ZIF-8 immobilized beta-agarase based on lysozyme surface modification and preparation method thereof

Abstract

The invention discloses ZIF-8 immobilized beta-agarase based on lysozyme surface modification and a preparation method of the ZIF-8 immobilized beta-agarase based on lysozyme surface modification, and the method comprises the following steps: using a lysozyme surface modification ZIF-8 carrier to obtain a phase change ZIF-8 carrier; under the mediation of glutaraldehyde, the phase change ZIF-8 carrier and beta-agarase are subjected to covalent crosslinking, and the immobilized beta-agarase is obtained. According to the method, the immobilized beta-agarase of which the protein loading rate and the recovery rate are 45.3% and 71.3% respectively can be obtained; the immobilized beta-agarase has more excellent environmental tolerance, the affinity to a substrate is reduced, but the catalytic activity is greatly improved, and the catalytic rate is greatly increased; 70% of enzyme activity is still maintained during the sixth repetition; the enzyme still has 70% enzyme activity after being placed at 25 DEG C for 25 days, and has excellent performance in the aspects of storage stability and operation repeatability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of immobilized enzyme, in particular to a lysozyme surface modified ZIF-8 immobilized beta-agarase and a preparation method thereof. BACKGROUND

[0002] Agar oligosaccharides have excellent properties in food, medicine and other fields. The most suitable method for obtaining agar oligosaccharides from agar is enzymatic preparation. However, the problems encountered in the production process of free enzyme catalysis greatly limit the production efficiency and the application of enzyme method. The current methods for immobilizing agarase include directed covalent immobilization, glutaraldehyde covalent crosslinking, chitosan immobilization and the like.

[0003] There are many commonly used carrier materials for immobilized enzyme technology, including nanomaterials, porous materials, magnetic materials and polymer matrices. As one of the MOFs materials, zeolite imidazolate framework (ZIF) has strong thermal stability and chemical stability with Zn or Co as metal source and imidazole or imidazole derivative as organic ligand. Therefore, there are studies on using ZIFs as a fixed carrier for different enzymes. Among them, ZIF-8 is a common representative of ZIFs materials. However, ZIF-8 is a highly hydrophobic substance, which will change the conformation of the enzyme when it interacts with the hydrophobic groups on the surface of the enzyme, resulting in enzyme inactivation.

[0004] Therefore, it is necessary to study a new type of agarase immobilization technology which is more efficient, more stable and more economical. SUMMARY

[0005] To solve the above problems, the present application provides a lysozyme surface modified ZIF-8 immobilized beta-agarase and a preparation method thereof. The method uses lysozyme surface modified ZIF-8, and then glutaraldehyde is used to mediate the immobilization of beta-agarase. The maximum reaction rate of the immobilized enzyme is significantly improved, and the repeatability is good.

[0006] The embodiment of the present application provides a method for immobilizing beta-agarase based on lysozyme surface modified ZIF-8, which comprises the following steps:

[0007] (1) using lysozyme surface modified ZIF-8 carrier to obtain a phase change ZIF-8 carrier;

[0008] (2) under the mediation of glutaraldehyde, covalently crosslinking the phase change ZIF-8 carrier with beta-agarase to obtain immobilized beta-agarase.

[0009] According to the method for immobilizing beta-agarase based on lysozyme surface modification ZIF-8, the lysozyme surface modification ZIF-8 is utilized, and then the beta-agarase is immobilized by glutaraldehyde mediation, so that the immobilized beta-agarase with a protein loading rate of 45.3% and a recovery rate of 71.3% is obtained; the optimal temperature of the immobilized beta-agarase is 50 DEG C, the optimal pH is 8.0, compared with the free enzyme, the pH stability of the immobilized enzyme is shifted to the alkaline region, and the enzyme activity is maintained above 60% in the thermal stability test at 35-60 DEG C, and the environment tolerance is more excellent. The affinity of the immobilized enzyme for the substrate decreases, but the catalytic activity is greatly improved, and the catalytic rate is greatly improved. The immobilized enzyme also has excellent performance in storage stability and operation repeatability; the immobilized enzyme still has 70% enzyme activity in the sixth cycle; the immobilized enzyme still has 70% enzyme activity after being placed for 25 days at 25 DEG C.

