Immobilized D-psicose-3-epimerase as well as preparation method and application thereof

Abstract

The invention provides a preparation method of immobilized D-psicose-3-epimerase, which comprises the following steps: firstly aminating inorganic silicon nanoflowers, then activating by using a cross-linking agent glutaraldehyde, finally adding enzyme liquid, reacting in a shaking table for a period of time, and centrifuging and washing the reactant to obtain the product, namely the immobilized D-psicose-3-epimerase. The enzymatic activity and the stability of the immobilized enzyme are obviously improved, the specific enzyme activity of the immobilized enzyme is 446.44-1033.09 U / g protein, the activity of the immobilized enzyme is 163.09% and 181.53% of that of free enzyme after the immobilized enzyme is incubated for 6 h under the condition that the temperature is 30 DEG C or the pH value is 5, 63.18-70.78% of the initial activity is still reserved after the immobilized enzyme is repeatedly used for 10 times, and 46.27-54.38% of the initial activity is still reserved after the immobilized enzyme is stored for 15 days. The immobilized enzyme can catalyze fructose to generate psicose, and a green and efficient preparation technology is provided for production of psicose.

Description

Technical Field

[0001] The invention belongs to the technical field of immobilized enzyme preparation, and specifically relates to a novel immobilized D-psicose-3-epimerase and a preparation method and application thereof. Background Art

[0002] As chronic diseases such as obesity, diabetes, and "three highs" spread around the world, "healthy intake" of sugar in the diet has become a new health issue, among which the development and application of low-calorie sweeteners is one of the most effective ways.

[0003] D-psicose is a rare sugar that has the characteristics of low calories (0.4kcal / g) and high sweetness (70% of the relative sweetness of sucrose). It has the potential to inhibit obesity and lower blood sugar, and has great application potential in the fields of food, medicine, and health products. However, it is rare in nature. The biocatalytic method of generating D-psicose by the diastereoisomerization reaction of D-fructose C-3 position catalyzed by D-psicose-3-dimerase has gradually attracted people's attention. D-psicose-3-dimerase can catalyze the mutual conversion between D-fructose and D-psicose. No other substances need to be added during the reaction, and there are few by-products after the reaction, which is conducive to reducing production costs and making the subsequent separation of D-psicose more convenient.

[0004] However, in the process of D-psicose 3-isomerase catalyzing the conversion of fructose into psicose, the enzyme cannot be reused, making the process cost relatively high. After the reaction, D-psicose-3-isomerase itself needs to be removed from the reaction system as a foreign protein, increasing the separation cost. Therefore, the development of immobilization technology for D-psicose 3-isomerase allows D-psicose 3-isomerase to be reused, which can not only greatly reduce the fermentation cost, but also reduce the subsequent separation cost, which is of great significance to the industrial development of psicose.

[0005] However, traditional carrier materials do not have good adjustability and crystallinity, which may lead to low protein loading efficiency, instability and easy leaching of enzymes, thus affecting the activity of immobilized enzymes. Embedding enzymes into nanomaterials has become a hot topic in recent years. Studies have shown that enzymes immobilized on nanomaterials have a wider pH and temperature adaptability range and higher thermal stability than natural enzymes. Since three-dimensional nanomaterials are superior to other nanomaterials in terms of surface area, reaction activity, stability, etc., three-dimensional nanomaterials have become a hot topic in enzyme immobilization research in recent years. Inorganic silicon nanoflowers (SNF) are a new type of three-dimensional nanomaterial with many advantages such as porosity, high specific surface area, and good chemical and thermal stability. It is expected to become a new type of excellent carrier for immobilized enzyme preparations. Summary of the invention

[0006] In view of the shortcomings of the prior art, the object of the present invention is to provide a novel immobilized D-psicose-3-epimerase and a preparation method and application thereof, so as to solve the problems raised in the above background technology.

[0007] In order to solve the above technical problems, the present invention proposes an immobilized D-psicose-3-epimerase, which uses D-psicose-3-epimerase as enzyme protein and inorganic silicon nanoflowers as immobilization carriers; the immobilized D-psicose-3-epimerase has a spherical morphology with a diameter of 340nm±15nm, and the sphere has pores, and the D-psicose-3-epimerase is fixed in the pores.

[0008] Furthermore, the specific enzyme activity of the immobilized D-psicose-3-epimerase of the present invention is 446.44-1033.09 U / g 蛋白 ; After incubation at 30°C for 6 hours, the immobilized D-psicose-3-epimerase retained 82.56-90.09% of the initial activity, and the free enzyme activity was 55.24%; after incubation at 70°C for 6 hours, the immobilized D-psicose-3-epimerase retained 5.18-9.72% of the initial activity, and the free enzyme activity was 0%; after incubation at pH = 5 for 6 hours, the immobilized D-psicose-3-epimerase retained 75.63-88.28% of the initial activity, and the free enzyme activity was 48.63%; after incubation at pH = 9 for 6 hours, the immobilized D-psicose-3-epimerase retained 94.73-97.86% of the initial activity, and the free enzyme activity was 69.56%.

[0009] The immobilized D-psicose-3-epimerase still retains 63.18-70.78% of the initial activity after being reused 10 times, and still retains 46.27-54.38% of the initial activity after being stored for 15 days.

[0010] At the same time, the present invention also proposes a method for preparing the above-mentioned immobilized lipase, which mainly comprises: using D-psicose-3-epimerase as the enzyme protein and using inorganic silicon nanoflowers as the immobilization carrier; firstly, the inorganic silicon nanoflowers are aminated with 3-aminopropyltriethoxysilane, then a cross-linking agent glutaraldehyde is added for activation, and finally, D-psicose-3-epimerase solution is added to solidify and react in a shaker for a period of time, and then the reactant is centrifuged and washed, and the obtained product is the immobilized D-psicose-3-epimerase.

