Biomineralized synthesis of luminescent protease-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanospheres and their applications as a stable and reusable enzyme reactor

Proteases, such as trypsin, are essential for extracting collagen in various industrial applications. The potential applications of rare earth nanomaterials, specifically yttrium nanoparticles, have attracted significant interest across various fields due to their distinctive characteristics, including high dielectric constant and thermal stability. Biomineraliza-tion has emerged as a promising approach to synthesize protein-inorganic nanomaterials with hierarchical structures and desired functions. In the present investigation, a novel protease-templated biomineralization strategy was developed for synthesizing protease-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanomaterials using a one-pot method under very mild conditions. For modifying the morphologies of (NH 4 ) 2 Y 3 F 11 •H 2 O throughout biomineralization, protease has been demonstrated to be a highly promising biotemplate. Protease was utilized as a template for morphological control in the biomineralization procedure, which resulted in a gradual transformation of the initially formed (NH 4 ) 2 Y 3 F 11 •H 2 O octahedral structures into uniform nanospheres. The applicability of this approach was supported by successfully utilizing various proteases to synthesize protease-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanospheres. In addition to a strong and desirable luminescent signal, these hybrid nanospheres demonstrated extensive recycling because of their high enzymatic activity, stability and durability. The protease-mediated biomineralization approach offers an easy and robust approach to develop innovative protease-inorganic composites. Its moderate reaction conditions and simple operation render it a viable tool for developing stable and reusable enzyme reactors in various industrial applications.


Introduction
Collagen is utilized in numerous industrial sectors, including leathers, films, cosmetics and pharmaceuticals [1,2].Collagen extraction in current industrial processes is effectively accomplished via enzymatic hydrolysis.Exclusive collagen, characterized by stable physical and chemical properties, high safety and purity, can be obtained via enzymatic extraction due to its resistance to proteases other than collagenase [3][4][5].Trypsin, being a significant protease utilized in collagen extraction, assumes an essential role in improving the structural integrity, stability and rate of collagen extraction [6].However, the use of free proteases as biocatalysts faces several limitations, including low stability, poor reusability and hindrance caused by reaction byproducts or contaminants.Immobilization techniques have been designed to produce protease-based biocatalysts that exhibit increased stability, reusability and selectivity, thereby overcoming these challenges and increasing the catalytic efficiency of proteases [7][8][9].Extensive research is being conducted on the immobilization of proteases onto various support materials to develop reusable and durable biocatalysts for a variety of industrial and nonindustrial applications [10].
Rare earth nanomaterials offer novel prospects to be implemented in various fields, including optics, biomedical and chemical industries [11][12][13][14].Among rare earth nanomaterials, yttrium nanoparticles have received significant attention due to their unique properties, including high stability and high dielectric constant [15,16].Yttrium nanoparticles acquire significant utility as constituents of luminescent materials employed in the field of biological imaging and diagnostics.For instance, yttrium-based up-conversion luminescence materials have demonstrated enormous potential in photodynamic therapy and cancer cell imaging [17,18].Furthermore, in numerous biomedical and environmental contexts, the detection of calcium ions with astounding sensitivity and selectivity has been accomplished via yttrium-mediated red fluorescent carbon dots [19].Moreover, yttrium nanoparticles have been utilized in various fields, including magnetic resonance imaging [20], solar cells [21,22] and electronic devices [23].Yttrium nanoparticles, owing to their exceptional biocompatibility and high stability, exhibit potential as a support material in the development of protease-based biocatalysts that are anticipated to exhibit improved selectivity, reusability and stability.Various methods have been reported to attain yttriumbased nanomaterials which include coprecipitation [24], emulsion [25], combustion [26], solvothermal [27], solgel [28,29] and hydrothermal methods [30,31].However, the biological applications of these techniques are severely constrained by their low yields, harsh reaction conditions and high toxicity.
Biomineralization is an essential process employed by organisms in the synthesis of inorganic nanomaterials and is extensively used in the production of hierarchically ordered functional materials.Biological macromolecules, including proteins, nucleic acids, polysaccharides and lipids, function as templates in nature to govern the formation of inorganic nanomaterials.This process results in the formation of multifunctional biomaterials such as bones, teeth and shells [32][33][34][35][36][37].Proteins are particularly attractive as biological templates among these macromolecules due to their high natural abundance, diversity and complex structures [38][39][40][41].A well-studied biomineral is the mollusk shell, in which a silk-like protease in the matrix is essential for the formation of aragonite crystals in the nacre sheets [42].By employing various proteases as templates, flower-like nanoparticles