[0010] Optionally, in step (1), the dispersed ZIF-8 carrier is directly immersed in a lysozyme solution, incubated for 40 min, and then centrifuged with deionized water multiple times to obtain the phase change ZIF-8 carrier.

[0011] Further, the lysozyme solution is prepared by mixing lysozyme buffer and TCEP buffer in equal volumes.

[0012] Optionally, in step (2), 4% glutaraldehyde solution is added to the phase change ZIF-8 carrier, crosslinked at 25 DEG C for 4 h, centrifuged to discard the supernatant, resuspended and washed the precipitate with deionized water twice, centrifuged to discard the supernatant, and obtained the precipitate.

[0013] Further, in step (2), beta-agarase solution is added to the precipitate, and then a dissolving buffer is added to make up the volume to 5 mL, and then the mixture is magnetically stirred at 300 rpm at 4 DEG C for 3 h, and then the precipitate is retained after centrifugation, and then the precipitate is washed with deionized water, and then the precipitate is centrifuged again, and then the washing is repeated twice, and then the obtained precipitate is the immobilized beta-agarase.

[0014] Further, the addition amount of the beta-agarase is 0.5 mL.

[0015] Further, the immobilization pH is 8.

[0016] Another embodiment of the present application provides an immobilized beta-agarase, which is prepared by the above-mentioned method for immobilizing beta-agarase based on lysozyme surface modification ZIF-8.

[0017] According to the immobilized beta-agarase of the embodiment of the present application, the enzyme activity recovery rate is 71.3%, and after six cycles of repeated use, the residual activity is still 70%, so that the immobilized beta-agarase has reusability and still has 70% enzyme activity after being placed for 25 days at 25 DEG C.

[0018] Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following and the attendant drawings or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Cross-linking time optimization graph according to an embodiment of the present application;

[0020] Figure 2 Glutaraldehyde concentration optimization graph according to an embodiment of the present application;

[0021] Figure 3 Cross-linking temperature optimization graph according to an embodiment of the present application;

[0022] Figure 4 Immobilization temperature optimization graph according to an embodiment of the present application;

[0023] Figure 5 Immobilization pH optimization graph according to an embodiment of the present application;

[0024] Figure 6 Immobilization time optimization graph according to an embodiment of the present application;

[0025] Figure 7 Immobilized enzyme addition amount optimization graph according to an embodiment of the present application;

[0026] Figure 8 Agitation speed optimization graph according to an embodiment of the present application;

[0027] Figure 9 Optimum temperature of free enzyme and immobilized β-agaro- blyase according to an embodiment of the present application;

[0028] Figure 10 Optimum pH of free enzyme and immobilized β-agaro- blyase according to an embodiment of the present application;

[0029] Figure 11 Thermal stability of free enzyme and immobilized β-agaro- blyase according to an embodiment of the present application;

[0030] Figure 12 pH stability of free enzyme and immobilized β-agaro- blyase according to an embodiment of the present application;

[0031] Figure 13 Operating stability of free enzyme and immobilized β-agaro- blyase according to an embodiment of the present application;

[0032] Figure 14 Storage stability of free enzyme and immobilized β-agaro- blyase according to an embodiment of the present application. DETAILED DESCRIPTION

[0033] The technical solutions of the present application are described below through specific examples. It should be understood that the one or more method steps mentioned in the present application do not exclude the presence of other method steps before and after the described combination steps or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool to identify each method step, and not a limitation on the arrangement order of each method step or a limitation on the scope of the present application that can be implemented, the change or adjustment of the relative relationship without substantial change of the technical content is also regarded as the scope of the present application that can be implemented.

[0034] In order to better understand the above technical solutions, the exemplary embodiments of the present application are described in more detail below. Although exemplary embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0035] The test materials used in the present application are all ordinary commercially available products and can be purchased in the market.

