A novel method for preparing sheet-like hollow porous chitin carriers and its application in immobilized enzymes.

Abstract

This invention discloses a novel sheet-like hollow porous chitin carrier preparation method and its application in immobilized enzymes. The invention utilizes shrimp powder-derived chitin (choline chloride:oxalic acid in a molar ratio of 1:3) to create a novel carrier MC-OA, which is then applied to immobilized enzymes. This chitin carrier exhibits superior protein loading performance, with a protein loading capacity 3.39 times that of natural chitin, and the enzyme activity is increased to 1.91 times that of the free enzyme BTL2. The novel carrier MC-OA provided by this invention has universal applicability to loading enzyme proteins from different sources, improving protein loading capacity while retaining the activation effect and stable catalytic performance of natural chitin on free enzymes, providing a good carrier source foundation for subsequent immobilized enzyme creation.

Description

Technical Field

[0001] This invention belongs to the field of immobilized enzyme technology, and relates to a method for preparing a novel sheet-like hollow porous chitin carrier and its application in immobilized enzymes. Background Technology

[0002] Natural structural supports are a superior enzyme immobilization carrier in industrial biocatalysis applications, possessing advantages such as higher surface area, ease of recovery, and reusability. Various nanomaterials, such as metal-organic frameworks, carbon nanotubes, silica-based nanoparticles, nanoflowers, nanofiber membranes, graphene oxide, and hybrid nanomaterials, are currently the most widely used carriers for enzyme immobilization. However, with in-depth research on carrier materials, compared to carriers prepared from nanomaterials, natural polymers have attracted widespread attention due to their low cost and a series of unique properties and advantages. For example, abundant biomass sources such as chitin have gained significant attention due to their non-toxicity, biodegradability, and biocompatibility. Chitin is the second most abundant polysaccharide on Earth, mainly found in the shells of crustaceans or in the cell walls of certain fungi and algae. Chitin, as the second most abundant biomass after cellulose, possesses excellent biocompatibility, biodegradability, and unique bioactive functions. However, chitin extracted in industrial environments undergoes severe reactions with strong acids and alkalis, resulting in damage to its microstructure and internal protein-binding groups. Therefore, further modification and processing are needed to prepare chitin immobilization carrier materials that meet practical application requirements.

[0003] Hollow, porous chitin sheet-like carriers have attracted widespread attention as enzyme immobilization carriers due to their high surface area-to-mass ratio and excellent mass transfer performance. However, current research shows that immobilization techniques relying on physical adsorption typically suffer from poor enzyme stability and low immobilization efficiency. Therefore, a more attractive immobilization scheme has emerged: covalent bonding of enzymes to modified chitin carriers. In practical applications, converting chitin into sheet-like, hollow, porous chitin carriers while maintaining their inherent properties is a significant challenge. During the drying process of chitin materials, the reduction in capillary force leads to chitin aggregation. This results in a compact chitin carrier structure with limited surface area, low porosity, and small pore size, thereby reducing the intraparticle diffusion rate of the adsorbate. Therefore, designing a carrier with high porosity and large pore size is currently a major research direction for chitin carriers. Chitin carriers with regular shapes and hollow porous structures can be prepared by repeated freeze-thaw cycles and depolymerization without the use of any toxic crosslinking agents. This carrier material has good biodegradability and is non-toxic, and can be used as a carrier for immobilized enzymes. Summary of the Invention

[0004] The first objective of this invention is to provide a method for preparing a sheet-like hollow porous chitin carrier MC-OA, the steps of which are as follows:

[0005] S1. Mix choline chloride and oxalic acid in a molar ratio of 1:3 and stir continuously at 80°C until the mixture becomes a stable suspension to obtain acidic NADES.

[0006] S2. Mix shrimp shell powder with acidic NADES prepared in step S1 at a mass ratio of 1:20, stir at 80°C for 2 hours, add deionized water to reduce the viscosity of the system, take out the sample, collect the solid components and wash and centrifuge repeatedly with deionized water until the pH of the supernatant is neutral, collect the solid components and decolorize them with 10% hydrogen peroxide at 80°C, wash with deionized water until the pH of the supernatant is neutral, and dry the obtained solid at 60°C to obtain chitin CCOA;

[0007] S3. Mix natural chitosan CCOA with NaOH / urea solution at a mass ratio of 3:97 until homogeneous. Freeze the mixture at -20°C, then thaw it at room temperature. Stir vigorously until the chitosan CCOA powder is completely and evenly distributed before the ice layer completely melts. Freeze the mixture again at -80°C, then thaw it at room temperature. Stir vigorously until the chitosan CCOA powder is completely and evenly distributed before the ice layer completely melts. Repeat the freezing, thawing, and stirring process until the precipitate is completely dissolved to obtain a chitosan solution.

[0008] S4. Add the chitin solution obtained in step S3 to the Tween 80-liquid paraffin system at a mass percentage of 30%, stir at low speed for 3 hours, and wash the precipitate with deionized water and anhydrous ethanol after stirring. After washing, recover the precipitate.

[0009] S5. The precipitate recovered in step S4 is frozen at -80℃ for more than 4 hours, and then freeze-dried into powder to obtain sheet-like hollow porous chitin carrier MC-OA.