Graphical Abstract
of Cu 3 (PO 4 ) 2 •3H 2 O were synthesized, facilitating anisotropic crystal growth.The immobilized proteases within the hybrid exhibit significantly higher catalytic efficacy and thermal stability compared to their free counterparts [43,44].The research demonstrated that proteins exerted a prominent influence on the control of multifunctional organic-inorganic composites.Therefore, the proteasetemplated biomineralization method may potentially be applied to the development of multifunctional organicinorganic composites with desirable properties.
Herein, a novel protease-templated biomineralization strategy was developed for the synthesis of (NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanospheres with biocatalyst as well as fluorescence properties.This approach exploited protease-templated biomineralization, which is a simple and eco-friendly process.It utilized the abundant amide and carboxyl groups present in protease molecules as ideal sites for the growth of rare earth fluoride crystals.The trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O nanospheres exhibited exceptional stability, allowing the immobilized enzymes to be reused numerous times without compromising enzymatic activity.Furthermore, the adaptability of this approach was validated through its successful application in the synthesis of protease-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanospheres utilizing various proteases, including lysozyme, glycoside hydrolase and pepsin.The promising potential of the synthesized luminescent protease-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanospheres for applications in various fields, including biocatalysis and biomedicine, is further demonstrated by their luminescent signals.

Synthesis of biomineralized hybrid nanospheres
A mixture of 500 μL trypsin solution (at a concentration of 8 mg/mL) and 100 μL Y(NO 3 ) 3 solution (at a concentration of 0.5 M) was utilized to synthesize trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanospheres.The mixture was stirred for 1 h to ensure the formation of a homogeneous solution.Following this, 285 μL of NH 4 F solution (0.5 M) was added and stirred for 10 min.After a 48-h incubation period at 25 °C, the mixture was centrifuged and the resulting solids were washed thrice with ethanol and subsequently air-dried at 25 °C.Various protein-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanospheres were synthesized using similar protocols by varying the protein concentrations (8.0 mg/mL BSA, 8.0 mg/mL pepsin, 4.0 mg/mL THRC and 20.0 mg/mL glycoside hydrolase).

Characterizations of trypsin-(NH
XRD, FT-IR, TGA, SEM, TEM, SAED and BET characterizations were performed according to previously reported protocols, and detailed descriptions are presented in supporting information [47].

Photoluminescence spectroscopy
Photoluminescence emission spectra were recorded using a Hitachi FLS920 fluorescence spectrophotometer with a xenon lamp as the excitation source.

Evaluation of the encapsulation yield of trypsin
The Branford method was employed to evaluate the efficacy of trypsin encapsulation.Coomassie brilliant blue G-250 (100 mg) was added into 50 mL of ethanol solution, followed by the incorporation of phosphoric acid solution (100 mL) and deionized water (200 mL).The solution was then diluted five times and stored at 4 °C.To construct the calibration curve for trypsin concentration, a series of PBS solutions (4 mL, 3 mM) was prepared containing varying concentrations of trypsin (0, 0.01, 0.1, 0.2 and 0.5 mg/mL) followed by the addition of 4 mL of Brandford assay.After 15 min, the absorbance of each solution was determined at 595 nm.The concentration of non-immobilized trypsin was determined using the following method.Supernatant (4 mL) was collected from each synthesis and added with 4 mL of Brandford assay.Subsequently, the absorbance of the mixed solution was monitored at 595 nm to determine the concentration of free trypsin using the calibration curve.The amount of encapsulated trypsin can be calculated by subtracting the amount of free trypsin from the total amount of trypsin introduced in the synthesis and the encapsulation yield of trypsin can be obtained by dividing the amount of encapsulated trypsin by the amount of total trypsin.

