Application of a rigid carrier-immobilized enzyme in D-tagatose conversion

Abstract

The present invention discloses an application of a rigid carrier-immobilized biological enzyme in D-tagatose conversion, comprising the following steps: S1, placing the immobilized enzyme in a D-galactose aqueous solution; S2, hydrating the boron atoms in the rigid carrier to [B(OH) 3 】or 【B(OH) 4 】 ‑ Complexing D-galactose; S3, isomerization reaction at 60°C to 70°C for 48 hours to 60 hours; S4, after the isomerization is completed, filtering and separating the L-arabinose isomerase immobilized on the rigid carrier to obtain D-tagatose; the present invention utilizes a rigid carrier with a curved surface and a network structure to effectively disperse the L-arabinose isomerase evenly and utilizes the stacking of the rigid carrier to form a spatial channel for accommodating D-galactose, thereby ensuring the effective complexation of D-galactose and promoting the conversion of D-tagatose.

Description

Technical Field

[0001] The invention relates to the technical field of immobilized enzymes, and in particular to application of a rigid carrier-immobilized biological enzyme in D-tagatose conversion. Background Art

[0002] D-tagatose is a rare hexose. In the prior art, there are two methods for preparing tagatose: chemical synthesis and biosynthesis. Among them, biosynthesis is a more commonly used method for converting D-galactose into D-tagatose. L-arabinose isomerase from microorganisms can catalyze the isomerization of D-galactose into D-tagatose. The technical problem is that when L-arabinose isomerase and D-galactose are combined, the catalytic efficiency and conversion efficiency are low due to the poor binding between D-galactose and isomerase. In addition, directly mixing L-arabinose isomerase from microorganisms with D-galactose for isomerization is not conducive to the separation of the two and the reuse of isomerase, which reduces production efficiency and increases production costs. Summary of the invention

[0003] The purpose of the present invention is to provide an application of a rigid carrier to fix a biological enzyme in the conversion of D-tagatose. The present invention uses a rigid carrier with a curved network structure to fix boron. The boron in the rigid carrier hydrolyzes and complexes D-galactose to cooperate with L-arabinose isomerase distributed on the surface of the rigid carrier to promote the effective isomerization. The rigid carrier with a curved surface and a network structure effectively disperses the L-arabinose isomerase evenly and forms a spatial channel for accommodating D-galactose by stacking the rigid carriers, thereby ensuring the effective complexation of D-galactose and promoting the conversion of D-tagatose.

[0004] To solve this technical problem, the technical solution of the present invention is: an application of a rigid carrier-immobilized enzyme in the conversion of D-tagatose, comprising the following steps:

[0005] S1, placing L-arabinose isomerase immobilized on a rigid carrier in a D-galactose aqueous solution having a pH value of 7.5 to 8.5;

[0006] S2. During the isomerization process, the boron atom in the rigid carrier is hydrated to [B(OH)3] or [B(OH)4] - , [B(OH)3] or [B(OH)4] - Complexing D-galactose: D-galactose as a reactant is adsorbed to the vicinity of L-arabinose isomerase on the rigid carrier through complexation;

[0007] S3, isomerization reaction at 60° C. to 70° C. for 48 to 60 hours;

[0008] S4. After the isomerization is completed, the L-arabinose isomerase immobilized on the rigid carrier is filtered and separated to obtain the D-tagatose solution.

[0009] Preferably, the method for preparing the rigid carrier comprises the following steps:

[0010] S11, stirring and mixing micron-sized silica particles, chitosan, boric acid, glutaraldehyde and hexadecyltrimethylammonium bromide, and placing in a reaction kettle for hydrothermal reaction;

[0011] S12, cooling after the hydrothermal reaction is completed, filtering the solid particles, and obtaining a carbon-boron oxide network structure surrounding the growth of the silicon dioxide particles;

[0012] Wash and dry;

[0013] S13, heat treating the carbon-boron oxide network structure surrounding the silicon dioxide particles obtained in S12 under nitrogen protection, so that the boron penetrates into the carbon to form a stable connection;

[0014] S14, placing the carbon-boron oxide network structure formed on the surface of the silica particles and interpenetrating into a stable skeleton in an alkaline solution to remove the silica particles to form a hollow network structure rigid carrier. The present invention effectively disperses boron and L-arabinose isomerase on the surface of the rigid carrier by constructing a hollow boron oxide network structure rigid carrier, thereby promoting the contact between D-galactose and L-arabinose isomerase, thereby improving the catalytic efficiency.