[0010] Preferably, the NaOH / urea solution in step S3 has the following composition: 8 parts by mass of NaOH, 4 parts by mass of urea, and 88 parts by mass of water; the Tween 80-liquid paraffin system in step S4 has the following composition: 99 parts by mass of liquid paraffin and 1 part by mass of Tween 80.

[0011] Preferably, the vigorous stirring in step S3 is a stirring speed of 4000 rpm or higher; the low-speed stirring in step S4 is a stirring speed of 2000 rpm or lower.

[0012] Preferably, the step S4 of washing the precipitate with deionized water and anhydrous ethanol is as follows: first, wash with deionized water and centrifuge to remove the liquid paraffin on the surface of the precipitate; then, wash with anhydrous ethanol and centrifuge to remove the liquid paraffin and Tween 80 inside the structure; finally, wash with deionized water to remove the liquid paraffin precipitated from inside the structure and the anhydrous ethanol remaining on the surface of the precipitate.

[0013] Preferably, the centrifugation is performed at 4000 rpm for 5 minutes.

[0014] A second objective of this invention is to provide a sheet-like hollow porous chitin carrier MC-OA prepared using the method described above.

[0015] A third objective of this invention is to provide a method for preparing immobilized lipases using the aforementioned sheet-like hollow porous chitin carrier MC-OA, comprising the following steps:

[0016] a. Take 0.05g of chitin carrier MC-OA and put it into a centrifuge tube. Add 3mL of pH=8.0 buffer solution and sonicate at low power for 10min to break the carrier into microparticles. After the breakdown is complete, centrifuge to remove the supernatant and keep the lower layer of carrier. The conditions for the low-power sonication are: power of 50W, 1s on and 2s off.

[0017] b. Mix free lipase, sheet-like hollow porous chitin carrier MC-OA, and pH=8.0 buffer solution at a ratio of 4mg:1g:10mL, stir for 1h under ice bath conditions, and place at 4℃ for 12h after stirring.

[0018] c. The precipitate was washed three times by centrifugation with a buffer solution of pH=8.0 to remove residual unfixed free lipase. The precipitate was recovered to obtain chitin-carrier MC-OA immobilized lipase.

[0019] Preferably, the free lipase is porcine pancreatic lipase PPL, Aspergillus niger lipase ANL, Pseudomonas fluorescens lipase PFL, Burkholderia cepacia lipase BCL, and Bacillus thermophilus lipase BTL2; the nucleotide sequence of the encoding gene of Bacillus thermophilus lipase BTL2 is shown in SEQ ID NO.1.

[0020] Preferably, the buffer solution is Tris-HCl buffer solution or PBS buffer solution; the stirring under ice bath conditions for 1 hour in step b is stirring at 90-100 rpm / min for 1 hour under ice bath conditions; the centrifugation in steps a and c is centrifugation at 4000 rpm for 5 minutes.

[0021] A fourth object of the present invention is to provide an immobilized lipase prepared using the method described above.

[0022] The beneficial effects of this invention are:

[0023] This invention mixes specific chitin (CCOA) with NaOH / urea / aqueous solution, dissolves it through a cyclic freeze-thaw method, and then prepares a fibrous porous carrier using the sol-gel method. It has advantages such as hierarchical pore structure, high specific surface area and good biocompatibility, and can be used as a carrier material for protein loading, adsorbent and drug delivery. Attached Figure Description

[0024] Figure 1 These are SEM images of different types of chitin vectors; where (a)-(f) are SEM images of MC, MC-LA, MC-CA, MC-MA, MC-TA, and MC-OA, respectively.

[0025] Figure 2 These are TEM images of different types of chitin vectors; where (a)-(f) are TEM images of MC, MC-LA, MC-CA, MC-MA, MC-TA, and MC-OA, respectively.

[0026] Figure 3 The results show the chemical structures of different types of chitin and the chitin carriers they are prepared from; (a) is the FT-IR result and (b) is the XRD result.

[0027] Figure 4 The results show the performance of BTL2 immobilized on different types of chitin vectors; (a) represents the loading content of BTL2 on different types of chitin vectors, and (b) represents the enzyme activity after loading BTL2 onto different types of chitin vectors.

[0028] Figure 5 The images show SEM analyses of BTL2 immobilized on different types of chitin vectors; where (a)-(f) are SEM images of BTL2 immobilized on MC, MC-LA, MC-CA, MC-MA, MC-TA, and MC-OA, respectively.

[0029] Figure 6 The images show the CLSM analysis of BTL2 immobilized on different types of chitin vectors; where (a)-(f) are CLSM diagrams of BTL2 immobilized on MC, MC-LA, MC-CA, MC-MA, MC-TA, and MC-OA, respectively.

[0030] Figure 7 The results show the general applicability of lipase immobilization on MC and MC-OA carriers; (a) shows the immobilization efficiency of different types of lipases immobilized on MC and MC-OA carriers, and (b) compares the enzyme activity of different types of lipases immobilized on MC and MC-OA carriers with that of the free enzyme.