[0036] Ultra-pure egg white lysozyme, tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were purchased from Shanghai Aladdin Biochem Technology Co., Ltd.; HEPES was purchased from Shanghai Shifeng Biological Technology Co., Ltd.; Tryptone, yeast extract powder were purchased from Guangdong Huan Kai Microbial Science and Technology Co., Ltd.; NaCl, NaH2PO4·2H2O, Na2HPO4·12H2O, imidazole, sodium hydroxide were analytical pure and purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.; Kanamycin sulfate, isopropyl-β-D-thiogalactopyranoside (IPTG), 5× coomassie brilliant blue G-250 solution (protein quantification special) were purchased from Beijing Solabio Science and Technology Co., Ltd.; DNS color developing solution was purchased from Feijie Biological Technology Co., Ltd.; glutaraldehyde was purchased from Shanghai Macklin Biochemical Technology Co., Ltd.; 2-methylimidazole was purchased from Shanghai Yuanye Biological Technology Co., Ltd.; D(+) galactose was purchased from Tianjin Guangfu Fine Chemical Research Institute; methanol, zinc nitrate hexahydrate, glycine were purchased from Xilong Chemical Co., Ltd.

[0037] The present application is described below with reference to specific examples, it should be noted that these examples are only descriptive and do not limit the present application in any way.

[0038] Example 1 Expression of recombinant β-carrageenase gene and extraction of crude enzyme solution

[0039] Inoculation activation: 50 μL of 50 mg / mL kanamycin was added to 50 mL of LB medium containing culture solution, 1 mL of E. coli BL21(DE3) engineering strain containing recombinant Guttatus AL1 β-agarase was inoculated, and the constant temperature shaker was placed in the culture solution. The culture was incubated at 37°C and 180 rpm overnight.

[0040] Expansion culture: 300 μL of 50 mg / mL kanamycin was added to 300 mL of culture medium containing culture solution, and 3 mL of the above cultured bacterial solution was inoculated. The constant temperature shaker was placed in the culture solution and incubated at 37°C and 180 rpm for 12 h.

[0041] IPTG induction: 30 μL of 0.5 mol / L IPTG was added to the above culture medium, and the constant temperature shaker was placed in the culture solution and induced at 16°C and 180 rpm for 12 h to express sufficient target protein.

[0042] Crude enzyme extraction: The overnight cultured bacterial solution was placed in a centrifuge bottle and balanced, and then placed in a high-speed refrigerated centrifuge at 4°C and 5000 rpm for 10 min. The supernatant was discarded and the precipitate was retained. 300 mL of bacterial solution was obtained. The precipitate was resuspended in 5 mL of lysis solution, and repeatedly blown with a pipette until the precipitate was completely dissolved, and then poured into a centrifuge tube. The solution was placed on ice and ultrasonically broken, with the ultrasonic conditions set at power 300 W, breaking time 5 s, interval time 5 s, and total working time 10 min.

[0043] Affinity chromatography purification of β-agarase: The protein was purified according to the Ni-NTA instructions of Qiagen company.

[0044] Preparation of Example 2 lysozyme surface modified ZIF-8

[0045] ZIF-8 synthesis: 50 mL of methanol solution was added to 1.485 g of zinc nitrate hexahydrate and 3.280 g of 2-methylimidazole, respectively, and the two were mixed. The white suspension was obtained by stirring at room temperature at a speed of 500 rpm for 2 h, and then centrifuged at 8000 rpm for 15 min. The supernatant was discarded, the precipitate was resuspended and washed with methanol, and then centrifuged again. The supernatant was discarded and the precipitate was washed twice. The obtained precipitate was placed in a 60°C oven and dried until constant weight, and then ground to obtain ZIF-8 powder.

[0046] Lysosozyme surface modification ZIF-8: Lysosozyme was dissolved in 10 mmol / L HEPES buffer at 2 mg / mL to obtain a lysosozyme buffer, pH 7.4; 50 mmol / L TCEP was dissolved in 10 mmol / L HEPES buffer, and the pH was adjusted to 5.0 with 5 mol / L NaOH to obtain a TCEP buffer. The lysosozyme buffer and the TCEP buffer were mixed in equal volumes to prepare a phase transition lysosozyme (PTL) solution. The dispersed ZIF-8 was directly immersed in the PTL solution (v / v: 1 / 10), and after incubation for 40 min, the PTL-coated ZIF-8 particles were obtained by centrifugation with deionized water several times, that is, lysosozyme surface modification ZIF-8.