[0015] The silicon dioxide particles of the present invention are nucleation centers, and rigid carriers are formed on the surfaces of the silicon dioxide particles, thereby limiting the distribution of boron oxide particles in shallow positions to a certain extent. The stacking of rigid carrier skeletons forms a three-dimensional spatial connection, and the liquid phase is dispersed in and between the connected spherical carbon skeletons. Boron oxide hydrolyzes and complexes D-galactose to increase the possibility of contact with D-galactose solidifying enzymes. Moreover, due to the stable structure of the rigid carrier skeleton, the immobilization degree of L-arabinose isomerase is effectively increased, and the obtained solidified enzyme can be repeatedly used, and the structure is stable and can be repeatedly used.

[0016] Preferably, L-arabinose isomerase, a rigid carrier and glutaraldehyde are cross-linked in an aqueous solution to obtain an immobilized enzyme. The specific process of the immobilized enzyme of the present invention comprises the following steps:

[0017] Step 1, placing the carbon skeleton carrier having an amino group obtained by hydrothermal reaction in a phosphate buffer in which free L-arabinose isomerase is dispersed, wherein the ratio of the carbon skeleton carrier to the free L-arabinose isomerase is 1 g: (1000 U to 1200 U);

[0018] After being uniformly dispersed and allowed to stand, L-arabinose isomerase was distributed on the surface of the carbon skeleton carrier through the adsorption of the carbon skeleton;

[0019] Step 2: Add glutaraldehyde solution to the mixed system obtained in step 1 to obtain an enzyme solidified material, and place the material system obtained in step 2 on a shaker at 130 rpm to 150 rpm and stir for 6 to 12 hours to perform a solidification reaction; the solidification temperature is 25° C. to 30° C.; the mass ratio of glutaraldehyde to the rigid carrier is (0.1 to 0.2):1;

[0020] Step 3: Filter and separate the solid particles obtained in step 2, wash them with phosphate buffer and then use deionized water to remove the unbound enzyme on the surface of the solid particles, and obtain L-arabinose isomerase immobilized on the carbon skeleton carrier.

[0021] Preferably, the L-arabinose isomerase is produced by biological fermentation and has an enzyme activity of 20,000 U / L to 30,000 U / L.

[0022] Preferably, the activity of the immobilized enzyme is 500U to 600U / g rigid carrier. The present invention effectively improves the enzyme activity of the immobilized enzyme by immobilizing L-arabinose isomerase on a rigid carrier with a three-dimensional structure.

[0023] The process conditions of the preferred S11 hydrothermal reaction are as follows:

[0024] Time 12 to 18 hours;

[0025] Temperature 180℃ to 220℃.

[0026] The present invention utilizes the network structure of chitosan gel to effectively disperse boric acid, and chitosan and boron oxide are mixed in a hydrothermal process to obtain a carbon-boron oxide network structure containing amino groups. Further, a subsequent heat treatment is performed to form a bond between carbon and B, thereby effectively fixing B on a rigid carrier and improving the catalytic stability of the obtained immobilized enzyme.

[0027] Preferably, the particle size of the silica particles is 20 μm to 100 μm;

[0028] The mass ratio of chitosan to boric acid is 1:(0.2 to 0.3);

[0029] The mass ratio of chitosan to glutaraldehyde is 1:(0.2 to 0.3);

[0030] The mass ratio of chitosan to hexadecyltrimethylammonium bromide is 1:(0.08 to 0.15).

[0031] The invention utilizes hexadecyltrimethylammonium bromide to cooperate with glutaraldehyde and chitosan to crosslink, thereby promoting the formation of coating on the surface of silicon dioxide particles, and forms a network skeleton structure in which boron oxide is fixed on carbon with carbonization through hydrothermal reaction.

[0032] Preferably, the temperature of S13 heat treatment is 600° C. to 700° C., and the heat treatment time is 3 hours to 6 hours. The present invention effectively achieves the bonding of BC and the effective fixation of B through heat treatment.

[0033] Preferably, the mass ratio of L-arabinose isomerase immobilized on the rigid carrier to D-galactose is (0.1 to 0.2):1.