[0031] Figure 8This is a graph showing the enzyme activity results of BTL2 immobilized using MC and MC-OA vectors after multiple applications. Detailed Implementation

[0032] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0033] Example 1: Screening of preparation conditions for porous fibrous chitin carriers

[0034] The chitin used in this embodiment is commercial chitin (Aladdin Biochemical Technology Co., Ltd.; C104157-100g).

[0035] (1) Screening of the ratio of chitin to NaOH / urea solution

[0036] Prepare a solution with a mass ratio of NaOH:urea:H2O = 8:4:88 (hereinafter referred to as NaOH / urea solution). Take 1g, 2g, 3g, 4g, 5g and 6g of chitin powder respectively according to solid-liquid mass ratios of 1%, 2%, 3%, 4%, 5% and 6% and add them to 99g, 98g, 97g, 96g, 95g and 94g of NaOH / urea solution. Freeze the solution at -20℃, thaw it at room temperature, and stir vigorously (above 4000rpm) until the chitin powder is evenly distributed before the ice layer is completely melted. Then freeze it again at -80℃. Repeat the freeze-thaw process (alternating between -20℃ and -80℃) until the precipitate is completely dissolved to obtain a chitin solution of a certain proportion. The number of freeze-thaw cycles depends on whether the chitin powder is completely dissolved.

[0037] To prepare a Tween 80-paraffin system, add 1g of Tween 80 to every 99g of liquid paraffin. Under ice bath conditions, slowly pour chitin solution into the Tween 80-paraffin system at a mass ratio of 30% and stir at low speed (below 2000rpm) for 3 hours. After stirring, remove the precipitate, wash it with deionized water, centrifuge at 4000rpm for 5 minutes to remove the liquid paraffin on the precipitate surface, then wash it with anhydrous ethanol and centrifuge at 4000rpm for 5 minutes to remove the liquid paraffin and Tween 80 inside the structure. Finally, wash with deionized water to remove the liquid paraffin precipitated from the structure and the anhydrous ethanol remaining on the precipitate surface. Freeze the prepared carrier sample at -80℃ for at least 4 hours, then freeze-dry it into powder (i.e., obtain the chitin carrier), which is used to immobilize BTL2 lipase.

[0038] Method for immobilizing BTL2 lipase: 0.05 g of the chitin carrier prepared under the different conditions described above was placed in a 10 mL centrifuge tube. 3 mL of Tris-HCl buffer solution (pH = 8.0) was added, and the carrier was disrupted by low-power sonication (50 W, 10 min, 1 s on, 2 s off) to form microparticles. After disruption, the supernatant was removed by centrifugation, retaining the lower carrier layer. The mixture was prepared according to a ratio of free lipase:carrier:Tris-HCl buffer solution (pH = 8.0) = 4 mg:1 g:10 mL. The mixture was stirred in an ice bath for 1 h (stirring speed 90-100 rpm / min). After stirring, the mixture was placed at 4℃ for 12 h to immobilize the free BTL2 lipase. After immobilization, the precipitate was washed three times by centrifugation at 4000 rpm for 5 min with Tris-HCl buffer solution (pH = 8.0) to remove any remaining unimmobilized free BTL2 lipase.

[0039] Table 1. Effects of different chitin contents in chitin solution on the performance of immobilized enzymes on the carrier.

[0040] Chitosan content (%) Enzyme loading (mg / g) Enzyme activity (U / mg) 1％ 3.51 164.77±2.92 2％ 3.46 159.29±2.07 3％ 4.51 242.43±2.33 4％ 3.36 15.06±2.94 5％ 4 75.92±2.84 6％ 3.56 37.51±3.06

[0041] The results in Table 1 show that the best enzyme immobilization performance was achieved when the mass ratio of chitin to NaOH / urea solution was 3:97, with a loading of 4.51 g / mg and an enzyme activity of 242.43 U / mg.

[0042] (2) Screening of chitin freezing temperature

[0043] Chitosan was mixed with NaOH / urea solution at a mass ratio of 3:97 and frozen at -20℃, -60℃, -70℃ and -80℃ respectively. Before the ice layer completely melted, the mixture was vigorously stirred (above 4000 rpm) until the chitosan powder was evenly distributed. Then, the mixture was frozen again at -20℃, -60℃, -70℃ and -80℃ respectively. The freeze-thaw stirring process was repeated until the precipitate was completely dissolved to obtain a chitosan solution of a certain proportion.

[0044] Chitosan solution was slowly added to the Tween 80-paraffin system at a mass ratio of 30% under ice bath conditions and stirred at low speed (below 2000 rpm) for 3 hours. After stirring, the precipitate was removed, washed with deionized water, and centrifuged at 4000 rpm for 5 minutes to remove the liquid paraffin on the surface of the precipitate. Then it was washed with anhydrous ethanol and centrifuged at 4000 rpm for 5 minutes to remove the liquid paraffin and Tween 80 inside the structure. Finally, it was washed with deionized water to remove the liquid paraffin precipitated from the inside of the structure and the anhydrous ethanol remaining on the surface of the precipitate. The prepared carrier sample was frozen at -80°C for at least 4 hours and then freeze-dried into powder for immobilization of BTL2 lipase (the method is the same as the method for immobilizing BTL2 lipase in Example 1 (1)).