[0047] Example 3 Preparation of immobilized β-agarase

[0048] In a 50 mL beaker, 0.05 g of lysosozyme surface modification ZIF-8 obtained in Example 2 was added, 5 mL of 5% glutaraldehyde solution was added, and magnetic stirring was performed at room temperature for 3 h. After centrifugation at 8000 rpm for 10 min, the supernatant was removed, and the precipitate was washed twice with deionized water.

[0049] In the above precipitate, an appropriate amount of dissolution buffer was first added and mixed, and an enzyme solution (obtained in Example 1) was added. The enzyme was fixed by rotating and mixing at 4°C for 3 h. After fixation, the mixture was mixed by blowing, and the precipitate was centrifuged at 12000 rpm for 1 min. The supernatant after washing was retained in another set of centrifuge tubes, and the protein concentration of each group was measured. The precipitate was subjected to enzyme activity determination.

[0050] β-Agarase immobilization condition optimization: The enzyme activity and enzyme activity recovery rate of the immobilized β-agarase were used as indicators to explore the effects of different factors on the properties of the immobilized β-agarase. The condition optimization included:

[0051] Glutaraldehyde concentration in cross-linking (1.0%, 2.0%, 3.0%, 4.0%, and 5.0%); glutaraldehyde cross-linking time (1 h, 2 h, 3 h, 4 h, and 5 h); glutaraldehyde cross-linking temperature (4°C, 25°C); enzyme addition amount in fixation (0.5, 0.7, 0.9, 1.1, 1.3 mL (3.6 U / mL)); immobilization temperature (4°C, 25°C); enzyme immobilization pH (6, 6.5, 7, 7.5, 8); enzyme immobilization time (1 h, 2 h, 4 h, 6 h, 8 h, and 10 h); stirring speed (100, 200, 300, 400, 500 rpm).

[0052] As Figure 1As shown, between 1 and 4 hours, the enzyme activity recovery rate and loading rate increased significantly with increasing cross-linking time, reaching 70.7% and 63.2%, respectively, at 4 hours. After 4 hours, these rates decreased. Initially, as cross-linking progressed, glutaraldehyde binding to the carrier gradually increased, reaching a peak, indicating saturation between the enzyme and carrier, and between the enzymes. Further increases in cross-linking time, due to environmental influences, could lead to glutaraldehyde dissociation from the cross-links.

[0053] like Figure 2 As shown, within the 1-4% concentration range, the recovery rate of immobilized enzyme activity and protein loading rate both increased with increasing glutaraldehyde concentration. Above 4%, both began to decline. The addition of glutaraldehyde affects loading rate and enzyme activity by forming a Schiff base structure through the binding of its aldehyde groups with the amino groups of the enzyme molecule. When the glutaraldehyde concentration is too high, it self-crosslinks, creating a dense supramolecular structure. This sterically hinders the binding of substrates to the active site, disrupting the enzyme's three-dimensional conformation and hindering the binding between the active site and substrate, thereby reducing enzyme activity.

[0054] like Figure 3 As shown in the figure, during the glutaraldehyde crosslinking process, 4°C and 25°C were selected as the crosslinking temperature to explore their effects on the recovery rate of immobilized enzyme activity and protein loading rate. At 25°C, the enzyme activity recovery rate and protein loading rate were slightly higher than those at 4°C, indicating that temperature does not significantly affect glutaraldehyde crosslinking. Based on factors such as economy and experimental simplicity, 25°C was selected as the crosslinking condition.

[0055] like Figure 4 As shown, during the immobilization process, the enzyme activity recovery rate and protein loading rate of the immobilized enzyme at 4°C were both above 50%, far exceeding those of the group with an immobilization temperature of 25°C.

[0056] like Figure 5 As shown in the figure, in the range of 6 to 8, with the increase of pH, the recovery rate of immobilized enzyme activity and protein loading rate gradually increased.