[0011] Furthermore, the preparation method of the present invention comprises the following specific steps:

[0012] Step 1) preparing inorganic silicon nanoflowers, comprising:

[0013] 1-1) mixing hexadecyltrimethylammonium bromide and a urea solution having a concentration of 0.1-1.0M at a mass ratio of 1:15-60, and then adding n-butanol to the mixed solution at a mass ratio of 1:15-60, and stirring until clear, to obtain a mixed solution A;

[0014] 1-2) adding cyclohexane to the mixed solution A at a mass ratio of 1:1-4, and stirring with ultrasound until clear, to obtain a mixed solution B;

[0015] 1-3) slowly adding ethyl silicate to the mixed solution B at a mass ratio of 1:10-40, and stirring with ultrasound until it becomes clear, then stirring under sealed conditions for 15-60 minutes, raising the temperature to 60-80° C., and continuing stirring for 12-48 hours to obtain a reaction mixture;

[0016] 1-4) The reaction mixture is centrifuged to obtain a white precipitate, and the precipitate is placed in a tubular calcining furnace and calcined at 450-650° C. for 3-7 hours. Air is passed throughout the calcination process. The calcined product is the inorganic silicon nanoflower;

[0017] Step 2) amination of nanoflowers, comprising:

[0018] 2-1) dispersing the inorganic silicon nanoflowers obtained in step 1) into n-hexane at a mass ratio of 1:100-400, and subjecting to ultrasonic oscillation for 15-60 minutes to obtain a mixed solution C;

[0019] 2-2) adding 3-aminopropyltriethoxysilane to the mixed solution C at a volume ratio of 1:50-200 to obtain a mixed solution D, and placing the mixed solution D under condensation reflux at 70-90° C. for 6-24 hours;

[0020] 2-3) The mixed solution D is centrifuged to obtain a white precipitate, the product is separated, washed with anhydrous ethanol for multiple times, and dried in an oven for 6-48 hours to obtain amino nanoflowers;

[0021] Step 3) activating the amino nanoflowers using glutaraldehyde as a crosslinking agent, comprising:

[0022] 3-1) adding the amino nanoflowers obtained in step 2) to a glutaraldehyde solution with a final concentration of 0.5-4% at a mass ratio of 1:200-800, and performing a cross-linking reaction on a shaking table at room temperature for 1.5-5 hours to obtain a mixed solution E;

[0023] 3-2) filtering the mixed solution E to obtain a reddish brown precipitate, and washing it three times with pure water to obtain glutaraldehyde-modified amino nanoflowers;

[0024] Step 4) preparing immobilized D-psicose-3-epimerase, the steps are as follows:

[0025] 4-1) dispersing the glutaraldehyde-modified amino nanoflowers obtained in step 3) into a PBS buffer solution at a mass ratio of 1:150-600, and uniformly dispersing by ultrasonication to obtain a mixed solution F;

[0026] 4-2) adding an enzyme solution having a D-psicose-3-epimerase protein concentration of 2.5-37.5 mg / mL to the mixed solution F to obtain a mixed solution, wherein the volume ratio of the mixed solution F to the enzyme solution is 3:1-4; placing the mixed solution on a shaker for a solidification reaction at 20-55° C. for 0.5-5 h, and after the reaction is completed, centrifuging to obtain a reddish-brown precipitate;

[0027] 4-3) The red-brown precipitate was washed three times with PBS buffer solution and freeze-dried for 6-24 hours to obtain immobilized D-psicose-3-epimerase.

[0028] Furthermore, in the preparation method of the present invention, the preferred process conditions are as follows:

[0029] In step 1-1), the concentration of the urea solution is 0.4M, the mass ratio of hexadecyltrimethylammonium bromide to the urea solution is 1:30, and the mass ratio of n-butanol added to the mixed solution is 1:31;

[0030] In step 1-2), the mass ratio of cyclohexane to mixed solution A is 3:8;

[0031] In step 1-3), the mass ratio of the mixed solution B to ethyl silicate is 22:1, the stirring time under sealed conditions is 30 min, the temperature after stirring is 70° C., and the stirring time after heating is 20 h;

[0032] In step 1-4), the conditions for centrifuging the reaction mixture are: centrifugation at 10000 rpm for 5 min; the calcination temperature in the tubular calcining furnace is 550° C., and the calcination time is 5 h;

[0033] In step 2-1), the inorganic silicon nanoflowers are dispersed in n-hexane and subjected to ultrasonic oscillation for 30 minutes, wherein the mass ratio of the inorganic silicon nanoflowers to n-hexane is 1:227;

[0034] In step 2-2), the volume ratio of the mixed solution C to 3-aminopropyltriethoxysilane is 100:1; the mixed solution D is placed under condensation reflux at 80° C. for 12 hours;

[0035] In step 2-3), the white precipitate obtained by centrifuging the mixed solution D was dried in an oven at 60° C. for 12 h;

[0036] In step 3-1), the amino nanoflowers are added to a glutaraldehyde solution with a final concentration of 1.5%, the mass ratio of the amino nanoflowers to the glutaraldehyde solution is 1:400, and the reaction is carried out under shaking conditions for 4 hours;

[0037] Step 3-2) Filter the mixed solution E to obtain a reddish brown precipitate, and wash it three times with pure water to obtain glutaraldehyde-modified amino nanoflowers;

[0038] In step 4-1), the concentration of the PBS buffer solution is 100 mM, pH=8.5, and the mass ratio of the glutaraldehyde-modified amino nanoflowers to the PBS buffer solution is 1:300;

[0039] In step 4-2), the D-psicose-3-epimerase protein concentration of the enzyme solution is 12.5 mg / mL, and the volume ratio of the mixed solution F to the enzyme solution is 3:2; the process conditions of the shaking table are: the rotation speed is 220 rpm, the curing reaction temperature is 40° C., and the curing reaction time is 3 h; after the reaction is completed, the reaction is centrifuged at 8000 rpm for 5 min.

[0040] The present invention also proposes using the prepared immobilized D-psicose-3-epimerase for the preparation of psicose, that is, adding the immobilized D-psicose-3-epimerase to a fructose solution, reacting the reaction mixture at a constant temperature, and separating and obtaining psicose after the reaction is completed.