[0034] By adopting the above technical solution, the beneficial effects of the present invention are:

[0035] The present invention proposes an application of a rigid carrier-immobilized biological enzyme in the conversion of D-tagatose. The rigid carrier-immobilized biological enzyme has a stable structure in a reaction solution with a pH value of 7.5 to 8.5 and can stably catalyze for 48 to 60 hours. During the catalytic process, the boron atoms in the rigid carrier are hydrated to [B(OH)3] or [B(OH)4]. - , [B(OH)3] or [B(OH)4] - The complexed D-galactose adsorbs D-galactose as a reactant to the vicinity of the L-arabinose isomerase on the rigid carrier through complexation to undergo an isomerization reaction at 60° C. to 70° C. After the isomerization is completed, the L-arabinose isomerase immobilized on the rigid carrier is filtered and separated to obtain a D-tagatose solution, and the D-tagatose is further concentrated and purified to obtain crystals of the target product. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a liquid chromatogram after isomerization in Example 2 of the present invention;

[0037] Figure 2 It is the liquid chromatogram after isomerization of the comparative example of the present invention. DETAILED DESCRIPTION

[0038] In order to further explain the technical solution of the present invention, the present invention is described in detail below through specific embodiments.

[0039] Example 1

[0040] This embodiment discloses a method for immobilizing a biological enzyme on a rigid carrier, wherein L-arabinose isomerase, a rigid carrier and glutaraldehyde are cross-linked in an aqueous solution to obtain an immobilized enzyme.

[0041] The specific process of the immobilized enzyme of the present invention comprises the following steps:

[0042] Step 1, placing the carbon skeleton carrier having an amino group obtained by hydrothermal reaction in a phosphate buffer in which free L-arabinose isomerase is dispersed, wherein the ratio of the carbon skeleton carrier to the free L-arabinose isomerase is 1 g: 1000 U;

[0043] After being uniformly dispersed and allowed to stand, L-arabinose isomerase was distributed on the surface of the carbon skeleton carrier through the adsorption of the carbon skeleton;

[0044] Step 2: Add glutaraldehyde solution to the mixed system obtained in step 1 to obtain enzyme solidified material, and place the material system obtained in step 2 on a shaker at 150 rpm for 12 hours to perform solidification reaction; the solidification temperature is 25° C.;

[0045] The volume concentration of glutaraldehyde used for cross-linking and solidifying L-arabinose isomerase was 1.0%;

[0046] Step 3: Filter and separate the solid particles obtained in step 2, wash them with phosphate buffer and then use deionized water to remove the unbound enzyme on the surface of the solid particles, and obtain L-arabinose isomerase immobilized on the carbon skeleton carrier.

[0047] The L-arabinose isomerase used in this example was prepared by biological fermentation, and the enzyme activity of L-arabinose isomerase per liter of fermentation broth was 22359 U using the carbazole method. The enzyme activity unit U is calculated as follows: Under the action of L-arabinose isomerase, the amount of enzyme that converts D-galactose to produce 1 μg of D-tagatose per minute is 1 unit of L-arabinose isomerase.

[0048] The activity of the immobilized enzyme in this example is 502 U / g rigid carrier. The present invention effectively improves the enzyme activity of the immobilized enzyme by immobilizing L-arabinose isomerase on a rigid carrier with a three-dimensional structure.

[0049] The method for preparing the rigid carrier described in this embodiment comprises the following steps:

[0050] S11, stirring and mixing micron-sized silica particles, chitosan, boric acid, glutaraldehyde and hexadecyltrimethylammonium bromide, the specific amounts of which are shown in Table 1, and placing in a reactor for hydrothermal reaction;

[0051] The process conditions of the S11 hydrothermal reaction in this embodiment are as follows:

[0052] Time: 18 hours; temperature: 180°C.

[0053] The present invention utilizes the network structure of chitosan gel to effectively disperse boric acid, and chitosan and boron oxide are mixed in a hydrothermal process to obtain a carbon-boron oxide network structure containing amino groups. Further, a subsequent heat treatment is performed to form a bond between carbon and B, thereby effectively fixing B on a rigid carrier and improving the catalytic stability of the obtained immobilized enzyme.

[0054] The invention utilizes hexadecyltrimethylammonium bromide to cooperate with glutaraldehyde and chitosan to crosslink, thereby promoting the formation of coating on the surface of silicon dioxide particles, and forms a network skeleton structure in which boron oxide is fixed on carbon with carbonization through hydrothermal reaction.