[0045] Table 2 Effect of different chitin freezing temperatures on the performance of immobilized enzymes on the carrier

[0046] Dissolution temperature (°C) Enzyme loading (mg / g) Enzyme activity (U / mg) -20 3.45 215.65±1.6 -60 1.22 56.39±0.81 -70 1.51 105.41±1.27 -80 1.18 53.45±0.56

[0047] The results in Table 2 show that the enzyme immobilization performance is best when the chitin dissolution temperature is -20℃, with a loading of 3.45 mg / g and an enzyme activity of 215.65 U / mg.

[0048] (3) Screening of chitin solution content in immobilization carrier preparation

[0049] Chitosan and NaOH / urea solution were mixed at a mass ratio of 3:97 and frozen at -20°C. Before the ice completely melted, the mixture was vigorously stirred (above 4000 rpm) until the chitosan powder was evenly distributed. Then, the mixture was frozen again at -80°C. The freeze-thaw process (alternating between -20°C and -80°C) was repeated until the precipitate was completely dissolved to obtain a chitosan solution of a certain proportion.

[0050] Chitosan solution was slowly added to the Tween 80-paraffin system at mass ratios of 10%, 20%, 30%, 40%, 50%, and 60% respectively under ice bath conditions, and stirred at low speed (below 2000 rpm) for 3 hours. After stirring, the precipitate was removed, washed with deionized water, and centrifuged at 4000 rpm for 5 minutes to remove the liquid paraffin on the surface of the precipitate. Then it was washed with anhydrous ethanol and centrifuged at 4000 rpm for 5 minutes to remove the liquid paraffin and Tween 80 inside the structure. Finally, it was washed with deionized water to remove the liquid paraffin precipitated from the inside of the structure and the anhydrous ethanol remaining on the surface of the precipitate. The prepared carrier sample was frozen at -80°C for at least 4 hours and then freeze-dried into powder for immobilization of BTL2 lipase (the method is the same as the method for immobilizing BTL2 lipase in Example 1 (1)).

[0051] Table 3 Effect of different chitin solution contents on the performance of immobilized enzymes on the carrier.

[0052] Quality percentage (%) Enzyme loading (mg / g) Enzyme activity (U / mg) 10 4 164.51±1.09 20 4 169.79±1.41 30 3.45 242.71±1.6 40 4 232.98±1.77 50 3.31 109.79±1.41 60 3.26 101.74±1.71

[0053] The results in Table 3 show that the enzyme immobilization performance was best when the chitin solution content was 30%, with a loading of 3.45 mg / g and an enzyme activity of 242.71 U / mg.

[0054] (4) Screening of stirring time in the preparation of immobilized carrier

[0055] Chitosan and NaOH / urea solution were mixed at a mass ratio of 3:97 and frozen at -20°C. Before the ice completely melted, the mixture was vigorously stirred (at a speed of over 4000 rpm) until the chitosan powder was evenly distributed. The mixture was then placed back into the ice at -80°C and frozen again. This freeze-thaw process (alternating between -20°C and -80°C) was repeated with stirring until the precipitate was completely dissolved to obtain a chitosan solution of a certain proportion.

[0056] Chitosan solution was slowly added to the Tween 80-paraffin system at a mass ratio of 30% under ice bath conditions and stirred at low speed (below 2000 rpm) for 1, 2, 3, 4, 5 and 6 hours respectively. After stirring, the precipitate was removed, washed with deionized water and centrifuged at 4000 rpm for 5 min to remove the liquid paraffin on the surface of the precipitate, washed with anhydrous ethanol and centrifuged at 4000 rpm for 5 min to remove the liquid paraffin and Tween 80 inside the structure, and finally washed with deionized water to remove the liquid paraffin precipitated from the inside of the structure and the anhydrous ethanol remaining on the surface of the precipitate. The prepared carrier sample was frozen at -80℃ for at least 4 hours and then freeze-dried into powder for immobilization of BTL2 lipase (the method is the same as the method for immobilizing BTL2 lipase in Example 1 (1)).

[0057] Table 4. Effects of different stirring times on the performance of immobilized enzymes on the carrier.

[0058] Stirring time (h) Enzyme loading (mg / g) Enzyme activity (U / mg) 1 4 160.98±2.42 2 4 165.88±2.07 3 3.36 242.71±2.33 4 4 236.42±2.64 5 3.48 165.50±2.84 6 3.56 245.22.±2.06

[0059] Table 4 shows that when the stirring time was 3 h, the enzyme loading was 3.36 mg / g and the enzyme activity was 242.71 U / mg; when the stirring time was 6 h, the enzyme loading was 3.56 mg / g and the enzyme activity was 245.22 U / mg. Both 3 h and 6 h stirring yielded immobilized enzymes with good performance, and the difference was not significant. Therefore, choosing 3 h for sample preparation saves time and costs.