[0041] In the above application, the conditions of the isothermal reaction are that the reaction temperature is 45-60°C, the reaction time is 0-20h and ≠0.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) The present invention proposes an immobilized D-psicose-3-epimerase, using SNF as an immobilization carrier. The SNF is a three-dimensional nanomaterial that is in the nanoscale range in three spatial dimensions and has a nanostructure and properties. Compared with nanomaterials such as carbon nanotubes and graphene, it has many advantages such as multiple pores, high specific surface area, and good chemical and thermal stability.

[0044] (2) The immobilized D-psicose-3-epimerase of the present invention uses SNF as an immobilization carrier and D-psicose-3-epimerase as an enzyme protein. According to the preparation method steps of the present invention, immobilized D-psicose-3-epimerase can be successfully prepared under normal temperature and pressure conditions. Compared with the free enzyme, the immobilized enzyme further improves the catalytic activity and stability, and has good reusability and storage stability. After testing, the specific enzyme activity of the immobilized D-psicose-3-epimerase is 446.44-1033.09 U / g 蛋白 , see Example 2; after incubation at 30°C for 6h, it retained 82.56-90.09% of the initial activity, and the free enzyme activity was 55.24, see Examples 12 and Fig.14 ; After incubation at 70°C for 6 h, 5.18-9.72% of the initial activity was retained, and the free enzyme activity was 0%, see Examples 13 and Fig.15 ; After incubation for 6 h at pH = 5, it retained 75.63-88.28% of the initial activity, and the free enzyme activity was 48.63%, see Examples 14 and Fig.16 ; After incubation for 6 h at pH = 9, 94.73-97.86% of the initial activity was retained, and the free enzyme activity was 69.56%, see Examples 15 and Fig.17 ; After repeated use 10 times, it still retains 63.18-70.78% of the initial activity, see Examples 17 and Fig.19 , after 15 days of storage, 46.27-54.38% of the initial activity was retained, see Examples 16 and Fig.18 .

[0045] (3) The preparation method of the present invention has mild synthesis conditions, short reaction time and simple operation, and is easy to promote, thus providing a green and efficient method for the preparation and production of psicose. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Determination of the glutaraldehyde concentration during the glutaraldehyde cross-linking process in the preparation method of the present invention;

[0047] Figure 2 Determination of the cross-linking time during the glutaraldehyde cross-linking process in the preparation method of the present invention;

[0048] Figure 3 Determination of the enzyme protein mass concentration during the enzyme immobilization process in the preparation method of the present invention;

[0049] Figure 4 The invention relates to the determination and optimization of the temperature during the enzyme immobilization process in the preparation method of the present invention.

[0050] Figure 5The present invention relates to the determination and optimization of pH during the enzyme immobilization process in the preparation method of the present invention.

[0051] Figure 6 The invention relates to the determination and optimization of the time in the enzyme immobilization process in the preparation method of the present invention.

[0052] Figure 7 This is a scanning electron microscope image of the SNF prepared in the preparation method of the present invention.

[0053] Figure 8 This is a transmission electron microscope image of the SNF prepared in the preparation method of the present invention.

[0054] Figure 9-1 and Figure 9-2 They are respectively the scanning transmission high-angle annular dark field and the corresponding mapping image of the SNF prepared in the preparation method of the present invention.

[0055] Figure 10-1 and Figure 10-2 are the N 2 Adsorption–desorption curves and pore size distribution diagrams.

[0056] Fig.11 This is the laser confocal analysis of the immobilized enzyme.

[0057] Fig.12 The invention relates to the determination and optimization of the temperature during the immobilized enzyme catalytic reaction in the preparation method of the present invention.

[0058] Fig.13 The invention relates to the determination and optimization of the pH value during the immobilized enzyme catalytic reaction in the preparation method of the present invention.

[0059] Fig.14 The figure is a comparison of the thermal stability of the free enzyme and the immobilized enzyme prepared by the present invention at 30°C.

[0060] Fig.15 The figure is a comparison of the thermal stability of the free enzyme and the immobilized enzyme prepared by the present invention at 70°C.

[0061] Fig.16 The figure is a comparison of the pH stability of the free enzyme and the immobilized enzyme prepared by the present invention under the condition of pH=5.

[0062] Fig.17 The figure is a comparison of the pH stability of the free enzyme and the immobilized enzyme prepared by the present invention under the condition of pH=9.

[0063] Fig.18 The storage stability of the immobilized enzyme prepared by the present invention.

[0064] Fig.19The immobilized enzyme prepared by the present invention is reusable.

[0065] Fig. 20 The value is the conversion rate of fructose to psicose catalyzed by the immobilized enzyme prepared by the present invention. DETAILED DESCRIPTION

[0066] The design concept of a novel immobilized D-psicose-3-epimerase proposed by the present invention is: using D-psicose-3-epimerase as enzyme protein and inorganic silicon nanoflower (SNF) as immobilization carrier. It mainly includes: firstly aminating the SNF with 3-aminopropyltriethoxysilane, then adding crosslinking agent glutaraldehyde for activation, and finally adding D-psicose-3-epimerase solution to solidify and react in a shaker for a period of time, centrifuging and washing the reactant, and the obtained product is immobilized D-psicose-3-epimerase. The immobilized D-psicose-3-epimerase has a spherical shape with a diameter of 300nm±10nm, and the sphere has pores, and the D-psicose-3-epimerase is fixed in the pores. The preparation method of the present invention gives full play to the advantages of SNF three-dimensional nanomaterials such as multiple pores, high specific surface area, and good chemical and thermal stability, so that the prepared immobilized enzyme has a stable nanostructure, can fix more enzyme proteins, so that the unit mass of the immobilized enzyme has higher enzymatic activity, and compared with the free enzyme, the immobilized enzyme prepared by the present invention has better thermal stability, pH stability, storage stability and reusability.

[0067] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the following embodiments are by no means intended to limit the present invention in any way.

