Immobilized trehalose synthase as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of trehalose preparation, and particularly discloses immobilized trehalose synthase as well as a preparation method and application thereof. The preparation method comprises the following steps: preparing silicon dioxide nanoparticles into silicon dioxide sol; mixing the silica sol with glutaraldehyde, and incubating to obtain activated silica sol; mixing the obtained sol with a trehalose synthase solution, and carrying out an immobilization reaction to obtain silicon dioxide sol containing trehalose synthase; and finally, mixing with glutaraldehyde, and carrying out cross-linking reaction to obtain the immobilized trehalose synthase. The method avoids the problems of large use amount of enzyme, high production cost and the like in the existing method for preparing trehalose, improves the utilization rate of trehalose synthase, reduces the production cost, and is suitable for industrial production of trehalose.

Description

Technical Field

[0001] The present invention relates to the technical field of trehalose preparation, and in particular to an immobilized trehalose synthase and a preparation method and application thereof. Background Art

[0002] Trehalose is a natural non-reducing disaccharide composed of two glucose residues connected by an α, α-1, 1 glycosidic bond. It has the special function of preserving biological activity. This unique property makes trehalose not only an excellent active protectant for protein drugs, enzymes, vaccines and other biological products, but also an important ingredient for maintaining cell activity and moisturizing cosmetics. It can also be used as a unique food ingredient to prevent food deterioration, maintain food freshness and flavor, and improve food quality. It has broad and huge application value in many fields.

[0003] Trehalose synthase is an intramolecular transglycosylase that can use maltose as a substrate to produce trehalose in a one-step transglycosylation process. In 2000, Nanning Zhongnuo Bioengineering Co., Ltd. successfully developed a process for enzymatic conversion of cassava starch to produce trehalose using this enzyme system, realizing the industrial production of trehalose by enzymatic method. This process uses cassava starch to be decomposed into short-chain dextrins by amylase and pullulanase, and then converted into trehalose by the action of MTSase and MTHase. After refining, it can produce food-grade crystalline trehalose with a content of 98.0% and high-purity crystalline trehalose with a content of more than 99.0% (Huang Ribo, Trehalose - A New Sugar in the 21st Century [M]. Beijing: Chemical Industry Press, 2010). However, the existing methods for preparing trehalose have the problems of large enzyme usage and high production costs.

[0004] The application of immobilized trehalose synthase improves its stability, reusability, and ease of substrate separation, providing a wider range of applications for bioenzyme preparations. Immobilized trehalose synthase production of trehalose combines the simplicity of enzymatic conversion with the advantages of immobilization technology, effectively reducing production costs and providing a new approach for the industrial production of trehalose.

[0005] Therefore, it is an urgent problem for those skilled in the art to develop a mature and stable method for immobilizing trehalose synthase to improve the utilization rate of trehalose synthase, reduce the amount of trehalose synthase used, and reduce production costs. Summary of the Invention

[0006] In view of this, the present invention provides an immobilized trehalose synthase and a preparation method and application thereof, in order to solve the problems of low trehalose synthase utilization, large amount of trehalose synthase used and high production cost in existing trehalose preparation methods.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for preparing immobilized trehalose synthase comprises the following steps:

[0009] 1) preparing silica nanoparticles into silica sol;

[0010] 2) mixing the silica sol with glutaraldehyde and incubating the mixture to obtain an activated silica sol;

[0011] 3) mixing the activated silica sol with a trehalose synthase solution to perform a trehalose synthase immobilization reaction to obtain a silica sol containing trehalose synthase;

[0012] 4) Mixing the silica sol containing trehalose synthase with glutaraldehyde and performing a cross-linking reaction to obtain immobilized trehalose synthase.

[0013] Preferably, the particle size of the silicon dioxide nanoparticles is 1 to 100 nm;

[0014] The mass concentration of silica in the silica sol is 0.045 to 0.056 g / mL. Preferably, the volume ratio of the silica sol to glutaraldehyde in step 2) is 100:3 to 5;

[0015] The incubation temperature in step 2) is 25-28° C., the incubation time is 6-10 h, and the incubation is carried out under stirring at a stirring rate of 150-200 rpm.

[0016] Preferably, the volume ratio of the activated silica sol to the trehalose synthase solution in step 3) is 1-2:5-10;

[0017] The mass concentration of silicon dioxide in the activated silicon dioxide sol is 0.045 to 0.056 g / mL;

[0018] The immobilization reaction temperature is 25-28° C., the time is 24-30 hours, and the immobilization reaction is carried out under stirring conditions at a stirring rate of 150-200 rpm.

[0019] Preferably, the volume ratio of the silica sol containing trehalose synthase to glutaraldehyde in step 4) is 100:0.05-0.5;

[0020] The mass concentration of the silica sol containing trehalose synthase is 0.045-0.056 g / mL;

[0021] The cross-linking reaction in step 4) is carried out at a temperature of 25 to 28° C. for 6 to 10 hours, and is carried out under stirring at a stirring rate of 150 to 200 rpm.

[0022] Another object of the present invention is to provide an immobilized trehalose synthase prepared by the above preparation method.

[0023] Another object of the present invention is to provide an application of an immobilized trehalose synthase in the production of trehalose, wherein the production method of trehalose is:

[0024] The maltose solution is mixed with immobilized trehalose synthase, and the reaction, centrifugal filtration, and concentration and crystallization are carried out in sequence to obtain trehalose;

[0025] Alternatively, immobilized trehalose synthase is loaded into a reaction column, and the maltose solution is circulated through the reaction column for reaction. After the reaction is completed, centrifugal filtration and concentration crystallization are sequentially performed to obtain trehalose.