[0055] S12, cooling after the hydrothermal reaction is completed, filtering the solid particles, and obtaining a carbon-boron oxide network structure surrounding the growth of the silicon dioxide particles;

[0056] Wash and dry;

[0057] S13, heat treating the carbon-boron oxide network structure surrounding the silicon dioxide particles obtained in S12 under nitrogen protection, so that the boron penetrates into the carbon to form a stable connection;

[0058] In this embodiment, the temperature of the heat treatment in S13 is 600° C. and the heat treatment time is 6 hours. The present invention effectively achieves the bonding of BC and the effective fixation of B through heat treatment.

[0059] S14, placing the carbon-boron oxide network structure formed on the surface of the silica particles and interpenetrating into a stable skeleton in an alkaline solution to remove the silica particles to form a hollow network structure rigid carrier. The present invention effectively disperses boron and L-arabinose isomerase on the surface of the rigid carrier by constructing a hollow boron oxide network structure rigid carrier, thereby promoting the contact between D-galactose and L-arabinose isomerase, thereby improving the catalytic efficiency.

[0060] Example 2

[0061] This embodiment discloses a method for immobilizing a biological enzyme on a rigid carrier, wherein L-arabinose isomerase, a rigid carrier and glutaraldehyde are cross-linked in an aqueous solution to obtain an immobilized enzyme.

[0062] The specific process of the immobilized enzyme of the present invention comprises the following steps:

[0063] Step 1, placing the carbon skeleton carrier having an amino group obtained by hydrothermal reaction in a phosphate buffer in which free L-arabinose isomerase is dispersed, wherein the ratio of the carbon skeleton carrier to the free L-arabinose isomerase is 1 g: 1000 U;

[0064] After being uniformly dispersed and allowed to stand, L-arabinose isomerase was distributed on the surface of the carbon skeleton carrier through the adsorption of the carbon skeleton;

[0065] Step 2: Add glutaraldehyde solution to the mixed system obtained in step 1 to obtain enzyme solidified material, and place the material system obtained in step 2 on a shaker at 150 rpm for 12 hours to perform solidification reaction; the solidification temperature is 25° C.;

[0066] The volume concentration of glutaraldehyde used for cross-linking and solidifying L-arabinose isomerase was 1.0%;

[0067] Step 3: Filter and separate the solid particles obtained in step 2, wash them with phosphate buffer and then use deionized water to remove the unbound enzyme on the surface of the solid particles, and obtain L-arabinose isomerase immobilized on the carbon skeleton carrier.

[0068] The L-arabinose isomerase used in this example was prepared by biological fermentation, and the enzyme activity of L-arabinose isomerase per liter of fermentation broth was 22359 U using the carbazole method. The enzyme activity unit U is calculated as follows: Under the action of L-arabinose isomerase, the amount of enzyme that converts D-galactose to produce 1 μg of D-tagatose per minute is 1 unit of L-arabinose isomerase.

[0069] The activity of the immobilized enzyme in this example is 568 U / g rigid carrier. The present invention effectively improves the enzyme activity of the immobilized enzyme by immobilizing L-arabinose isomerase on a rigid carrier with a three-dimensional structure.

[0070] The method for preparing the rigid carrier described in this embodiment comprises the following steps:

[0071] S11, stirring and mixing micron-sized silica particles, chitosan, boric acid, glutaraldehyde and hexadecyltrimethylammonium bromide, the specific amounts of which are shown in Table 1, and placing in a reactor for hydrothermal reaction;

[0072] The process conditions of the S11 hydrothermal reaction in this embodiment are as follows:

[0073] Time: 12 hours; temperature: 200°C.

[0074] The present invention utilizes the network structure of chitosan gel to effectively disperse boric acid, and chitosan and boron oxide are mixed in a hydrothermal process to obtain a carbon-boron oxide network structure containing amino groups. Further, a subsequent heat treatment is performed to form a bond between carbon and B, thereby effectively fixing B on a rigid carrier and improving the catalytic stability of the obtained immobilized enzyme.

[0075] The invention utilizes hexadecyltrimethylammonium bromide to cooperate with glutaraldehyde and chitosan to crosslink, thereby promoting the formation of coating on the surface of silicon dioxide particles, and forms a network skeleton structure in which boron oxide is fixed on carbon with carbonization through hydrothermal reaction.