[0068] Example 1: Preparation of immobilized D-psicose-3-epimerase, comprising the following steps:

[0069] 1) Preparation of SNF: 1 g of hexadecyltrimethylammonium bromide (CTAB), 1 g of n-butanol, and 30 g of 0.4 M urea solution were added to a round-bottom flask and stirred until clear; 12 g of cyclohexane was added and ultrasonically stirred until the mixed solution became clear, then transferred to an oil bath and slightly stirred, 2 g of tetraethyl orthosilicate (TEOS) was added dropwise with a syringe, and then the bottle mouth was sealed, the temperature was raised to 70°C, and stirring was continued for 20 h in a sealed environment. The mixed solution was centrifuged at 10,000 rpm for 5 min to obtain a white precipitate, which was placed in a tubular calcining furnace and calcined at 550°C for 5 h. Air was passed throughout the calcination process, and the calcined product was SNF.

[0070] 2) Amino modification of SNF: 0.29 g of SNF was placed in a 250 mL round-bottom flask, dispersed in 100 mL of n-hexane and subjected to ultrasonic oscillation for 30 min. Then, 1 mL of 3-aminopropyltriethoxysilane (APTES) was added. The round-bottom flask was then placed in a water bath and refluxed at 80 °C for 12 h. The resulting product was recorded as amino nanoflowers and NH 2 -SNF, the product was separated by centrifugation at 8000 rpm for 5 min, washed several times with anhydrous ethanol, and dried in an oven at 60°C for 12 h.

[0071] 3) Activation of NH by glutaraldehyde 2 -SNF: 50 mg NH 2 -SNF was added into 20 mL of glutaraldehyde solution with a final concentration of 1.5%, and the mixed solution was reacted for 4 h at room temperature and 220 rpm on a shaker. The precipitate was separated by filtration and washed three times with pure water.

[0072] 4) Preparation of immobilized D-psicose-3-isomerase (DAEase@SNF): 10 mg of glutaraldehyde-modified NH 2 -SNF was dispersed in 3 mL of PBS buffer solution (100 mM, pH = 7.5) and evenly dispersed by ultrasonication. Then, 2 mL of D-psicose-3-isomerase (DAEase) solution with a protein concentration of 12.5 mg / mL was added to the mixed solution. The mixed solution was then placed on a shaker and reacted at 220 rpm at 40 ° C for 3 h. After the reaction was completed, centrifuged at 8000 rpm for 5 min to obtain a reddish-brown precipitate. The reddish-brown precipitate was washed three times with PBS buffer solution and freeze-dried for 12 h. The resulting product was immobilized D-psicose-3-isomerase, recorded as DAEase@SNF.

[0073] Example 2: Determination of catalytic activity

[0074] The method for determining the catalytic activity in the present invention is to determine the catalytic activity of the free enzyme and the immobilized D-psicose-3-epimerase DAEase@SNF prepared in Example 1 based on the amount of psicose produced per unit time. One enzyme activity unit (U) is defined as the amount of enzyme that produces 1 μmoL of psicose per unit time under the conditions of the optimal pH (7.5 for both the free enzyme and the immobilized enzyme) and 55°C. The standard reaction system is 2 mL, including 40 μL of DAEase with a concentration of 1 mg / mL or DAEase@SNF with an equivalent enzyme amount, 1 mL of 200 g / L fructose and 960 μL of PBS buffer solution (100 mM, pH = 7.5). The mixture is mixed evenly and incubated in a 55°C water bath for 10 min. The reaction is accurately timed in a 55°C water bath for 10 min. After the reaction is completed, it is immediately transferred to a 100°C dry thermostat and heated for 5 min to terminate the reaction. After centrifugation, 1 mL of the supernatant is filtered using an organic filter membrane with a pore size of 0.22 μm and detected using liquid phase detection.

[0075] The catalytic activity of DAEase@SNF in Example 1 was measured and its specific enzyme activity was 1033.09 U / g 蛋白 .

[0076] Example 3: Optimization of the cross-linking agent glutaraldehyde concentration for immobilized D-psicose-3-epimerase

[0077] The preparation process is basically the same as that of Example 1, except that in step 3) glutaraldehyde is used to activate NH 2 In the -SNF step, seven DAEase@SNF samples with different final concentrations of glutaraldehyde solution were prepared, namely, 7 portions of 50 mg NH 2 -SNF was added to 20 mL of glutaraldehyde solution with final concentrations of 0.5%, 1.0%, 1.5%, 2.5%, 3%, 4%, and 6%, respectively, and the mixed solution was reacted for 4 h at room temperature and 220 rpm on a shaker, and the precipitate was separated by filtration and washed three times with pure water. The catalytic activity of the 7 DAEase@SNF samples finally obtained was determined by the catalytic activity determination method in Example 2.

[0078] The catalytic activities of the seven DAEase@SNF samples prepared in Example 3 are shown in Table 1. Figure 1 As shown, when the final concentration of the cross-linking agent glutaraldehyde was 1.5%, the relative enzyme activity of DAEase@SNF was the highest and the immobilization effect was the best.

[0079] Example 4: Optimization of cross-linking time of immobilized D-psicose-3-epimerase

[0080] The preparation process is basically the same as that of Example 1, except that in step 3) glutaraldehyde is used to activate NH 2 -In the SNF step, select NH2 The mixed solution of -SNF and glutaraldehyde was shaken at room temperature for different periods of time, and finally 7 samples of DAEase@SNF were prepared, that is, 7 portions of 50 mg NH 2 -SNF was added to 20 mL of glutaraldehyde solution with a final concentration of 1.5%, and the mixed solution was shaken at room temperature and 220 rpm on a shaker for 0.5 h, 1.5 h, 2.5 h, 3 h, 4 h, 4.5 h, and 5 h, respectively. The catalytic activity of the 7 DAEase@SNF samples finally obtained was determined by the catalytic activity determination method in Example 2, so that the highest enzyme activity was determined as 100%, and the relative enzyme activity was calculated.

[0081] The catalytic activities of the seven DAEase@SNF samples prepared in Example 4 are as follows: Figure 2 As shown in the figure, when the cross-linking time was 4 h, the relative enzyme activity of DAEase@SNF was the highest and the immobilization effect was the best.

