5030031
Chaikina MV, Bulina NV, Vinokurova OB, Gerasimov KB, Prosanov IY, Kompankov NB, Lapina OB, Papulovskiy ES, Ishchenko AV, Makarova SV. Possibilities of Mechanochemical Synthesis of Apatites with Different Ca/P Ratios. Ceramics. 2022;5(3):404–22.10.3390/ceramics5030031
70. Kottegoda N Munaweera I Madusanka N Karunaratne V A green slow-release fertilizer composition based on urea-modified hydroxyapatite nanoparticles encapsulated wood Curr Sci 2011 101 1 73 78
Kottegoda N, Munaweera I, Madusanka N, Karunaratne V. A green slow-release fertilizer composition based on urea-modified hydroxyapatite nanoparticles encapsulated wood. Curr Sci. 2011;101(1):73–8.
71. Ma MG Hierarchically nanostructured hydroxyapatite: hydrothermal synthesis, morphology control, growth mechanism, and biological activity Int J Nanomedicine 2012 7 1781 1791 10.2147/IJN.S29884 22619527
Ma MG. Hierarchically nanostructured hydroxyapatite: hydrothermal synthesis, morphology control, growth mechanism, and biological activity. Int J Nanomedicine. 2012;7:1781–91.22619527 10.2147/IJN.S29884
72. Catros S Guillemot F Lebraud E Chanseau C Perez S Bareille R Amédée J Fricain JC Physico-chemical and biological properties of a nano-hydroxyapatite powder synthesized at room temperature IRBM 2010 31 4 226 233 10.1016/j.irbm.2010.04.002
Catros S, Guillemot F, Lebraud E, Chanseau C, Perez S, Bareille R, Amédée J, Fricain JC. Physico-chemical and biological properties of a nano-hydroxyapatite powder synthesized at room temperature. IRBM. 2010;31(4):226–33.10.1016/j.irbm.2010.04.002
73. Sanosh KP Chu M-C Balakrishnan A Kim TN Cho S-J Preparation and characterization of nano-hydroxyapatite powder using sol-gel technique Bull Mater Sci 2009 32 5 465 470 10.1007/s12034-009-0069-x
Sanosh KP, Chu M-C, Balakrishnan A, Kim TN, Cho S-J. Preparation and characterization of nano-hydroxyapatite powder using sol-gel technique. Bull Mater Sci. 2009;32(5):465–70.10.1007/s12034-009-0069-x
74. Munir MU Ihsan A Sarwar Y Bajwa SZ Bano K Tehseen B Zeb N Hussain I Ansari MT Saeed M Hollow mesoporous hydroxyapatite nanostructures; smart nanocarriers with high drug loading and controlled releasing features Int J Pharm 2018 544 1 112 120 10.1016/j.ijpharm.2018.04.029 29678543
Munir MU, Ihsan A, Sarwar Y, Bajwa SZ, Bano K, Tehseen B, Zeb N, Hussain I, Ansari MT, Saeed M, et al. Hollow mesoporous hydroxyapatite nanostructures; smart nanocarriers with high drug loading and controlled releasing features. Int J Pharm. 2018;544(1):112–20.29678543 10.1016/j.ijpharm.2018.04.029
75. Wijesinghe WPSL Mantilaka MMMGPG, Peiris TAN, Rajapakse RMG, Wijayantha KGU, Pitawala HMTGA, Premachandra TN, Herath HMTU, Rajapakse RPVJ: Preparation and characterization of mesoporous hydroxyapatite with non-cytotoxicity and heavy metal adsorption capacity New J Chem 2018 42 12 10271 10278 10.1039/C8NJ00673C
Wijesinghe WPSL. Mantilaka MMMGPG, Peiris TAN, Rajapakse RMG, Wijayantha KGU, Pitawala HMTGA, Premachandra TN, Herath HMTU, Rajapakse RPVJ: Preparation and characterization of mesoporous hydroxyapatite with non-cytotoxicity and heavy metal adsorption capacity. New J Chem. 2018;42(12):10271–8.10.1039/C8NJ00673C
76. Pota G, Gallucci N, Cavasso D, Krauss IR. Controlling the adsorption of β-Glucosidase onto wrinkled SiO(2) nanoparticles to boost the yield of immobilization of an efficient biocatalyst. Langmuir. 2023;39(4):1482–94.