[0057] like Figure 6 As shown in the figure, within the free enzyme immobilization time range of 1 to 3 hours, both the enzyme activity recovery rate and protein loading rate showed an upward trend with increasing time. Beyond 3 hours, the enzyme activity recovery rate and protein loading rate decreased. Immobilization is not complete before 3 hours, and as time passes, unimmobilized free enzyme gradually binds to the support material. After 3 hours, immobilization is essentially complete; further incubation may cause the immobilized enzyme to detach due to external factors or disrupt its spatial structure. Furthermore, the binding between the enzyme and the support may be loose or tight at different times, affecting the cross-linking effect.

[0058] like Figure 7As shown in the figure, within the enzyme addition amount range of 0.5-1.3 mL (3.6 U / mL), with the increase of enzyme addition amount, the enzyme activity recovery rate and the protein loading rate of the immobilized enzyme both showed a downward trend. Due to the limited number of carriers, with the increase of enzyme molecules, when the immobilized enzyme is saturated, the excess free enzyme cannot be fixed, resulting in waste of enzyme. Considering the enzyme activity recovery rate and the loading rate, 0.5 mL was selected as the optimal addition amount for further discussion.

[0059] As shown in the figure, within the range of 100-300 rpm, with the increase of the rotation speed in the immobilization step, the enzyme activity recovery rate and the protein loading rate of the immobilized enzyme gradually increased. Within the range of 300-500 rpm, it began to decrease. Appropriate stirring speed can prevent the carrier material from sinking to the bottom, increase the contact area with free enzyme, and thus increase the immobilization efficiency. Lower stirring speed may lead to uneven distribution of reactants in the reaction system, insufficient contact between immobilized enzyme and reaction substrate, thereby affecting the cross-linking degree of the material and reducing its stability. If the stirring speed is too high, it may affect the morphology and distribution of the cross-linking reaction products, and even cause mechanical damage and inactivation of the immobilized enzyme. Figure 8

[0060] Example 4 Enzymatic properties of immobilized β-agarase

[0061] Accurately weigh 0.05 g of lysozyme surface modified ZIF-8 in a 50 mL beaker, then add 5 mL of glutaraldehyde solution with a concentration of 4.0%, and crosslink at 25°C for 4 h. After crosslinking is completed, centrifuge at 8000 rpm and 25°C for 10 min, discard the supernatant, and retain the precipitate. Wash the precipitate with deionized water, and centrifuge again to obtain the precipitate. Repeat the washing step twice. Then add 500 μL of β-agarase solution (obtained in Example 1) to the precipitate, and add the dissolution buffer to make up the volume to 5 mL. Magnetically stir at 300 rpm and 4°C for 3 h. After the immobilization is completed, centrifuge at 8000 rpm and 25°C for 10 min, discard the supernatant, and retain the precipitate. Wash the precipitate with deionized water, and centrifuge again to obtain the precipitate; repeat the washing step twice to obtain the immobilized β-agarase. After vacuum freeze-drying, store it at 4°C for future use.

[0062] (1) β-agarase activity determination: Dinitrosalicylic acid (DNS) and reducing sugar produce an oxidation-reduction reaction, which can synthesize 3-amino-5-nitrosalicylic acid. After boiling, it presents a brown-red color, and the color depth within a certain range is proportional to the amount of reducing sugar. Through the degree of color depth, the content of reducing sugar can be quantitatively calculated.

[0063] ​Take 10 mg of immobilized β-agarase (or 10 μL of free β-agarase), add 490 μL of 0.2% mass fraction of agarose solution (dissolved with 50 mmol / L NaH2PO4-Na2HPO4 buffer at pH 7.0), and react in a constant temperature mixer for 15 min. Immediately after the reaction time is over, place on ice, add 500 μL of DNS, mix, color in a boiling water bath for 10 min, centrifuge, and measure the absorbance at 520 nm. Calculate the reducing sugar concentration by the standard curve. The enzyme activity of β-agarase is defined as follows: 1 μmol of galactose released per minute under the above conditions is one enzyme activity unit (U).