[0082] Example 5: Optimization of immobilized D-psicose-3-epimerase enzyme protein concentration

[0083] The preparation process is basically the same as that of Example 1, except that in step 4) the preparation step of DAEase@SNF) is to add NH 2 -SNF and PBS buffer solution, 2 mL of DAEase enzyme solution with different protein concentrations was added to prepare 5 DAEase@SNF samples. 2 -SNF, each was dispersed into 3mL of PBS buffer solution (100mM, pH=7.5), ultrasonically dispersed evenly, and then 2mL of D-psicose-3-isomerase (DAEase) enzyme solution was added to the mixed solution; in each sample, the final concentration of the mixed solution enzyme protein was 2.5mg / mL, 5.0mg / mL, 7.5mg / mL, 10.0mg / mL, and 15.0mg / mL, and finally 5 DAEase@SNF samples were obtained. The catalytic activity was determined by the catalytic activity determination method in Example 2, so that the highest enzyme activity was determined to be 100%, and the relative enzyme activity was calculated.

[0084] The catalytic activities of the five DAEase@SNF samples prepared in Example 5 are shown in Figure 3 As shown in the figure, when the immobilized enzyme protein concentration was 5.0 mg / mL, the relative enzyme activity of DAEase@SNF was the highest and the immobilization effect was the best.

[0085] Example 6: Optimization of the immobilization temperature of immobilized D-psicose-3-epimerase

[0086] The preparation process was basically the same as that of Example 1, except that in step 4) DAEase@SNF preparation, the mixed solution with enzyme solution was reacted on a shaker at 220 rpm at different immobilization temperatures for 3 h, and finally 8 DAEase@SNF samples were prepared. That is, 8 portions of 10 mg glutaraldehyde-modified NH 2 -SNF was dispersed into 3 mL of PBS buffer solution (100 mM, pH = 7.5), and ultrasonically dispersed uniformly. Then, 2 mL of D-psicose-3-isomerase (DAEase) enzyme solution with a protein concentration of 12.5 mg / mL was added to the mixed solution, and then the mixed solution was placed on a shaker at 220 rpm at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, and 55°C for 3 h, and finally 8 DAEase@SNF samples were obtained. The catalytic activity was determined by the catalytic activity determination method in Example 2, so that the highest enzyme activity was determined to be 100%, and the relative enzyme activity was calculated.

[0087] The catalytic activities of the eight DAEase@SNF samples prepared in this example are shown in Figure 2. Figure 4 As shown in the figure, when the immobilization temperature was 40 °C, the relative enzyme activity of DAEase@SNF was the highest and the immobilization effect was the best.

[0088] Example 7: Optimization of pH value for immobilization of immobilized D-psicose-3-epimerase

[0089] The preparation process is basically the same as that of Example 1, except that in step 4) the preparation of DAEase@SNF), NH 2 -SNF was dispersed in 3 mL of PBS buffer solution with different concentrations and pH values. After adding the enzyme solution, the final pH value of the mixed solution was controlled to prepare 6 DAEase@SNF samples. 2 -SNF was dispersed into 3 mL of PBS buffer solution with pH values ​​of 6.0, 7.0, 8.0, 8.5, 9.0, and 10.0, and finally 6 DAEase@SNF samples were obtained. The catalytic activity was determined by the catalytic activity determination method in Example 2, and the highest enzyme activity was determined as 100%, and the relative enzyme activity was calculated.

[0090] The catalytic activities of the six DAEase@SNF samples prepared in this example are shown in Figure 2. Figure 5 As shown, when the immobilization pH = 8.5, the relative enzyme activity of DAEase@SNF is the highest and the immobilization effect is the best.

[0091] Example 8: Optimization of immobilization time of immobilized D-psicose-3-epimerase

[0092] The preparation process is basically the same as that of Example 1, except that in step 4) DAEase@SNF preparation, the mixed solution with enzyme solution is placed on a shaker at 220 rpm at 40°C, and different curing reaction times are selected to finally prepare 6 DAEase@SNF samples. That is, 6 portions of 10 mg glutaraldehyde-modified NH 2 -SNF were dispersed into 3mL of PBS buffer solution (100mM, pH=7.5), ultrasonically dispersed evenly, and then 2mL of D-psicose-3-isomerase (DAEase) enzyme solution with a protein concentration of 12.5mg / mL was added to the mixed solution, and then the mixed solution was placed on a shaker at 220rpm and reacted at 40°C for 0.5h, 1.0h, 1.5h, 2.0h, 2.5h, 3.0h; finally, 6 DAEase@SNF samples were obtained. The catalytic activity was determined by the catalytic activity determination method in Example 2, so that the highest enzyme activity was determined to be 100%, and the relative enzyme activity was calculated.

[0093] The catalytic activities of the six DAEase@SNF samples prepared in this example are shown in Figure 2. Figure 6 As shown in the figure, when the immobilization time was 3.0 h, the relative enzyme activity of DAEase@SNF was the highest and the immobilization effect was the best.

[0094] Example 9: Characterization of SNF and DAEase@SNF

[0095] Scanning electron microscope (SEM), transmission electron microscope (TEM), N 2 SNFs were characterized by adsorption-desorption and confocal laser scanning microscopy (CLSM).

[0096] Figure 7 This is a scanning electron microscope image of the inorganic silicon nanoflower (SNF) prepared in Example 1. It can be seen that the SNF surface is rough and porous, has an obvious flower-like structure, and the particle size is about 340 nm.

[0097] Figure 8This is a transmission electron micrograph of the inorganic silicon nanoflower (SNF) prepared in Example 1. The morphology of the SNF was analyzed using a transmission electron microscope, showing that the SNF has a radial pore structure leading to the center. This structure greatly increases the specific surface area of ​​the material, provides a large number of attachment sites for the enzyme, and improves the mass transfer efficiency.

[0098] FIG9 is a scanning transmission high-angle annular dark field and corresponding mapping image of the inorganic silicon nanoflower (SNF) prepared in Example 1, wherein: Figure 9-1 It is to scan and transmit high-angle annular dark field, and further detect the elements of SNF to obtain the corresponding mapping image; Figure 9-2 It shows that C, N, O and Si elements are evenly distributed in the SNF material, confirming the successful preparation of inorganic silicon nanoflowers.