hybrid nanospheres and free trypsin
The enzymatic activity of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanospheres was evaluated using the Folin assay, according to the previously reported protocol [47,48].Briefly, 10 mg of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O was added to 1 mL of 10 mg/mL casein solution (PBS, 50 mM, pH 7.4) and incubated at 30 °C for 30 min.Following centrifugation for 5 min at 9000 rpm, the supernatant was collected which was then added with an aliquot of 2 mL of TCA solution (0.4 M) and subjected to a 5-min incubation period.After centrifugation, the supernatant was harvested.Na 2 CO 3 solution (5 mL, 0.4 M) and Ciocalteu's phenol reagent (1 mL) were mixed with 1 mL of the supernatant.The mixture was then incubated at 40 °C for 20 min to complete the color development reaction.An evaluation of the casein hydrolysis performance was conducted by measuring the absorbance at 763 nm.The activity of free trypsin was evaluated using the same procedure by supplementing the hybrid nanospheres with an equivalent quantity of trypsin.The determination of the trypsin content in the composite nanomaterials was further evaluated via thermogravimetric analysis (TGA) (Fig. 1c).The results of the analysis revealed two distinct weight-loss stages of TEM images also revealed a uniform spherical structure of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O crystals (Fig. 2d).The highly ordered structure of these nanoparticles was further supported by the HRTEM image, which displayed the characteristic lattice spacing (Fig. 2e).A highly oriented assembly of the primary protease-(NH 4 ) 2 Y 3 F 11 •H 2 O composites was indicated by the symmetry observed in the single-crystal diffraction pattern (Fig. 2f ).
Further, the Barrett-Joyner-Halenda (BJH) model was utilized to ascertain the average pore size and Brunauer-Emmett-Teller (BET) surface area of the trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O crystals.The outcomes of this analysis revealed the porous characteristics of the nanospheres, as illustrated in Figure S1, with values of around 4 nm and 13.6 m 2 /g, respectively.Metal ions are capable of forming coordinate bonds with trypsin due to the variety of functional groups it possesses, such as carboxyl and amine groups.The interactions between these coordinates are essential in affecting the nucleation and development of metal nanoparticles, resulting in the formation of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O nanospheres.These findings collectively demonstrated that trypsin was capable of producing mesocrystals with well-organized structures and that it was an essential component in the biomineralization process.

Synthesis of protein-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanospheres
Various protein categories (trypsin, glycoside hydrolase, pepsin, BSA and THRC) were examined as the templates during the biomineralization process for the synthesis of protein-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanomaterials.SEM was utilized to perform the morphological analysis of the as-prepared hybrid nanomaterials (Fig. 3).It is noteworthy that the (NH 4 ) 2 Y 3 F 11 •H 2 O materials revealed octahedral nanostructures in the absence of protein (Fig. 3a).The synthesized nanomaterials exhibited the formation of small nanospheres featuring convex and concave gradations, rough surfaces, and uniform spherical diameters of approximately 1.0 μm when exposed to 4.0 mg trypsin (Fig. 3b).Upon the addition of 10.0 mg glycoside hydrolase, the generated nanomaterials exhibited relatively smooth surfaces with a uniform size of around 0.95 μm in diameter (Fig. 3c).The surfaces of the nanomaterials produced were comparatively smooth following the addition of 4.0 mg pepsin, as illustrated in Fig. 3d The formation of (NH 4 ) 2 Y 3 F 11 •H 2 O nanoparticles over time was monitored using UV-Vis spectroscopy, both with and without trypsin (Figure S2).Increasing UV absorbance at 313 nm with increasing incubation time suggested the gradual production of these nanoparticles.Significantly, the inclusion of trypsin led to increased UV 313 values at every time interval, indicating that the trypsin facilitated the formation of (NH 4 ) 2 Y 3 F 11 •H 2 O nanoparticles and performed an essential role as a biotemplate.(Figure S4b).Similarly, calcination-synthesized trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanospheres also demonstrated less organized nanostructures (Figure S4c).The results indicated that proteins influenced the development of the supramolecular architecture of nanomaterials.Based on the XRD spectrum of calcined trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanospheres, it is possible to disregard the negligible effect of calcination on the crystalline phase of the composite material (Figure S5).In summary, these findings suggested that the protein and Y 3+ ions were uniformly dispersed throughout the hybrid nanomaterials and collaborated to precisely control the spherical structure.
Based on the results, a proposed biomineralization approach utilizes protease as a biological template in the preparation of (NH 4 ) 2 Y 3 F 11 •H 2 O nanospheres.The biomineralization process relies on the coordinated interactions that occur between rare earth ions and functional groups located on the protease molecule, thereby facilitating subsequent crystal growth (Fig. 5).Notably, the abundance of amide and carboxyl groups in protease molecules provides favorable conditions for the formation and growth of rare earth fluoride crystals.During the primary growth phase, coordinated interactions occur between the functional groups on the protease molecules and rare earth ions, resulting in the development of primary crystals.Following this, further nucleation events involving these primary crystals result in the formation of nanospheres during the secondary growth stage.Therefore, proteases serve as pivotal mediators, orchestrating the creation of spherical nanomaterials through their intricate interactions with rare earth ions even under mild biogenic conditions.To examine the behavior of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O in organic solvents, casein solution was supplemented with increasing concentrations of ethanol (Fig. 6f ).Free trypsin activity decreased rapidly in an ethanol solution with a concentration of 20% and reached a lower value of 0.23 in a 70% ethanol solution.In contrast, trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O nanomaterials exhibited high relative activity and stability in ethanol, indicating an increased tolerance to organic solvents.The increased stability observed in immobilized trypsin can be possibly ascribed to protein conformational stabilization, which is achieved through coordinate interactions between the trypsin amino groups and the carrier material.