77. Zhang Y Gao F Zhang S-P Su Z-G Ma G-H Wang P Simultaneous production of 1,3-dihydroxyacetone and xylitol from glycerol and xylose using a nanoparticle-supported multi-enzyme system with in situ cofactor regeneration Biores Technol 2011 102 2 1837 1843 10.1016/j.biortech.2010.09.069
Zhang Y, Gao F, Zhang S-P, Su Z-G, Ma G-H, Wang P. Simultaneous production of 1,3-dihydroxyacetone and xylitol from glycerol and xylose using a nanoparticle-supported multi-enzyme system with in situ cofactor regeneration. Biores Technol. 2011;102(2):1837–43.10.1016/j.biortech.2010.09.069
78. Rios NS Pinheiro BB Pinheiro MP Bezerra RM dos Santos JCS Barros Gonçalves LR Biotechnological potential of lipases from Pseudomonas: Sources, properties and applications Process Biochem 2018 75 99 120 10.1016/j.procbio.2018.09.003
Rios NS, Pinheiro BB, Pinheiro MP, Bezerra RM, dos Santos JCS, Barros Gonçalves LR. Biotechnological potential of lipases from Pseudomonas: Sources, properties and applications. Process Biochem. 2018;75:99–120.10.1016/j.procbio.2018.09.003
79. Faulón Marruecos D Schwartz DK Kaar JL Impact of surface interactions on protein conformation Curr Opin Colloid Interface Sci 2018 38 45 55 10.1016/j.cocis.2018.08.002
Faulón Marruecos D, Schwartz DK, Kaar JL. Impact of surface interactions on protein conformation. Curr Opin Colloid Interface Sci. 2018;38:45–55.10.1016/j.cocis.2018.08.002
80. Ge J Lei J Zare RN Protein–inorganic hybrid nanoflowers Nat Nanotechnol 2012 7 7 428 432 10.1038/nnano.2012.80 22659609
Ge J, Lei J, Zare RN. Protein–inorganic hybrid nanoflowers. Nat Nanotechnol. 2012;7(7):428–32.22659609 10.1038/nnano.2012.80
81. Ofudje EA Akande JA Sodiya EF Ajayi GO Ademoyegun AJ Al-Sehemi AG Kavil YN Bakheet AM Bioactivity properties of hydroxyapatite/clay nanocomposites Sci Rep 2023 13 1 19896 10.1038/s41598-023-45646-7 37963905
Ofudje EA, Akande JA, Sodiya EF, Ajayi GO, Ademoyegun AJ, Al-Sehemi AG, Kavil YN, Bakheet AM. Bioactivity properties of hydroxyapatite/clay nanocomposites. Sci Rep. 2023;13(1):19896.37963905 10.1038/s41598-023-45646-7
82. Sailuam W Phacheerak K Atipong b, Fongkaew I, Limpijumnong S: Elastic and mechanical properties of hydroxyapatite under pressure: A first-principles investigation Computational Condensed Matter 2020 24 e00481 10.1016/j.cocom.2020.e00481
Sailuam W, Phacheerak K. Atipong b, Fongkaew I, Limpijumnong S: Elastic and mechanical properties of hydroxyapatite under pressure: A first-principles investigation. Computational Condensed Matter. 2020;24: e00481.10.1016/j.cocom.2020.e00481
83. Abellanas-Perez P Carballares D Fernandez-Lafuente R Rocha-Martin J Glutaraldehyde modification of lipases immobilized on octyl agarose beads: Roles of the support enzyme loading and chemical amination of the enzyme on the final enzyme features Int J Biol Macromol 2023 248 125853 10.1016/j.ijbiomac.2023.125853 37460068
Abellanas-Perez P, Carballares D, Fernandez-Lafuente R, Rocha-Martin J. Glutaraldehyde modification of lipases immobilized on octyl agarose beads: Roles of the support enzyme loading and chemical amination of the enzyme on the final enzyme features. Int J Biol Macromol. 2023;248: 125853.37460068 10.1016/j.ijbiomac.2023.125853
84. Dal Magro L Kornecki JF Klein MP Rodrigues RC Fernandez-Lafuente R Pectin lyase immobilization using the glutaraldehyde chemistry increases the enzyme operation range Enzyme Microb Technol 2020 132 109397 10.1016/j.enzmictec.2019.109397 31731972
Dal Magro L, Kornecki JF, Klein MP, Rodrigues RC, Fernandez-Lafuente R. Pectin lyase immobilization using the glutaraldehyde chemistry increases the enzyme operation range. Enzyme Microb Technol. 2020;132: 109397.31731972 10.1016/j.enzmictec.2019.109397
85. Gestrelius S Mattiasson B Mosbach K On the Regulation of the Activity of Immobilized Enzymes Eur J Biochem 1973 36 1 89 96 10.1111/j.1432-1033.1973.tb02888.x 4732921