[0099] Figure 10-1 and Figure 10-2 are respectively N and N of the immobilized enzyme prepared in Example 1. 2 Adsorption-desorption curve and pore size distribution diagram 10-1 show that the SNF prepared in Example 1 has a typical IV type adsorption-desorption isotherm, proving that SNF is a typical mesoporous material. Figure 10-2 The average pore size of SNF pores is 10-15 nm. The BET and BJH results show that the specific surface area and pore volume of SNF are 587.0 m 2 / g and 1.78cm 3 / g, indicating that SNF has a large specific surface area and pore volume, which helps to improve the fixation efficiency and reduce the diffusion limitation of substrates and products during the catalytic process. 2 -The specific surface area and pore volume of SNF are 159.9 m 2 / g and 0.99cm 3 / g. Compared with SNF, NH 2 -The specific surface area and pore volume of SNF were reduced to a certain extent, proving that the pore structure of SNF was covered to a certain extent, which also indirectly confirmed the successful amination of SNF. The specific surface area and pore volume of DAEase@SNF prepared in Example 1 were 99.5 m 2 / g and 0.62cm 3 / g, compared with NH 2 -SNF, the specific surface area and pore volume of DAEase@SNF are reduced to a certain extent, proving that the pore structure of SNF is covered to a certain extent, which also indirectly confirms the successful preparation of DAEase@SNF.

[0100] Fig.11This is a confocal laser analysis of the immobilized enzyme prepared in Example 1. DAEase was fluorescently labeled with rhodamine B. Immobilized enzymes were prepared using fluorescently labeled proteins, and DAEase@SNF was observed under a confocal laser microscope. Under the action of excitation light of a specific wavelength, the fluorescently labeled enzyme molecules appeared red. This proves that DAEase@SNF was successfully immobilized on the carrier.

[0101] Example 10: Effect of reaction temperature on the DAEase@SNF enzyme activity of Example 1

[0102] Under the condition of pH=7.5, the activity of DAEase@SNF in the temperature range of 30-80°C was determined using a standard reaction system (the standard reaction system is the same as that in Example 2), and the relative enzyme activity was calculated with the enzyme activity at the optimum temperature as 100%. Fig.12 As shown in the figure, the optimal reaction temperature of DAEase@SNF is 55℃.

[0103] Example 11: Effect of reaction pH on the DAEase@SNF enzyme activity of Example 1

[0104] At 55°C, the activity of DAEase@SNF in the pH range of pH = 4-10 was measured using a standard reaction system (the standard reaction system is the same as in Example 2), and the relative enzyme activity was calculated with the enzyme activity at the optimum pH as 100%. Fig.13 As shown, the optimal reaction pH of DAEase@SNF is 7.5.

[0105] Example 12: Stability of DAEase and DAEase@SNF at 30°C

[0106] DAEase and DAEase@SNF of Example 1 were prepared into solutions using a pH 7.5 buffer solution, and the solutions were placed in a 30°C water bath. Samples were taken at regular intervals, and the activities of DAEase and DAEase@SNF were measured using a standard reaction system (the standard reaction system was the same as that of Example 2). The relative enzyme activity was calculated with the enzyme activity without heat treatment as 100%. Fig.14 As shown in the figure, DAEase@SNF has good thermal stability, and after incubation at 30℃ for 6h, it still retains 90.09% of the initial activity, while the free enzyme activity is only 55.24%. The stability experiment at 30℃ was repeated with DAEase@SNF activated with 4% final concentration of glutaraldehyde in Example 3, and it was measured that the initial activity retained by DAEase@SNF after incubation at 30℃ for 6h was 5.18%.

[0107] Example 13: Stability of DAEase and DAEase@SNF at 70°C

[0108] DAEase and DAEase@SNF of Example 1 were prepared into solutions using a buffer solution of pH=7.5, and the solutions were placed in a 70°C water bath. Samples were taken at regular intervals, and the activities of DAEase and DAEase@SNF were measured using a standard reaction system (the standard reaction system was the same as that of Example 2). The relative enzyme activity was calculated with the enzyme activity without heat treatment as 100%. The results are shown in Fig.15 As shown in the figure, DAEase@SNF has good thermal stability, and after incubation at 70℃ for 6h, it still retains 9.72% of the initial activity, while the free enzyme activity is 0%. The stability experiment at 70℃ was repeated with DAEase@SNF activated with 4% final concentration of glutaraldehyde in Example 3, and it was measured that the initial activity of DAEase@SNF retained after incubation at 70℃ for 6h was 82.56%.

[0109] Example 14: Stability of DAEase and DAEase@SNF at pH = 5.0

[0110] DAEase and DAEase@SNF of Example 1 were prepared into solutions using a pH 5.0 buffer solution, and the solutions were stored at 4°C. Samples were taken at regular intervals, and the activities of DAEase and DAEase@SNF were measured using a standard reaction system (the standard reaction system was the same as that of Example 2). The relative enzyme activity was calculated with the enzyme activity without acidic environment treatment as 100%. Fig.16 As shown, DAEase@SNF has good pH stability, and after incubation at pH = 5.0 for 6 hours, it still retains 88.28% of the initial activity, while the free enzyme activity is 48.63%. The DAEase@SNF activated with 4% final concentration of glutaraldehyde in Example 3 was used to repeat the stability experiment at pH = 5.0, and it was measured that the DAEase@SNF retained 75.63% of the initial activity after incubation at pH = 5.0 for 6 hours.

[0111] Example 15: Stability of DAEase and DAEase@SNF at pH = 9.0

[0112] DAEase and DAEase@SNF of Example 1 were prepared into solutions using a pH 9.0 buffer solution, and the solutions were stored at 4°C. Samples were taken at regular intervals, and the activities of DAEase and DAEase@SNF were measured using a standard reaction system (the standard reaction system was the same as that of Example 2). The relative enzyme activity was calculated with the enzyme activity without alkaline environment treatment as 100%. Fig.17As shown, DAEase@SNF has good pH stability, and after incubation for 6 hours at pH = 5.0, it still retains 97.86% of the initial activity, while the free enzyme activity is 69.56%. The stability experiment at pH = 9.0 was repeated with DAEase@SNF activated with 4% final concentration of glutaraldehyde in Example 3, and it was measured that the initial activity of DAEase@SNF after incubation for 6 hours at pH = 9.0 was 94.73%.