[0060] By dissolving commercial chitin with different contents and preparing chitin carriers, and then attempting to optimize the immobilization enzyme, the optimal scheme for preparing chitin carriers was determined as follows: The solid-liquid mass ratio of chitin to NaOH / urea solution (calculated by mass ratio, NaOH:urea:H2O=8:4:88) is 3:97 (i.e., the chitin content is 3%). The solution is frozen at -20℃, and before the ice layer completely melts, it is vigorously stirred (above 4000 rpm) until the chitin powder is evenly distributed. Then, it is frozen again at -20℃. The freeze-thaw process (alternating between -20℃ and -80℃) and stirring process are repeated until the precipitate is completely dissolved to obtain a chitin solution. 30g of chitin solution was slowly poured into the mixture under ice bath conditions and stirred at low speed (below 2000rpm) for 3 hours. After stirring, the precipitate was removed and washed with deionized water and centrifuged at 4000rpm for 5 minutes to remove liquid paraffin from the precipitate surface. Then, it was washed with anhydrous ethanol and centrifuged at 4000rpm for 5 minutes to remove liquid paraffin and Tween 80 from the interior of the structure. Finally, it was washed with deionized water to remove liquid paraffin precipitated from the interior of the structure and anhydrous ethanol remaining on the precipitate surface. The resulting precipitate was frozen at -80℃ for at least 4 hours and then freeze-dried into powder to obtain the chitin carrier. Immobilization performance analysis showed a maximum protein loading of 4.51mg / g and a maximum enzyme activity of 242.71±2.33U / mg.

[0061] Example 2: Preparation of shrimp shell-derived carrier

[0062] Acidic natural eutectic solvent (NADES) was prepared by continuously stirring choline chloride-lactic acid (CCLA, molar ratio 1:1), choline chloride-malic acid (CCMA, molar ratio 1:3), choline chloride-citric acid (CCCA, molar ratio 1:3), choline chloride-tartaric acid (CCTA, molar ratio 1:3), and choline chloride-oxalic acid (CCOA, molar ratio 1:3) at 80°C until the mixture became a stable suspension. The prepared acidic NADES was then used to pretreat shrimp shell powder to extract chitin. The steps for extracting chitin are as follows: Shrimp shell powder and acidic NADES are mixed at a mass ratio of 1:20 and stirred at 80°C for 2 hours. After adding deionized water to reduce the viscosity of the system, the sample is taken out, the solid components are collected, and the solid components are repeatedly washed with deionized water and centrifuged until the pH of the supernatant is neutral. The collected solid components are decolorized with 10% hydrogen peroxide at 80°C. After decolorization, the solid components are washed with deionized water until the pH of the supernatant is neutral. The obtained solid is dried at 60°C to obtain chitin.

[0063] Chitin solutions were prepared using the different types of chitin and commercial chitin according to the optimal scheme screened in Example 1. Chitin carriers were prepared by repeating the optimal carrier preparation method. The carriers prepared from different types of chitin were named MC-LA, MC-MA, MC-CA, MC-TA, and MC-OA, respectively, and the carrier prepared using commercial chitin was named MC.

[0064] SEM results of different types of chitin vectors are as follows: Figure 1 As shown, all six carriers exhibit a microscopic state of nanoparticle aggregation or entanglement into intertwined fibers. Figure 1 As shown in (a), due to the high purity of the commercial chitin, impurities have less interference with the carrier (MC) formation process, resulting in uniform particle distribution and the carrier being formed by the aggregation of nanoparticles. Figure 1 The carriers in (b)-(e) are MC-LA, MC-CA, MC-MA, and MC-TA, respectively, and are entangled in a fibrous form. This may be because the chitin used is extracted using an acidic natural eutectic solvent (NADES), which has a lower purity compared to commercial chitin. The chitin nanoparticles are affected by impurities, forming chitin fibers first, and then entangled into carrier particles through intermolecular forces such as electrostatic adsorption. Figure 1 The carrier in (f) is MC-OA, exhibiting an irregular polyhedral morphology. This is likely due to the presence of calcium oxalate crystals in the chitin, causing chitin nanoparticles to aggregate around these crystals, forming irregular polyhedral shapes clustered along their edges. Notably, CCOA chitin containing calcium oxalate impurities produces carriers with even smaller particle sizes. This phenomenon is presumably attributed to the presence of calcium oxalate crystals, which to some extent control the direction of carrier formation. Calcium oxalate, acting as the core of the carrier, guides the reaction to produce carriers with smaller particle sizes.

[0065] The results of TEM are as follows Figure 2 It can be observed that there is a large amount of space in the inner structure of all six carriers. Figure 2 Results (a) show that the internal space of the carrier MC contains a large number of pores that penetrate the entire carrier. The formation of these internal spaces may be due to the more uniform dispersion of the dissolved high-purity chitin during carrier preparation. When the reaction system begins to aggregate and form the carrier, the nanoparticles aggregate according to the directionality of chitin molecule crystallization. In nature, α-chitin molecules tend to crystallize into strip-shaped or rod-shaped crystals. Therefore, the carrier MC may be composed of multiple α-chitin crystals linked together, resulting in a structure with numerous internal pores. Figure 2In (b)-(e), we can see that these four carriers (MC-LA, MC-CA, MC-MA, and MC-TA) also have pores that penetrate the carrier, but the number of pores is less. This is because the presence of impurities affects the aggregation of nanoparticles. During the carrier formation stage, chitin nanoparticles aggregate prematurely, generating carriers with larger particle sizes, which restricts the formation of internal pores. Figure 2 Similarly, in (f), numerous pores can be observed within the MC-OA carrier. Combined with SEM analysis, it is clear that calcium oxalate crystals act as the core of the carrier formation, triggering the aggregation of nanoparticles. However, the core calcium oxalate crystal particles are relatively small and insufficient to support the chitin agglomeration into a larger carrier, thus preserving a large number of pores within the internal space.