Gestrelius S, Mattiasson B, Mosbach K. On the Regulation of the Activity of Immobilized Enzymes. Eur J Biochem. 1973;36(1):89–96.4732921 10.1111/j.1432-1033.1973.tb02888.x
86. Tischer W Kasche V Immobilized enzymes: crystals or carriers? Trends Biotechnol 1999 17 8 326 335 10.1016/S0167-7799(99)01322-0 10407405
Tischer W, Kasche V. Immobilized enzymes: crystals or carriers? Trends Biotechnol. 1999;17(8):326–35.10407405 10.1016/S0167-7799(99)01322-0
87. Chandra P Enespa, Singh R, Arora PK: Microbial lipases and their industrial applications: a comprehensive review Microb Cell Fact 2020 19 1 169 10.1186/s12934-020-01428-8 32847584
Chandra P. Enespa, Singh R, Arora PK: Microbial lipases and their industrial applications: a comprehensive review. Microb Cell Fact. 2020;19(1):169.32847584 10.1186/s12934-020-01428-8
88. Filho DG Silva AG Guidini CZ Lipases: sources, immobilization methods, and industrial applications Appl Microbiol Biotechnol 2019 103 18 7399 7423 10.1007/s00253-019-10027-6 31375880
Filho DG, Silva AG, Guidini CZ. Lipases: sources, immobilization methods, and industrial applications. Appl Microbiol Biotechnol. 2019;103(18):7399–423.31375880 10.1007/s00253-019-10027-6
89. Basso A Serban S Industrial applications of immobilized enzymes—A review Molecular Catalysis 2019 479 110607 10.1016/j.mcat.2019.110607
Basso A, Serban S. Industrial applications of immobilized enzymes—A review. Molecular Catalysis. 2019;479: 110607.10.1016/j.mcat.2019.110607
90. Rafiee F Rezaee M Different strategies for the lipase immobilization on the chitosan based supports and their applications Int J Biol Macromol 2021 179 170 195 10.1016/j.ijbiomac.2021.02.198 33667561
Rafiee F, Rezaee M. Different strategies for the lipase immobilization on the chitosan based supports and their applications. Int J Biol Macromol. 2021;179:170–95.33667561 10.1016/j.ijbiomac.2021.02.198
91. Bolivar JM Woodley JM Fernandez-Lafuente R Is enzyme immobilization a mature discipline? Some critical considerations to capitalize on the benefits of immobilization Chem Soc Rev 2022 51 15 6251 6290 10.1039/D2CS00083K 35838107
Bolivar JM, Woodley JM, Fernandez-Lafuente R. Is enzyme immobilization a mature discipline? Some critical considerations to capitalize on the benefits of immobilization. Chem Soc Rev. 2022;51(15):6251–90.35838107 10.1039/D2CS00083K
92. Bullo GT, Marasca N, Almeida FLC, Forte MBS. Lipases: market study and potential applications of immobilized derivatives. Biofuels, Bioproducts and Biorefining. 2024. 10.1002/bbb.2607.
93. Çakıroğlu B Jabiyeva N Holzinger M Photosystem II as a chemiluminescence-induced photosensitizer for photoelectrochemical biofuel cell-type biosensing system Biosens Bioelectron 2023 226 115133 10.1016/j.bios.2023.115133 36773487
Çakıroğlu B, Jabiyeva N, Holzinger M. Photosystem II as a chemiluminescence-induced photosensitizer for photoelectrochemical biofuel cell-type biosensing system. Biosens Bioelectron. 2023;226: 115133.36773487 10.1016/j.bios.2023.115133
94. Çakıroğlu B Chauvin J Le Goff A Gorgy K Özacar M Holzinger M Photoelectrochemically-assisted biofuel cell constructed by redox complex and g-C3N4 coated MWCNT bioanode Biosens Bioelectron 2020 169 112601 10.1016/j.bios.2020.112601 32931991
Çakıroğlu B, Chauvin J, Le Goff A, Gorgy K, Özacar M, Holzinger M. Photoelectrochemically-assisted biofuel cell constructed by redox complex and g-C3N4 coated MWCNT bioanode. Biosens Bioelectron. 2020;169: 112601.32931991 10.1016/j.bios.2020.112601
95. Li Y-Y, Ma X-X, Song X-Y, Ma L-L, Li Y-Y, Meng X, Chen Y-J, Xu K-X, Moosavi-Movahedi AA, Xiao B-L, et al. Glucose biosensor based on glucose oxidase immobilized on BSA cross-linked nanocomposite modified glassy carbon electrode. Sensors 2023;23(6):3209.