[0076] S12, cooling after the hydrothermal reaction is completed, filtering the solid particles, and obtaining a carbon-boron oxide network structure surrounding the growth of the silicon dioxide particles;

[0077] Wash and dry;

[0078] S13, heat treating the carbon-boron oxide network structure surrounding the silicon dioxide particles obtained in S12 under nitrogen protection, so that the boron penetrates into the carbon to form a stable connection;

[0079] In this embodiment, the temperature of the heat treatment in S13 is 700° C. and the heat treatment time is 6 hours. The present invention effectively achieves the bonding of BC and the effective fixation of B through heat treatment.

[0080] S14, placing the carbon-boron oxide network structure formed on the surface of the silica particles and interpenetrating into a stable skeleton in an alkaline solution to remove the silica particles to form a hollow network structure rigid carrier. The present invention effectively disperses boron and L-arabinose isomerase on the surface of the rigid carrier by constructing a hollow boron oxide network structure rigid carrier, thereby promoting the contact between D-galactose and L-arabinose isomerase, thereby improving the catalytic efficiency.

[0081] Example 3

[0082] This embodiment discloses a method for immobilizing a biological enzyme on a rigid carrier, wherein L-arabinose isomerase, a rigid carrier and glutaraldehyde are cross-linked in an aqueous solution to obtain an immobilized enzyme.

[0083] The specific process of the immobilized enzyme of the present invention comprises the following steps:

[0084] Step 1, placing the carbon skeleton carrier having an amino group obtained by hydrothermal reaction in a phosphate buffer dispersed with free L-arabinose isomerase, wherein the ratio of the carbon skeleton carrier to the free L-arabinose isomerase is 1 g: 1200 U;

[0085] After being uniformly dispersed and allowed to stand, L-arabinose isomerase was distributed on the surface of the carbon skeleton carrier through the adsorption of the carbon skeleton;

[0086] Step 2: Add glutaraldehyde solution to the mixed system obtained in step 1 to obtain enzyme solidified material, and place the material system obtained in step 2 on a shaker at 150 rpm for 12 hours to perform solidification reaction; the solidification temperature is 25° C.;

[0087] The volume concentration of glutaraldehyde used for cross-linking and solidifying L-arabinose isomerase was 1.0%;

[0088] Step 3: Filter and separate the solid particles obtained in step 2, wash them with phosphate buffer and then use deionized water to remove the unbound enzyme on the surface of the solid particles, and obtain L-arabinose isomerase immobilized on the carbon skeleton carrier.

[0089] The L-arabinose isomerase used in this example was prepared by biological fermentation, and the enzyme activity of L-arabinose isomerase per liter of fermentation broth was 22359 U using the carbazole method. The enzyme activity unit U is calculated as follows: Under the action of L-arabinose isomerase, the amount of enzyme that converts D-galactose to produce 1 μg of D-tagatose per minute is 1 unit of L-arabinose isomerase.

[0090] The activity of the immobilized enzyme in this example is 589 U / g rigid carrier. The present invention effectively improves the enzyme activity of the immobilized enzyme by immobilizing L-arabinose isomerase on a rigid carrier with a three-dimensional structure.

[0091] The method for preparing the rigid carrier described in this embodiment comprises the following steps:

[0092] S11, stirring and mixing micron-sized silica particles, chitosan, boric acid, glutaraldehyde and hexadecyltrimethylammonium bromide, the specific amounts of which are shown in Table 1, and placing in a reactor for hydrothermal reaction;

[0093] The process conditions of the S11 hydrothermal reaction in this embodiment are as follows:

[0094] Time: 18 hours; temperature: 220°C.

[0095] The present invention utilizes the network structure of chitosan gel to effectively disperse boric acid, and chitosan and boron oxide are mixed in a hydrothermal process to obtain a carbon-boron oxide network structure containing amino groups. Further, a subsequent heat treatment is performed to form a bond between carbon and B, thereby effectively fixing B on a rigid carrier and improving the catalytic stability of the obtained immobilized enzyme.

[0096] The invention utilizes hexadecyltrimethylammonium bromide to cooperate with glutaraldehyde and chitosan to crosslink, thereby promoting the formation of coating on the surface of silicon dioxide particles, and forms a network skeleton structure in which boron oxide is fixed on carbon with carbonization through hydrothermal reaction.