[0064] The lysozyme surface modified ZIF-8 prepared in the preparation is used to obtain a multifunctional interface ZIF-8 material. After 4% mass fraction glutaraldehyde room temperature treatment for 4 h, and 0.5 mL enzyme solution is added at 4°C for 3 h, the environmental pH is kept at 8.0, the immobilized β-agarase with a protein loading rate and recovery rate of 45.3% and 71.3% can be obtained.

[0065] (2) β-agarase optimum reaction temperature determination: 0.01 g of immobilized enzyme and 10 μL of free enzyme are added to a centrifuge tube of 1 mL system respectively, and 490 μL of 0.2% mass fraction of agarose solution is measured. After setting three parallels, place in different temperature gradients (35, 40, 45, 50, 55 and 60°C) constant temperature water bath pot for reaction for 15 min. Then cool on ice and add DNS solution, boil for 10 min. Measure the absorbance at 520 nm, and calculate the enzyme activity. The optimum reaction temperature of β-agarase and immobilized β-agarase is explored. Take the highest enzyme activity as 100%.

[0066] As shown in Figure 9 , the optimum reaction temperature of lysozyme surface modified ZIF-8 immobilized β-agarase and free β-agarase is 50°C. The immobilized enzyme has good catalytic activity in the range of 35-60°C, compared with 50°C, it still has good enzyme activity at 45-60°C, and the enzyme activity decreases at 35-40°C, but still maintains more than 85% of the enzyme activity. Compared with 50°C, the enzyme activity of free β-agarase gradually decreases with the decrease of temperature, and decreases with the increase of temperature. This shows that the technology of lysozyme surface modification of ZIF-8 material for glutaraldehyde covalent crosslinking immobilization of β-agarase can well protect the spatial structure stability of the enzyme by covalent combination between free enzyme and glutaraldehyde, and well reduce the distortion and damage of enzyme conformation caused by heat exchange.

[0067] (3) The determination of the optimum pH of β-gelling enzyme: 0.01 g of immobilized enzyme and 10 μL of free enzyme were added to a centrifuge tube of 1 mL system, 490 μL of 0.2% mass fraction of different pH buffer (6, 7, 8, 9, 10, 11 and 12) configured agarose solution was measured, and it was placed in the optimum temperature constant water bath pot for reaction for 15 min. Then it was cooled on ice and DNS solution was added, and it was boiled for 10 min. The absorbance was measured at 520 nm, and the enzyme activity was calculated. The optimum reaction pH of β-gelling enzyme and immobilized β-gelling enzyme was explored. The highest enzyme activity was taken as 100%.

[0068] As shown in Figure 10 , after the lysozyme surface modification of ZIF-8 immobilized β-gelling enzyme, the optimum pH was 10.0, and the optimum pH of free β-gelling enzyme was 8.0. The enzyme activity of the immobilized β-gelling enzyme was greatly affected in the acidic buffer (pH 6-8), which was lower than that of the free β-gelling enzyme, and the enzyme activity was significantly higher than that of the free β-gelling enzyme in the alkaline buffer (pH 9-12). It was shown that the optimum reaction pH of the immobilized β-gelling enzyme shifted to the alkaline direction. The aldehyde group of glutaraldehyde and the amino group of enzyme molecules and the carrier formed Schiff base, and the deprotonation of amino group was more conducive to the stability of the product under alkaline conditions.

[0069] (4) Thermal stability of β-gelling enzyme: 10 mg of immobilized β-gelling enzyme and 10 μL of free β-gelling enzyme were respectively incubated at different temperatures (35, 40, 45, 50, 55 and 60℃) for 1 h, and then 490 μL of 0.2% mass fraction of agarose solution was added to determine the enzyme activity. The data were compared to explore the thermal stability of immobilized β-gelling enzyme and free enzyme. The enzyme activity of the unincubated sample was taken as 100%.