[0066] The FT-IR results of the vector are as follows Figure 3 As shown in (a), the experiment compared and investigated the differences in functional groups between the selected chitin raw materials and the prepared carriers. The results showed that the optimal preparation method of the carrier in Example 1 did not affect the properties of chitin, and the carriers retained the stable physicochemical properties of chitin. Furthermore, the crystal structure information of five chitin samples (CCLA, CCMA, CCCA, CCTA, CCOA) and the carriers (MC-LA, MC-MA, MC-CA, MC-TA, MC-OA) were compared by XRD to compare the changes in the crystal form of chitin during the carrier preparation process. The results are shown in Figure 1. Figure 3 As shown in (b) above, the carriers prepared for the chitin samples do not affect the crystal form of chitin, and the XRD patterns of all carriers show the characteristic peaks of α-chitin. It is noteworthy that the MC-OA carrier shows results consistent with the raw material CCOA, also exhibiting the characteristic peaks of calcium oxalate crystals. Therefore, this carrier sample retains, to some extent, the unique synergistic effect of CCOA in immobilized enzymes.

[0067] Table 5 shows the zeta potential and particle size analysis results of different types of carrier samples. The absolute value of the zeta potential can be used to determine the dispersion stability of the particles. The absolute values ​​of the zeta potentials of carriers MC, MC-CA, and MC-TA are around 5, indicating that these three carriers will rapidly aggregate or agglomerate in solution. The absolute values ​​of the zeta potentials of the other three carriers are greater than ±10, indicating that these three carriers require a longer standing time in solution before aggregation or agglomeration occurs, and their dispersion stability is better than that of MC, MC-CA, and MC-TA. The particle size analysis results show that the carrier preparation method can produce micron-sized carriers. The particle size of MC-TA and MC-OA is smaller because the chitin raw materials CCTA and CCOA are more broken down during extraction, resulting in a more complete chitin dissolution reaction, which makes them easier to disperse and form smaller micron-sized particles during carrier preparation. The other four carrier samples, due to their relatively low solubility, agglomerate earlier during preparation, resulting in larger particles.

[0068] Table 5. Zeta potential and particle size of chitin carriers

[0069] Sample name Zeta potential (mV) Particle size (nm) MC -5.92±1.63 2484.66±366.14 MC-LA 13.56±1.11 4423.00±721.94 MC-CA 6.87±1.03 1360.33±74.77 MC-MA 11.47±3.63 5884.33±1075.40 MC-TA 5.28±2.50 926.70±20.31 MC-OA 16.13±1.18 979.76±47.38

[0070] Example 3: Enzyme Immobilized on Chitosan Vector

[0071] Take 0.05g of each type of chitin carrier and place it into a 10mL centrifuge tube. Add 3mL of Tris-HCl buffer solution (pH=8.0) and sonicate at low power (50W, 10min, 1s on, 2s off) to break the carrier into microparticles. After disruption, centrifuge at 4000rpm for 5min to remove the supernatant and retain the lower carrier layer. Mix the free lipase, carrier, and Tris-HCl buffer solution (pH=8.0) at a ratio of 4mg:1g:10mL and stir on ice for 1h (stirring speed 90-100rpm / min). After stirring, incubate at 4℃ for 12h to immobilize the free lipase. After immobilization, wash the precipitate three times with Tris-HCl buffer solution (pH=8.0) at 4000rpm for 5min to remove residual unimmobilized free lipase. Collect the supernatant for later use (to determine the content of unimmobilized protein and enzyme activity). Store the immobilized enzyme at 4℃.

[0072] The free lipases used in the above steps are porcine pancreatic lipase PPL, Aspergillus niger lipase ANL, Candida albicans lipase CAL, Pseudomonas fluorescens lipase PFL, Burkholderia cepacia lipase BCL, and Bacillus thermophilus lipase BTL2. Specific information is shown in Table 6.

[0073] Table 6. Information related to free lipase

[0074]

[0075] (1) Effects of different types of chitin carriers on the loading capacity and enzyme activity of Bacillus thermophilus lipase BTL2

[0076] Different types of chitin-based carriers correspond to different immobilized lipase properties.

[0077] like Figure 4 As shown in (a), the MC and MC-OA vectors exhibited excellent immobilization efficiency for BTL2. The immobilization capacity (enzyme protein loading capacity) of these two vectors for BTL2 was 3.37 times (2.80 mg / g) and 3.39 times (2.82 mg / g) that of CCOA (0.83 mg / g), respectively. Furthermore, except for the MC-CA vector, whose immobilization efficiency was similar to CCOA, the immobilized enzyme performance of the MC-LA, MC-MA, and MC-TA vectors was improved to some extent, with increases ranging from 1.71 to 2.79 times to 2.05 mg / g, 2.32 mg / g, and 1.42 mg / g, respectively.