[0097] S12, cooling after the hydrothermal reaction is completed, filtering the solid particles, and obtaining a carbon-boron oxide network structure surrounding the growth of the silicon dioxide particles;

[0098] Wash and dry;

[0099] S13, heat treating the carbon-boron oxide network structure surrounding the silicon dioxide particles obtained in S12 under nitrogen protection, so that the boron penetrates into the carbon to form a stable connection;

[0100] In this embodiment, the temperature of the heat treatment in S13 is 700° C. and the heat treatment time is 6 hours. The present invention effectively achieves the bonding of BC and the effective fixation of B through heat treatment.

[0101] S14, placing the carbon-boron oxide network structure formed on the surface of the silica particles and interpenetrating into a stable skeleton in an alkaline solution to remove the silica particles to form a hollow network structure rigid carrier. The present invention effectively disperses boron and L-arabinose isomerase on the surface of the rigid carrier by constructing a hollow boron oxide network structure rigid carrier, thereby promoting the contact between D-galactose and L-arabinose isomerase, thereby improving the catalytic efficiency.

[0102] Table 1 Raw material composition for preparing rigid carriers in Examples 1 to 3

[0103] project Example 1 Example 2 Example 3 Silica particle size 20 to 100 μm mixed 50μm 50μm <![CDATA[m 壳聚糖 :m 二氧化硅 ]]> 1:20 1:25 1:30 <![CDATA[m 壳聚糖 :m 硼酸 ]]> 1:0.2 1：0.3 1:0.2 <![CDATA[m 壳聚糖 :m 戊二醛 ]]> 1:0.2 1：0.3 1:0.2 <![CDATA[m 壳聚糖 :m CTAB ]]> 1:0.15 1:0.08 1:0.10

[0104] The specific surface areas of the rigid carriers obtained in Examples 1 to 3 are shown in Table 2.

[0105] Table 2 Specific surface area of ​​rigid carriers prepared in Examples 1 to 3

[0106]

[0107]

[0108] Example 4

[0109] This example discloses the use of the rigid carrier prepared in Example 1 to immobilize the enzyme in the conversion of D-tagatose, comprising the following steps:

[0110] S1, placing the L-arabinose isomerase immobilized on the rigid carrier in a D-galactose aqueous solution with a pH value of 7.5 and a concentration of 50 mmol / L;

[0111] In this embodiment, the mass ratio of L-arabinose isomerase immobilized on the rigid carrier to D-galactose is 0.1:1.

[0112] S2. During the isomerization process, the boron atom in the rigid carrier is hydrated to [B(OH)3] or [B(OH)4] - , [B(OH)3] or [B(OH)4] - Complexing D-galactose: D-galactose as a reactant is adsorbed to the vicinity of L-arabinose isomerase on the rigid carrier through complexation;

[0113] S3, isomerization reaction at 60°C for 60 hours;

[0114] S4. After the isomerization is completed, the L-arabinose isomerase immobilized on the rigid carrier is filtered and separated to obtain the D-tagatose solution.

[0115] Example 5

[0116] This example discloses the use of the rigid carrier prepared in Example 2 to immobilize the enzyme in the conversion of D-tagatose. Figure 1 As shown, the following steps are included:

[0117] S1, placing the L-arabinose isomerase immobilized on the rigid carrier in a D-galactose aqueous solution with a pH value of 8.0 and a concentration of 50 mmol / L;

[0118] In this embodiment, the mass ratio of L-arabinose isomerase immobilized on the rigid carrier to D-galactose is 0.2:1.

[0119] S2. During the isomerization process, the boron atom in the rigid carrier is hydrated to [B(OH)3] or [B(OH)4] - , [B(OH)3] or [B(OH)4] - Complexing D-galactose: D-galactose as a reactant is adsorbed to the vicinity of L-arabinose isomerase on the rigid carrier through complexation;

[0120] S3, isomerization reaction at 65°C for 55 hours;

[0121] S4. After the isomerization is completed, the L-arabinose isomerase immobilized on the rigid carrier is filtered and separated to obtain the D-tagatose solution.