[0070] As shown in Figure 11 , after the immobilized β-gelling enzyme and the free β-gelling enzyme were incubated at different temperatures (35-60℃) for 1 h, the immobilized enzyme always maintained more than 90% of the enzyme activity in the whole temperature range of 35-55℃, and the enzyme activity remained more than 80% under the incubation condition of 60℃. The free enzyme could also maintain more than 90% of the enzyme activity after incubation at 35-50℃, but the enzyme activity sharply decreased at 55-60℃, and the residual enzyme activity was 30% and 28%, respectively. This phenomenon showed that the enzyme was immobilized on the carrier, and the spatial conformation change of the enzyme was limited due to the covalent bond and the influence of steric hindrance, which could prevent the denaturation and inactivation of the enzyme protein caused by the destruction of the spatial structure under high temperature.

[0071] (5) pH stability of β-gelling enzyme: 10 mg of immobilized β-gelling enzyme and 10 μL of free β-gelling enzyme were weighed and added to 200 μL and 40 μL of buffer with different pH (6, 7, 8, 9, 10, 11 and 12) respectively. After 1 h of incubation, 490 μL of 0.2% agarose solution was added to measure the enzyme activity under the optimal reaction conditions. The pH stability of immobilized β-gelling enzyme and free enzyme was compared by taking the data of the sample without incubation as the benchmark, and the enzyme activity was taken as 100%.

[0072] As shown in FIG. 6, after 1 h of incubation of immobilized β-gelling enzyme and free β-gelling enzyme in different pH (6-12) buffer at the optimal reaction temperature, the enzyme activity of immobilized β-gelling enzyme remained above 80% in the entire pH range, showing excellent pH stability. The enzyme activity of free β-gelling enzyme remained above 80% in the pH range of 6-8, and then dropped sharply to below 70% in the pH range of 9-12. This shows that the use of lysozyme surface modified ZIF-8 to immobilize β-gelling enzyme weakens the influence of external environmental pH changes on the microenvironment around the active center of the enzyme, so that the active site of the enzyme protein is not damaged, and the immobilized enzyme can exhibit high catalytic activity in a wider pH environment. Figure 12 (6) Determination of kinetic parameters of β-gelling enzyme: A series of gradient (0.05%-0.5%) agarose solutions were prepared as substrates for immobilized β-gelling enzyme and free enzyme, and their enzyme activities were measured under the same conditions. According to the obtained data, double-reciprocal plots were made to calculate the kinetic parameters (V max , K m ) of immobilized β-gelling enzyme and free enzyme.

[0073] As shown in Table 1, the K m of immobilized enzyme was 7.5 mg / mL, and the V max was 217.39 U / mg; the K m of free enzyme was 5.2 mg / mL, and the V max was 144.92 U / mg. This shows that the immobilized enzyme has a great improvement in catalytic rate compared to the free enzyme, but the affinity for the substrate is reduced. It is speculated that the pores and groups of the carrier material may cover the active site of the enzyme or hinder the entry of the substrate to some extent. In general, the immobilized enzyme has a significant improvement in catalytic activity. Lysozyme surface modified ZIF-8 provides numerous attachment points for free enzyme, increases its hydrophilicity, and strengthens the directional action and enrichment between enzyme and substrate, increasing the contact opportunity, thereby increasing the maximum reaction rate.

[0074]

[0075] ​Table 1 Constants and correlation coefficients used in the Michaelis equation

[0076]

[0077] (7) Analysis of the operational stability of immobilized β-agarase: At the optimum temperature, 0.2% mass fraction of agarose solution was used as the substrate, and the immobilized β-agarase was placed in a constant temperature water bath for 15 min, and then centrifuged in a high-speed centrifuge, and the supernatant was reserved. The precipitate was continuously reacted according to the above steps for six times. DNS solution was added to the six reaction solutions, and boiled in a boiling water bath for 10 min. 200 μL of the reaction solution was taken to a 96-well plate, and the absorbance was measured at a wavelength of 520 nm. The residual enzyme activity was calculated, and the operational stability was evaluated.

[0078] As shown in Figure 13 , the enzyme activity loss of the immobilized enzyme was within 3% in the first repetition, and still maintained a high enzyme activity, and the enzyme activity was still 70% of the original enzyme activity in the sixth time. This is in sharp contrast to the waste of one-time use of free enzymes in industrial production due to the inability to recover them. The use of immobilized enzymes can greatly reduce costs and increase economic benefits.