[0078] After immobilizing BTL2 free enzyme on different types of chitin carriers, enzyme activity was tested under optimal reaction conditions (pH = 8.0, Tris-HCl buffer solution). The results are as follows: Figure 4 As shown in (b), the enzyme activity of BTL2 immobilized on different types of chitin carriers is weaker than that of the free enzyme. This is presumably because the lipase is encapsulated within the internal structure of the carrier, preventing sufficient contact between the substrate and the active site of the enzyme protein during the enzymatic digestion reaction, thus reducing enzyme activity. The MC-OA carrier exhibits the same defect, but notably, when the reaction conditions are changed to PBS buffer solution (pH = 8.0), the enzyme activity of MC-OA is improved. This phenomenon may be due to certain functional groups in the MC-OA carrier affecting the enzyme protein's structure during immobilization, altering the enzyme's requirements for reaction conditions, thus requiring a PBS buffer system to exhibit activity.

[0079] Figure 5 and Figure 6 The immobilization of the enzyme protein on the chitin support was characterized. The results showed that the protein was successfully loaded into the chitin support structure, with a large number of BTL2 protein crystals attached to the sheet-like stacked structure of the support. The presence of this sheet-like stacked structure significantly improved the immobilization effect of the lipase. However, the enzyme protein's loading into the interior of the support, to some extent, hindered the contact between the substrate and the enzyme's active site, resulting in an inhibition of enzyme activity recovery.

[0080] (2) Effects of optimized chitin carrier on the immobilization performance and enzyme activity of different types of lipases

[0081] Immobilization efficiency of commercial lipases immobilized on carriers, such as Figure 7 As shown in (a), the immobilization efficiency of lipase by the MC-OA carrier is superior to that by the MC carrier. This phenomenon may be due to the smaller particle size of the MC-OA carrier, resulting in a larger specific surface area, thus allowing it to contact more enzyme proteins and load more lipases. Another reason may be the high enzyme protein loading characteristics inherent in the chitin CCOA raw material used to prepare MC-OA. Notably, the MC-OA carrier cannot load lipase CAL, possibly because the prepared carrier is incompatible with the molecular size or surface groups of this lipase, preventing covalent cross-linking. The reason why the MC carrier cannot load lipase PPL, while the MC-OA carrier successfully loads PPL, is speculated to be that the calcium oxalate contained in the chitin CCOA undergoes a directional morphological change, making it compatible with lipase PPL. Figure 7 Figure (b) shows the enzyme activities of enzymes immobilized on MC and MC-OA vectors under their respective optimal reaction conditions for commercial lipases. The results indicate that, due to the masking of the active site of the commercial lipase, it is still unable to effectively initiate the enzymatic hydrolysis reaction even under optimal reaction conditions. Therefore, the MC-OA vector has a certain degree of universality for loading lipases, but for the enzyme activity of free BTL2 (128.49 U / mg) and the enzyme activity of BTL2 immobilized on the MC vector (216.71 ± 2.47 U / mg), the MC-OA vector effectively increases the enzyme activity of BTL2 when loaded, with an activity of 245.43 ± 2.51 U / mg. Figure 7 (b) in the middle.

[0082] (3) Performance evaluation of the reusability of the optimized chitin carrier immobilized enzyme

[0083] Reusability is a key parameter in industrial biocatalysis, referring to the ability of immobilized enzymes to maintain catalytic activity and structural stability across multiple reaction cycles. This capability minimizes enzyme waste, reduces production costs, and solves problems such as inactivation and recovery difficulties associated with free enzymes. Figure 8The reproducibility of immobilized enzyme activity was verified, thus assessing its recycling potential. Although immobilized BTL2 exhibited good stability during repeated use, its activity gradually decreased over time and with increasing frequency of use. This decline in enzyme activity can be attributed to two main factors: (1) structural changes or denaturation of the enzyme due to repeated use; and (2) detachment or degradation of the immobilization site, which reduces the number of enzyme molecules with catalytic activity. When the enzyme was immobilized using MC and MC-OA carriers, immobilized BTL2 retained more than 50% of its relative activity after reaching the maximum number of cycles at 10 and 12 cycles, respectively, with enzyme activities decreasing to 121.5 and 125.01 U / mg, representing 56.34% and 51.51% of the initial enzyme activity, respectively. Further recovery of enzyme activity after this point was minimal, indicating a threshold for the reusability of immobilized enzymes.

[0084] In summary, the chitin-based immobilized lipase loading efficiency of this invention is significantly improved, with the MC and MC-OA carriers increasing the enzyme immobilization efficiency by 3.37 times and 3.39 times, respectively. The BTL2 enzyme activity immobilized by MC-OA can reach up to 1.91 times that of free BTL2 enzyme under optimal reaction conditions, demonstrating that the carrier is a good immobilized lipase carrier. The MC-OA carrier, prepared using chitin CCOA, outperformed the MC carrier in all performance indicators, proving that the MC-OA carrier retains the lipase-enhancing effect of natural chitin and has great potential as an immobilized lipase carrier. The results of immobilization of commercial lipases show that the MC-OA carrier has universal applicability to loading various lipase proteins, but exhibits excellent performance in enhancing BTL2 enzyme activity.