[0122] Example 6

[0123] This example discloses the use of the rigid carrier prepared in Example 3 to immobilize the enzyme in the conversion of D-tagatose. Figure 1 As shown, the following steps are included:

[0124] S1, placing the L-arabinose isomerase immobilized on the rigid carrier in a D-galactose aqueous solution with a pH value of 8.5 and a concentration of 50 mmol / L;

[0125] In this embodiment, the mass ratio of L-arabinose isomerase immobilized on the rigid carrier to D-galactose is 0.15:1.

[0126] S2. During the isomerization process, the boron atom in the rigid carrier is hydrated to [B(OH)3] or [B(OH)4] - , [B(OH)3] or [B(OH)4] - Complexing D-galactose: D-galactose as a reactant is adsorbed to the vicinity of L-arabinose isomerase on the rigid carrier through complexation;

[0127] S3, isomerization reaction at 70°C for 48 hours;

[0128] S4. After the isomerization is completed, the L-arabinose isomerase immobilized on the rigid carrier is filtered and separated to obtain the D-tagatose solution.

[0129] Comparative Example

[0130] This comparative example discloses a rigid carrier without using undoped boron oxide for immobilizing enzymes for the preparation of D-tagatose.

[0131] This comparative example discloses a method for fixing L-arabinose isomerase using sodium alginate:

[0132] Mix 10mL of fully dissolved sodium alginate solution (mass fraction 2.0%) with 5mL of free enzyme solution, then add 5mL of gelatin solution (mass fraction 2.0%), and mix and emulsify for 30min. Use a syringe with a needle with an inner diameter of 0.6mm to drip the above mixture into a CaCl2 (mass fraction 4.0%) solution at a rate of 60 drops / min to form smooth particles of uniform size, which will stand and harden at 0-4℃. Wash the immobilized particles, filter and dry them. Add them to a glutaraldehyde solution with a mass fraction of 0.02%, oscillate and cross-link at 25℃ for 1h, pour off the solution, wash the immobilized particles with 0.9% NaCl solution and deionized water respectively, filter and dry them to obtain the immobilized enzyme.

[0133] The L-arabinose isomerase prepared by biological fermentation used in this comparative example is the same as that in Examples 1 to 3.

[0134] The immobilized L-arabinose isomerase of this comparative example was placed in a D-galactose solution with a concentration of 50 mmol / L, and the pH value of the D-galactose aqueous solution was 8.0;

[0135] In this comparative example, the mass ratio of L-arabinose isomerase immobilized on the rigid carrier to D-galactose was 0.2:1.

[0136] The isomerization reaction was carried out at 65° C. for 55 hours. After the isomerization was completed, the L-arabinose isomerase immobilized on the sodium alginate was filtered and separated to obtain the D-tagatose solution.

[0137] The conversion rates of D-tagatose in the first catalysis, the fifth catalysis and the twelfth catalysis of Test Examples 4 to 6 and the Comparative Example are shown in Table 3.

[0138] Table 3 Conversion rate of immobilized enzymes for D-galactose in Examples 4 to 6 and Comparative Examples

[0139]

[0140]

[0141] Combined with Table 3, it can be seen that the present invention uses silica particles as nucleation centers, and rigid carriers are formed on the surface of silica particles, thereby limiting the distribution of boron oxide particles in shallow positions to a certain extent. The stacking of rigid carrier skeletons forms a three-dimensional space connection, and the liquid phase is dispersed in and between the connected spherical carbon skeletons. Boron oxide hydrolyzes and complexes D-galactose to increase the possibility of contact with D-galactose solidifying enzyme; and because the rigid carrier skeleton has a stable structure, it effectively improves the immobilization degree of L-arabinose isomerase, and the obtained solidified enzyme can be used repeatedly, and the structure is stable and can be used repeatedly.

[0142] The carbon skeleton of the present invention effectively combines the boron element. Combined with the elemental analysis in Table 4, it can be seen that BC bonds are formed by penetration under heating during the heat treatment process.