[0079] (8) Analysis of the storage stability of immobilized and free β-agarase: The β-agarase was stored at room temperature (25°C) for 25 days. Every 5 days, the enzyme activity of the stored sample was measured, and the initial activity was taken as 100%. The activity of the subsequent samples was compared with the initial activity, and the relative activity was calculated. The storage stability of β-agarase was investigated.

[0080] As shown in Figure 14 , the lysozyme surface modified immobilized β-agarase had good storage stability, and still had more than 70% enzyme activity after 25 days of placement. While the free β-agarase had only about 65% enzyme activity after 25 days of placement. In comparison, the immobilized enzyme showed more excellent enzyme activity storage stability. The interaction between the groups of the enzyme and the carrier can neutralize the charged groups, so that the carrier can stabilize the spatial structure of the enzyme during storage.

[0081] According to an embodiment of the present invention, the method uses lysozyme to modify the surface of ZIF-8 composite nanomaterials to prepare phase-change ZIF-8, and then uses glutaraldehyde to covalently cross-link the carrier material and the free enzyme to prepare lysozyme surface-modified ZIF-8 immobilized β-lysozyme; wherein, the prepared ZIF-8 is surface-modified with lysozyme to obtain a ZIF-8 material with a multifunctional interface, which is treated with 4% mass fraction glutaraldehyde at room temperature for 4 hours and fixed with 0.5mL enzyme solution at 4°C for 3 hours. The environmental pH is maintained at 8.0, and an immobilized β-agarase with a protein loading rate and recovery rate of 45.3% and 71.3% respectively can be obtained. The optimal temperature of immobilized β-agarase is 50°C and the optimal pH is 8.0. Compared with the free enzyme, the pH stability after immobilization shifts to the alkaline region, and the enzyme activity is maintained at more than 60% in the thermal stability test at 35-60°C. The affinity of the immobilized enzyme for the substrate is reduced, but the catalytic activity is greatly improved, and the catalytic rate is greatly improved. It also has excellent performance in storage stability and operational repeatability.

[0082] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification.

[0083] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for immobilizing β-agarase based on surface modification of lysozyme with ZIF-8, characterized in that: The following steps are involved: (1) Using lysozyme to modify the surface of ZIF-8 carrier to obtain a phase-change ZIF-8 carrier; (2) Under the mediation of glutaraldehyde, the phase-changed ZIF-8 carrier and β-agarase are covalently cross-linked to obtain immobilized β-agarase.

2. The method according to claim 1, wherein In step (1), the dispersed ZIF-8 carrier is directly immersed in a lysozyme solution, incubated for 40 minutes, and then centrifuged multiple times with deionized water to obtain the phase-change ZIF-8 carrier.

3. The method according to claim 2, wherein The lysozyme solution is prepared by mixing equal volumes of lysozyme buffer and TCEP buffer.

4. The method according to claim 1, wherein In step (2), 4% glutaraldehyde solution was added to the phase-change ZIF-8 carrier, cross-linked at 25° C. for 4 h, centrifuged and the supernatant was discarded, and the precipitate was resuspended and washed twice with deionized water, centrifuged and the supernatant was discarded to obtain a precipitate.

5. The method according to claim 4, wherein In step (2), β-agarase solution is added to the precipitate, and then dissolution buffer is added to make the volume up to 5 mL. The mixture is fixed with magnetic stirring at 300 rpm at 4°C for 3 h. After centrifugation, the precipitate is retained, washed with deionized water, and centrifuged again to obtain a precipitate. The washing is repeated twice to obtain the immobilized β-agarase.

6. The method according to claim 5, wherein The added amount of the β-agarase was 0.5 mL.

7. The method according to claim 5, wherein The immobilization pH was 8.

8. An immobilized β-agarase, characterized in that The method for immobilizing β-agarase based on surface modification of lysozyme on ZIF-8 is adopted as described in any one of claims 1 to 7.

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