Reusability of the trypsin-(NH
Reusability is one of the best advantages of enzyme immobilization.As an excellent model system for visualizing the enzymatic cleavage activity of trypsin, triple helix recombinant collagen (THRC) with a non-triple helical domain and a collagen domain was selected to assess the reusability.When subjected to temperature conditions below the melting point of THRC (37 ℃), trypsin exhibited a significant ability to digest the non-triple helical domain.On the contrary, the collagen domain demonstrated considerable resistance to trypsin cleavage.Therefore, after subjecting the digested THRC product to SDS-PAGE analysis, a single collagen band was detected.This may be considered a convenient approach for evaluating the activity and reusability of immobilized trypsin.The THRC protein from the control sample migrated and organized into a single band at an estimated molecular weight of 45 kDa, as determined by SDS-PAGE (Fig. 7a).In contrast, the digestion product collagen exhibited migration as a single band upon the addition of trypsin-(NH 4 ) 2 Y 3 F    7b).The nanospheres exhibited a remarkable capacity to retain their activity even after ten cycles.However, the remaining enzymatic activity of these nanospheres towards the substrate THRC decreased marginally after multiple cycles.The observed decrease in enzymatic efficiency may be attributable to the partial loss of nanospheres during the elution processes.These results demonstrated that immobilized trypsin has the potential to be an effective and durable biocatalytic agent for a range of biochemical uses, because of its remarkable reusability and a highly valued attribute among immobilized enzymes.were successfully developed by doping rare earth ions, specifically Eu 3+ , into the rare earth ion matrix.The composite materials exhibited distinct luminescent characteristics that are intrinsic to rare earth ions.The current research highlights the innovative potential of bio-mineralization as a versatile method for the development of novel protease-inorganic composites, with promising applications in biosensors and biocatalysts.