Claims

1. A method for preparing a sheet-like hollow porous chitin carrier MC-OA, characterized in that, Here are the steps: S1. Mix choline chloride and oxalic acid in a molar ratio of 1:3 and stir continuously at 80°C until the mixture becomes a stable suspension to obtain acidic NADES. S2. Mix shrimp shell powder with acidic NADES prepared in step S1 at a mass ratio of 1:20, stir at 80°C for 2 h, add deionized water to reduce the viscosity of the system, take out the sample, collect the solid components and wash and centrifuge repeatedly with deionized water until the pH of the supernatant is neutral, collect the solid components and decolorize them with 10% hydrogen peroxide at 80°C, wash with deionized water until the pH of the supernatant is neutral, and dry the obtained solid at 60°C to obtain chitin CCOA; S3. Mix chitin CCOA with NaOH / urea solution at a mass ratio of 3:97 until homogeneous. Freeze the mixture at -20°C, then thaw it at room temperature. Stir vigorously until the chitin CCOA powder is completely and evenly distributed before the ice layer completely melts. Freeze the mixture again at -80°C, then thaw it at room temperature. Stir vigorously until the chitin CCOA powder is completely and evenly distributed before the ice layer completely melts. Repeat the freezing, thawing, and stirring process until the precipitate is completely dissolved to obtain a chitin solution. S4. Add the chitin solution obtained in step S3 to the Tween 80-liquid paraffin system at a mass percentage of 30%, stir at low speed for 3 h, and wash the precipitate with deionized water and anhydrous ethanol after stirring. After washing, recover the precipitate. S5. The precipitate recovered in step S4 is frozen at -80℃ for more than 4 hours, and then freeze-dried into powder to obtain the sheet-like hollow porous chitin carrier MC-OA.

2. The method according to claim 1, characterized in that, The NaOH / urea solution in step S3 has the following composition: 8 parts by mass of NaOH, 4 parts by mass of urea, and 88 parts by mass of water; the Tween 80-liquid paraffin system in step S4 has the following composition: 99 parts by mass of liquid paraffin and 1 part by mass of Tween 80.

3. The method according to claim 1, characterized in that, The vigorous stirring in step S3 refers to a stirring speed of 4000 rpm or higher; the low-speed stirring in step S4 refers to a stirring speed of 2000 rpm or lower.

4. The method according to claim 1, characterized in that, The step S4 of washing the precipitate with deionized water and anhydrous ethanol is as follows: first, wash with deionized water and centrifuge to remove the liquid paraffin on the surface of the precipitate; then, wash with anhydrous ethanol and centrifuge to remove the liquid paraffin and Tween 80 inside the structure; finally, wash with deionized water to remove the liquid paraffin precipitated from inside the structure and the anhydrous ethanol remaining on the surface of the precipitate.

5. The method according to claim 4, characterized in that, The centrifugation was performed at 4000 rpm for 5 minutes.

6. The sheet-like hollow porous chitin carrier MC-OA prepared by the method according to any one of claims 1-5.

7. A method for preparing immobilized lipase using the sheet-like hollow porous chitin carrier MC-OA as described in claim 6, characterized in that, Here are the steps: a. Take 0.05 g of sheet-like hollow porous chitin carrier MC-OA and place it in a centrifuge tube. Add 3 mL of pH=8.0 buffer solution and sonicate at low power for 10 min to break the carrier into microparticles. After breaking, centrifuge to remove the supernatant and retain the lower layer of carrier. The conditions for low-power sonication are: power of 50 W, 1 s on and 2 s off. b. Mix the free lipase, the sheet-like hollow porous chitin carrier MC-OA treated in step a, and the buffer solution at pH=8.0 in a ratio of 4 mg:1 g:10 mL, stir in an ice bath for 1 h, and place at 4℃ for 12 h after stirring. c. The precipitate was washed three times by centrifugation with a buffer solution of pH=8.0 to remove residual unfixed free lipase. The precipitate was recovered to obtain chitin-carrier MC-OA immobilized lipase.

8. The method according to claim 7, characterized in that, The free lipases mentioned are porcine pancreatic lipase PPL, Aspergillus niger lipase ANL, Pseudomonas fluorescens lipase PFL, Burkholderia cepacia lipase BCL, and Bacillus thermophilus lipase BTL2; the nucleotide sequence of the encoding gene of Bacillus thermophilus lipase BTL2 is shown in SEQ ID NO.

1.

9. The method according to claim 7, characterized in that, The buffer solution is Tris-HCl buffer solution or PBS buffer solution; the stirring under ice bath conditions for 1 h in step b means stirring at 90-100 rpm / min for 1 h under ice bath conditions; the centrifugation in steps a and c means centrifugation at 4000 rpm for 5 min.

10. An immobilized lipase prepared using the method described in any one of claims 7-9.

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