96. Ge L Hou R Cao Y Tu J Wu Q Photoelectrochemical enzymatic sensor for glucose based on Au@C/TiO2 nanorod arrays RSC Adv 2020 10 72 44225 44231 10.1039/D0RA08920F 35517172
Ge L, Hou R, Cao Y, Tu J, Wu Q. Photoelectrochemical enzymatic sensor for glucose based on Au@C/TiO2 nanorod arrays. RSC Adv. 2020;10(72):44225–31.35517172 10.1039/D0RA08920F
97. Li J Liu Y Tang X Xu L Min L Xue Y Hu X Yang Z Multiwalled carbon nanotubes coated with cobalt(II) sulfide nanoparticles for electrochemical sensing of glucose via direct electron transfer to glucose oxidase Microchim Acta 2020 187 1 80 10.1007/s00604-019-4047-8
Li J, Liu Y, Tang X, Xu L, Min L, Xue Y, Hu X, Yang Z. Multiwalled carbon nanotubes coated with cobalt(II) sulfide nanoparticles for electrochemical sensing of glucose via direct electron transfer to glucose oxidase. Microchim Acta. 2020;187(1):80.10.1007/s00604-019-4047-8
98. Baghayeri M Veisi H Ghanei-Motlagh M Amperometric glucose biosensor based on immobilization of glucose oxidase on a magnetic glassy carbon electrode modified with a novel magnetic nanocomposite Sens Actuators, B Chem 2017 249 321 330 10.1016/j.snb.2017.04.100
Baghayeri M, Veisi H, Ghanei-Motlagh M. Amperometric glucose biosensor based on immobilization of glucose oxidase on a magnetic glassy carbon electrode modified with a novel magnetic nanocomposite. Sens Actuators, B Chem. 2017;249:321–30.10.1016/j.snb.2017.04.100
99. Temoçin Z Designing of a stable and selective glucose biosensor by glucose oxidase immobilization on glassy carbon electrode sensitive to H2O2 via nanofiber interface J Appl Electrochem 2021 51 2 283 293 10.1007/s10800-020-01502-4
Temoçin Z. Designing of a stable and selective glucose biosensor by glucose oxidase immobilization on glassy carbon electrode sensitive to H2O2 via nanofiber interface. J Appl Electrochem. 2021;51(2):283–93.10.1007/s10800-020-01502-4
100. Huang C Hao Z Qi T Pan Y Zhao X An integrated flexible and reusable graphene field effect transistor nanosensor for monitoring glucose Journal of Materiomics 2020 6 2 308 314 10.1016/j.jmat.2020.02.002
Huang C, Hao Z, Qi T, Pan Y, Zhao X. An integrated flexible and reusable graphene field effect transistor nanosensor for monitoring glucose. Journal of Materiomics. 2020;6(2):308–14.10.1016/j.jmat.2020.02.002
101. Zhang C Wei C Chen D Xu Z Huang X Construction of inorganic-organic cascade enzymes biosensor based on gradient polysulfone hollow fiber membrane for glucose detection Sens Actuators, B Chem 2023 385 133630 10.1016/j.snb.2023.133630
Zhang C, Wei C, Chen D, Xu Z, Huang X. Construction of inorganic-organic cascade enzymes biosensor based on gradient polysulfone hollow fiber membrane for glucose detection. Sens Actuators, B Chem. 2023;385: 133630.10.1016/j.snb.2023.133630
102. Wang Q Chen M Xiong C Zhu X Chen C Zhou F Dong Y Wang Y Xu J Li Y Dual confinement of high–loading enzymes within metal–organic frameworks for glucose sensor with enhanced cascade biocatalysis Biosens Bioelectron 2022 196 113695 10.1016/j.bios.2021.113695 34688111
Wang Q, Chen M, Xiong C, Zhu X, Chen C, Zhou F, Dong Y, Wang Y, Xu J, Li Y, et al. Dual confinement of high–loading enzymes within metal–organic frameworks for glucose sensor with enhanced cascade biocatalysis. Biosens Bioelectron. 2022;196: 113695.34688111 10.1016/j.bios.2021.113695