2. 7 4 ) 2 Y 3 F3 Results and discussion 3 . 1 4 ) 2 Y 3 F
Reusability of trypsin-(NH 11 •H 2 O hybrid nanospheresTo examine the reusability of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O, the digestion of triple helix recombinant collagen (THRC) was conducted following the previously reported methods[47,48].In brief, 2 mg of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O was incubated at 25 °C with THRC for 12 h.Following centrifugation, the supernatant was harvested and stored at a temperature of -20 °C.The precipitates were subsequently prepared for processing in another cycle following a triple-washing procedure using glycine buffer.All the collected supernatant from 10 cycles was analyzed for SDS-PAGE.Characterization of trypsin-(NH 11 •H 2 O hybrid nanomaterials Proteases, a class of proteins, are essential for the regulation of a wide variety of biological processes in living organisms.The synthesis of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanomaterials involved a 12-h incubation of a mixture comprising trypsin (4.5 mg/mL), Y(NO 3 ) 3 (56 mM) and NH 4 F (161 mM) at 25 °C.The crystallinity of the prepared nanomaterials was determined using X-ray diffraction (XRD).The diffraction profiles were found to be consistent with the standard XRD spectrum of (NH 4 ) 2 Y 3 F 11 •H 2 O (JCPDS No. 28-97) (Fig. 1a).The absence of additional diffraction peaks, except for those corresponding to (NH 4 ) 2 Y 3 F 11 •H 2 O, signified that the trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanomaterials were free of any additional impurities.This result demonstrated that under moderate incubation conditions, trypsin functions is an effective bio-template for the production of pure yttrium-based crystals (NH 4 ) 2 Y 3 F 11 •H 2 O.The FT-IR spectrum of the (NH 4 ) 2 Y 3 F 11 •H 2 O exhibited a strong vibrational peak at 1400 cm −1 , which was attributed to the Y-O bond.The FT-IR spectrum of trypsin revealed distinct vibration peaks at 2964 and 1537 cm −1 corresponding to the C-H and N-H bonds, respectively.In the FT-IR spectrum of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanomaterials, characteristic vibration peaks of the C-H and N-H bonds appeared at 2964 and 1537 cm −1 , along with the vibration peak at 1420 cm −1 corresponding to the Y-O bond.These results indicated the presence of trypsin within the synthesized (NH 4 ) 2 Y 3 F 11 •H 2 O crystal (Fig. 1b).

Fig. 2
Fig. 2 Morphological characterization of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanomaterials.a, b SEM images of hybrid nanomaterials; c EDS spectrum of the hybrid nanomaterials; d TEM images of hybrid nanomaterials; e High-resolution TEM image of the crystal lattice structure of the composite; f The SAED pattern of the composite . In the presence of 4.0 mg BSA or 2.0 mg THRC, (NH 4 ) 2 Y 3 F 11 •H 2 O hybrid composites developed a spherical structure with a diameter of approximately 0.5 μm, convex and concave rank, and adequate dispersion (Fig.3e, f).In summary, various proteins could serve as biotemplates for the synthesis of protein-(NH 4 ) 2 Y 3 F 11 •H 2 O nanospheres.The results demonstrated the wide range of applications of proteins as templates for the production of (NH 4 ) 2 Y 3 F 11 •H 2 O nanospheres.

Fig. 6
Fig. 6 Enzymatic activity of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O composites. a Different pH; b Various incubation temperatures; c Different incubation times at 45 ℃; d Various concentrations of urea; (e) Different concentrations of acetonitrile (ACN); f Various concentrations of ethanol

3. 6
Photoluminescence of the trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanospheres Luminescent materials incorporating lanthanide ions have been widely utilized in the field of bioimaging and optoelectronics due to their advantageous properties-such as minimal toxicity, significant Stokes shift and exceptional photodegradation resistance.Solid-state fluorescence was employed for the characterization of the photoluminescent features of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanospheres doped with Eu 3+ (Fig. 8).The fluorescence emission spectrum revealed the distinctive peaks of Eu 3+ at 593 nm and 620 nm, which occurred at the excitation wavelength of 318 nm.This result showed that the trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O: Eu 3+ nanomaterials exhibited favorable photoluminescent characteristics.

4 ConclusionFig. 7 Fig. 8
Fig. 7 Reusability of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O hybrid nanospheres.a SDS-PAGE analysis of the cleavage of triple helix recombinant collagen (THRC) via repeated use of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O composites.b Relative activity of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O nanospheres over 10 consecutive cycles The enzymatic properties of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O and free trypsin under various pH, temperatures, incubation time and denatured conditions (urea, acetonitrile or ethanol) were detected by the Folin method.The enzymatic properties of trypsin-(NH 4 ) 2 Y 3 F 11 •H 2 O and free trypsin were evaluated at different pH values in 50 mM glycine following a 12-h incubation at 37 ℃. 11