[0113] Example 16: Storage stability of DAEase@SNF

[0114] The DAEase@SNF of Example 1 was stored at 4°C. An equal amount of DAEase@SNF was taken every other day and stored at 55°C, pH=7.5 in a standard reaction system (the standard reaction system is the same as that of Example 2) to detect the activity of DAEase@SNF. The above operation was repeated for 15 days. The catalytic activity after each day's reaction was recorded. The enzyme activity of the first day's reaction was taken as 100%, and the relative enzyme activity of the subsequent reaction process was calculated to determine the storage stability of DAEase@SNF. The results are shown in FIG. Fig.18 As shown, DAEase@SNF has good storage stability, and still retains 54.38% of the initial activity after storage for 15 days. The storage stability experiment was repeated with DAEase@SNF activated with 4% final concentration of glutaraldehyde in Example 3, and it was measured that the initial activity retained by DAEase@SNF after storage for 15 days was 46.27%.

[0115] Example 17: Number of times DAEase@SNF is reused

[0116] At 55°C and pH=7.5, the activity of DAEase@SNF of Example 1 was detected in a standard reaction system (the standard reaction system is the same as that of Example 2). After the reaction was completed, the immobilized enzyme was separated from the reaction solution by centrifugation at 10,000 rpm for 5 minutes. The reaction was then washed once with ultrapure water and the above reaction process was repeated again. The catalytic activity after each reaction was recorded, and the enzyme activity of the first reaction was taken as 100%. The relative enzyme activity of the subsequent reaction process was calculated to determine the reuse rate of DAEase@SNF. The results are shown in FIG. Fig.19 As shown, DAEase@SNF has good reusability, and after being reused 10 times, it still retains 70.78% of the initial activity. The DAEase@SNF activated with 4% final concentration of glutaraldehyde in Example 3 was repeatedly used for several times, and it was measured that the initial activity retained by the DAEase@SNF after being reused 10 times was 70.78%.

[0117] Example 18: Immobilized enzyme conversion capacity of DAEase@SNF catalyzing fructose to psicose

[0118] 10 mg of DAEase@SNF of Example 1 was added to 2 mL of 100 g / L fructose solution, mixed evenly, and reacted in a 55°C water bath for 2 h. After the reaction was completed, the mixture was immediately transferred to a 100°C dry thermostat and heated for 5 min to terminate the reaction. After centrifugation, 1 mL of the supernatant was filtered with an organic filter membrane with a pore size of 0.22 μm and detected by liquid phase. The above process was repeated, and the reaction time was adjusted to 4, 6, 8, 10, 12, 14, 16, 18, and 20 h, respectively, and then liquid phase detection was used for each.

[0119] Test results such as Fig. 20 As shown in the figure, DAEase@SNF can catalyze the preparation of allulose from fructose. The conversion rate of allulose increases rapidly during the reaction time of 2-10 h. The conversion rate is 31.42% at 12 h and 31.27% at 20 h. The curve is relatively straight. Even if the reaction time is extended, the conversion rate does not change significantly, indicating that the conversion capacity of the immobilized enzyme is stable at around 31%. It also shows that the enzyme has no activity in promoting the reverse reaction.

[0120] Although the present invention has been described above in conjunction with the accompanying drawings, the present invention is not limited to the above-mentioned specific embodiments, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in the field should regard the specification as a whole and make many improvements and changes without departing from the purpose of the present invention. For example, the technical solutions in the various embodiments may also be appropriately combined, all of which are within the protection of the present invention.

Claims

1. An immobilized D-psicose-3-epimerase, characterized in that: D-psicose-3-epimerase is used as the enzyme protein, and inorganic silicon nanoflowers are used as the immobilization carrier; the immobilized D-psicose-3-epimerase is spherical in shape with a diameter of 340nm±15nm, and the sphere has pores, in which the D-psicose-3-epimerase is immobilized.

2. The immobilized D-psicose-3-epimerase according to claim 1, characterized in that: The specific enzyme activity of the immobilized D-psicose-3-epimerase is 446.44-1033.09 U / g 蛋白 ; The immobilized D-psicose-3-epimerase retained 82.56-90.09% of the initial activity after incubation at 30° C. for 6 hours, and the free enzyme activity was 55.24%; after incubation at 70° C. for 6 hours, the immobilized D-psicose-3-epimerase retained 5.18-9.72% of the initial activity, and the free enzyme activity was 0%; The immobilized D-psicose-3-epimerase retains 75.63-88.28% of the initial activity after incubation for 6 hours under pH=5, and the free enzyme activity is 48.63%; and retains 94.73-97.86% of the initial activity after incubation for 6 hours under pH=9, and the free enzyme activity is 69.56%.

3. The immobilized D-psicose-3-epimerase according to claim 1, characterized in that: The immobilized D-psicose-3-epimerase still retains 63.18-70.78% of the initial activity after being reused 10 times, and still retains 46.27-54.38% of the initial activity after being stored for 15 days.

4. The method for preparing the immobilized lipase according to any one of claims 1 to 3, characterized in that: D-psicose-3-epimerase is used as the enzyme protein and inorganic silicon nanoflowers are used as immobilization carriers. The inorganic silicon nanoflowers are firstly aminated with 3-aminopropyltriethoxysilane, and then a cross-linking agent glutaraldehyde is added for activation. Finally, D-psicose-3-epimerase solution is added to solidify and react in a shaker for a period of time, and then the reactant is centrifuged and washed to obtain the immobilized D-psicose-3-epimerase.