[0143] Table 4 Elemental composition (mass percentage) of the rigid carrier obtained in Examples 1 to 3

[0144] project C O H B N Example 1 78.0 3.5 7.2 4.6 6.7 Example 2 77.3 2.9 7.1 5.2 7.5 Example 3 79.5 2.1 6.4 4.7 7.3

[0145] The present invention uses a rigid carrier to fix the biological enzyme for D-tagatose conversion. The rigid carrier-fixed biological enzyme is structurally stable in a reaction solution with a pH value of 7.5 to 8.5, and can stably catalyze for 48 to 60 hours. During the catalytic process, the boron atom in the rigid carrier is hydrated to [B(OH)3] or [B(OH)4]. - , [B(OH)3] or [B(OH)4] - Complex D-galactose: D-galactose as a reactant is adsorbed to the vicinity of the L-arabinose isomerase on the rigid carrier through complexation and isomerized at 60°C to 70°C; after the isomerization is completed, the L-arabinose isomerase immobilized on the rigid carrier is filtered and separated to obtain a D-tagatose solution, and the D-tagatose is further concentrated and purified to obtain the target product. Liquid phase spectra after separation of immobilized enzymes in Example 2 and Comparative Example Figure 1 and Figure 2 It can be seen that the present invention facilitates the conversion of D-galactose and D-tagatose, and the use of a rigid skeleton to fix the boron element also facilitates the separation and purification of D-galactose and D-tagatose. The rigid carrier used in the present invention has a large specific surface area. As the number of uses increases, on the one hand, the pollutants loaded on the surface increase, and on the other hand, there is a certain degree of enzyme loss during use, resulting in a slow decrease in the conversion rate of D-galactose.

Claims

1. Application of a rigid carrier-immobilized enzyme in D-tagatose conversion, characterized in that: The following steps are involved: S1, placing L-arabinose isomerase immobilized on a rigid carrier in a D-galactose aqueous solution having a pH value of 7.5 to 8.5; S2. During the isomerization process, the boron atom in the rigid carrier is hydrated to [B(OH)3] or [B(OH)4] - , [B(OH)3] or [B(OH)4] - Complexing D-galactose: D-galactose as a reactant is adsorbed to the vicinity of L-arabinose isomerase on the rigid carrier through complexation; S3, isomerization reaction at 60° C. to 70° C. for 48 to 60 hours; S4, after the isomerization is completed, the L-arabinose isomerase immobilized on the rigid carrier is filtered and separated to obtain D-tagatose; The method for preparing the rigid carrier comprises the following steps: S11, stirring and mixing micron-sized silica particles, chitosan, boric acid, glutaraldehyde and hexadecyltrimethylammonium bromide, and placing in a reaction kettle for hydrothermal reaction; The process conditions of S11 hydrothermal reaction are as follows: Time 12 to 18 hours; Temperature 180°C to 220°C; The particle size of the silica particles is 20 μm to 100 μm; The mass ratio of chitosan to boric acid is 1:(0.2 to 0.3); The mass ratio of chitosan to glutaraldehyde is 1:(0.2 to 0.3); The mass ratio of chitosan to hexadecyltrimethylammonium bromide is 1:(0.08 to 0.15); S12, cooling after the hydrothermal reaction is completed, filtering the solid particles, and obtaining a carbon-boron oxide network structure surrounding the growth of the silicon dioxide particles; Wash and dry; S13, heat treating the carbon-boron oxide network structure surrounding the silicon dioxide particles obtained in S12 under nitrogen protection, so that the boron penetrates into the carbon to form a stable connection; S13: the temperature of the heat treatment is 600° C. to 700° C., and the heat treatment time is 3 hours to 6 hours; S14, placing the carbon-boron oxide network structure formed on the surface of the silica particles and mutually penetrating into a stable skeleton in an alkaline solution to remove the silica particles to form a hollow network structure rigid carrier.

2. The use of a rigid carrier-immobilized enzyme in D-tagatose conversion according to claim 1, characterized in that: L-arabinose isomerase, a rigid carrier and glutaraldehyde are cross-linked in an aqueous solution to obtain an immobilized enzyme.

3. The use of a rigid carrier-immobilized enzyme in D-tagatose conversion according to claim 2, characterized in that: L-arabinose isomerase is produced by biological fermentation, and the enzyme activity is 20000U / L to 30000U / L.

4. The use of a rigid carrier-immobilized enzyme in D-tagatose conversion according to claim 2, characterized in that: The activity of the immobilized enzyme is 500U to 600U / g rigid carrier.

5. The use of a rigid carrier-immobilized enzyme in D-tagatose conversion according to any one of claims 1 to 4, characterized in that: The mass ratio of L-arabinose isomerase immobilized on the rigid carrier to D-galactose is rigid carrier: D-galactose (0.1 to 0.2):1.

Images