5. The method for preparing immobilized lipase according to any one of claim 4, characterized in that: The specific steps are as follows: Step 1) preparing inorganic silicon nanoflowers, comprising: 1-1) mixing hexadecyltrimethylammonium bromide and a urea solution having a concentration of 0.1-1.0M at a mass ratio of 1:15-60, and then adding n-butanol to the mixed solution at a mass ratio of 1:15-60, and stirring until clear, to obtain a mixed solution A; 1-2) adding cyclohexane to the mixed solution A at a mass ratio of 1:1-4, and stirring with ultrasound until clear, to obtain a mixed solution B; 1-3) slowly adding ethyl silicate to the mixed solution B at a mass ratio of 1:10-40, and stirring with ultrasound until it becomes clear, then stirring under sealed conditions for 15-60 minutes, raising the temperature to 60-80° C., and continuing stirring for 12-48 hours to obtain a reaction mixture; 1-4) The reaction mixture is centrifuged to obtain a white precipitate, and the precipitate is placed in a tubular calcining furnace and calcined at 450-650° C. for 3-7 hours. Air is passed throughout the calcination process. The calcined product is the inorganic silicon nanoflower; Step 2) amination of nanoflowers, comprising: 2-1) dispersing the inorganic silicon nanoflowers obtained in step 1) into n-hexane at a mass ratio of 1:100-400, and subjecting to ultrasonic oscillation for 15-60 minutes to obtain a mixed solution C; 2-2) adding 3-aminopropyltriethoxysilane to the mixed solution C at a volume ratio of 1:50-200 to obtain a mixed solution D, and placing the mixed solution D under condensation reflux at 70-90° C. for 6-24 hours; 2-3) The mixed solution D is centrifuged to obtain a white precipitate, the product is separated, washed with anhydrous ethanol for multiple times, and dried in an oven for 6-48 hours to obtain amino nanoflowers; Step 3) activating the amino nanoflowers using glutaraldehyde as a crosslinking agent, comprising: 3-1) adding the amino nanoflowers obtained in step 2) to a glutaraldehyde solution with a final concentration of 0.5-4% at a mass ratio of 1:200-800, and performing a cross-linking reaction on a shaking table at room temperature for 1.5-5 hours to obtain a mixed solution E; 3-2) filtering the mixed solution E to obtain a reddish brown precipitate, and washing it three times with pure water to obtain glutaraldehyde-modified amino nanoflowers; Step 4) preparing immobilized D-psicose-3-epimerase, the steps are as follows: 4-1) dispersing the glutaraldehyde-modified amino nanoflowers obtained in step 3) into a PBS buffer solution at a mass ratio of 1:150-600, and uniformly dispersing by ultrasonication to obtain a mixed solution F; 4-2) adding an enzyme solution having a D-psicose-3-epimerase protein concentration of 2.5-37.5 mg / mL to the mixed solution F to obtain a mixed solution, wherein the volume ratio of the mixed solution F to the enzyme solution is 3:1-4; placing the mixed solution on a shaker for a solidification reaction at 20-55° C. for 0.5-5 h, and after the reaction is completed, centrifuging to obtain a reddish-brown precipitate; 4-3) The red-brown precipitate was washed three times with PBS buffer solution and freeze-dried for 6-24 hours to obtain immobilized D-psicose-3-epimerase.

6. The method for preparing immobilized D-psicose-3-epimerase according to claim 5, characterized in that: In step 1-1), the concentration of the urea solution is 0.4M, the mass ratio of hexadecyltrimethylammonium bromide to the urea solution is 1:30, and the mass ratio of n-butanol added to the mixed solution is 1:31; In step 1-2), the mass ratio of cyclohexane to mixed solution A is 3:8; In step 1-3), the mass ratio of the mixed solution B to ethyl silicate is 22:1, the stirring time under sealed conditions is 30 min, the temperature after stirring is 70° C., and the stirring time after heating is 20 h; In step 1-4), the conditions for centrifuging the reaction mixture are: centrifugation at 10000 rpm for 5 min; the calcination temperature in the tubular calcining furnace is 550° C., and the calcination time is 5 h; In step 2-1), the inorganic silicon nanoflowers are dispersed in n-hexane and subjected to ultrasonic oscillation for 30 minutes, wherein the mass ratio of the inorganic silicon nanoflowers to n-hexane is 1:227; In step 2-2), the volume ratio of the mixed solution C to 3-aminopropyltriethoxysilane is 100:1; the mixed solution D is placed under condensation reflux at 80° C. for 12 hours; In step 2-3), the white precipitate obtained by centrifuging the mixed solution D was dried in an oven at 60° C. for 12 h; In step 3-1), the amino nanoflowers are added to a glutaraldehyde solution with a final concentration of 1.5%, the mass ratio of the amino nanoflowers to the glutaraldehyde solution is 1:400, and the reaction is carried out under shaking conditions for 4 hours; Step 3-2) Filter the mixed solution E to obtain a reddish brown precipitate, and wash it three times with pure water to obtain glutaraldehyde-modified amino nanoflowers; In step 4-1), the concentration of the PBS buffer solution is 100 mM, pH=8.5, and the mass ratio of the glutaraldehyde-modified amino nanoflowers to the PBS buffer solution is 1:300; In step 4-2), the D-psicose-3-epimerase protein concentration of the enzyme solution is 12.5 mg / mL, and the volume ratio of the mixed solution F to the enzyme solution is 3:2; the process conditions of the shaking table are: the rotation speed is 220 rpm, the curing reaction temperature is 40° C., and the curing reaction time is 3 h; after the reaction is completed, the reaction is centrifuged at 8000 rpm for 5 min.

7. Use of the immobilized D-psicose-3-epimerase prepared by the preparation method according to claim 5 or 6 in the preparation of psicose, characterized in that: The immobilized D-psicose-3-epimerase is added to the fructose solution, the reaction mixture is reacted at a constant temperature, and after the reaction is completed, psicose is separated and obtained.

8. Use of the immobilized D-psicose-3-epimerase in the preparation of psicose according to claim 7, characterized in that: The conditions of the isothermal reaction are that the reaction temperature is 45-60°C, the reaction time is 0-20h and ≠0.

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