[0026] Preferably, the pH value of the maltose solution is independently 6.3 to 6.7;

[0027] The reaction temperature is independently 31 to 35° C., and the reaction time is independently 24 to 26 hours.

[0028] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects:

[0029] The invention provides a method for producing trehalose by immobilizing trehalose synthase using silicon dioxide as a carrier. The method uses silicon dioxide nanoparticles as a carrier, performs activation, covalent and cross-linking reactions with glutaraldehyde of appropriate concentration and trehalose synthase, and the immobilized trehalose synthase obtained after the reaction has the advantage of being reusable and can also be used for column filling to prepare a trehalose synthase immobilized enzyme column. The trehalose synthase immobilized enzyme column can be repeatedly used for trehalose production according to the process requirements of trehalose production by enzymatic conversion. The method avoids the problems of large enzyme usage and high production cost in existing methods for preparing trehalose, improves the utilization rate of trehalose synthase, reduces production cost, and is suitable for industrialized production of trehalose. DETAILED DESCRIPTION

[0030] The present invention provides a method for preparing immobilized trehalose synthase, comprising the following steps:

[0031] 1) preparing silica nanoparticles into silica sol;

[0032] 2) mixing the silica sol with glutaraldehyde and incubating the mixture to obtain an activated silica sol;

[0033] 3) mixing the activated silica sol with a trehalose synthase solution to perform a trehalose synthase immobilization reaction to obtain a silica sol containing trehalose synthase;

[0034] 4) Mixing the silica sol containing trehalose synthase with glutaraldehyde and performing a cross-linking reaction to obtain immobilized trehalose synthase.

[0035] In the present invention, the particle size of the silicon dioxide nanoparticles is 1 to 100 nm, specifically 15 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, or 80 nm.

[0036] In the present invention, the mass concentration of silica in the silica sol is 0.045-0.056 g / mL, specifically 0.046 g / mL, 0.048 g / mL, 0.050 g / mL, 0.052 g / mL, 0.054 g / mL, and 0.055 g / mL.

[0037] In the present invention, the silica sol is preferably prepared by mixing and dissolving silica nanoparticles with a phosphate buffer solution to form a stable translucent colloidal dispersion (sol) having a Tyndall effect, and then centrifuging to remove excess buffer solution to obtain the silica sol.

[0038] In the present invention, the mass ratio of silica nanoparticles to phosphate buffer is 1:15 to 20, preferably 1:16 to 18, and more preferably 1:17.

[0039] In the present invention, the pH value of the phosphate buffer is preferably 6.5, and the concentration is preferably 0.05 mol / L.

[0040] In the present invention, the centrifugal speed is preferably 4000-5000 rpm, specifically 4200 rpm, 4400 rpm, 4500 rpm, 4600 rpm, 4800 rpm; the centrifugal time is preferably 5-10 min, specifically 6 min, 7 min, 8 min, 9 min.

[0041] In the present invention, the volume ratio of the silica sol to glutaraldehyde in step 2) is 100:3-5, preferably 100:4-5, and more preferably 100:5.

[0042] In the present invention, the incubation temperature in step 2) is 25-28°C, specifically 25.5°C, 26°C, 26.5°C, 27°C, and 27.5°C; the incubation time is 6-10h, specifically 7h, 8h, and 9h; the incubation is carried out under stirring conditions, and the stirring rate is 150-200rpm, specifically 160rpm, 170rpm, 180rpm, and 190rpm.

[0043] In the present invention, the volume ratio of the activated silica sol to the trehalose synthase solution in step 3) is 1-2:5-10, preferably 1.5-2:8-10, and more preferably 2:10.

[0044] In the present invention, the trehalose synthase solution in step 3) is preferably obtained by fermenting Bacillus subtilis that integrates and expresses trehalose synthase, followed by isolation, purification, and concentration, and has an enzyme activity ≥ 2000, specifically 2000, 2100, 2200, 2500, or 3000. The trehalose synthase solution is tested for enzyme activity by the DNS colorimetric method. 1 mL of enzyme solution converts 1 μg / mL of maltose solution to 1 μg / mL of non-reducing sugar at 37° C. and pH 6.5 in 1 minute, which is 1 unit of enzyme activity, expressed as U / mL.

[0045] In the present invention, the mass concentration of silica in the activated silica sol is 0.045-0.056 g / mL, specifically 0.046 g / mL, 0.048 g / mL, 0.050 g / mL, 0.052 g / mL, 0.054 g / mL, and 0.055 g / mL.

[0046] In the present invention, the temperature of the immobilization reaction is 25-28°C, specifically 25.5°C, 26°C, 26.5°C, 27°C, and 27.5°C; the time is 24-30h, specifically 25h, 26h, 27h, 28h, and 29h; the immobilization reaction is carried out under stirring conditions, and the stirring rate is 150-200rpm, specifically 160rpm, 170rpm, 180rpm, and 190rpm.

[0047] In the present invention, the volume ratio of the silica sol containing trehalose synthase to glutaraldehyde in step 4) is 100:0.05-0.5, preferably 100:0.1-0.4, more preferably 100:0.15-0.2, and further preferably 100:0.15.

[0048] In the present invention, the mass concentration of the silica sol containing trehalose synthase is 0.045-0.056 g / mL, specifically 0.046 g / mL, 0.048 g / mL, 0.050 g / mL, 0.052 g / mL, 0.054 g / mL, and 0.055 g / mL.

[0049] In the present invention, the temperature of the cross-linking reaction in step 4) is 25-28°C, specifically 25.5°C, 26°C, 26.5°C, 27°C, and 27.5°C; the time is 6-10h, specifically 7h, 8h, and 9h; the cross-linking reaction is carried out under stirring conditions, and the stirring rate is 150-200rpm, specifically 160rpm, 170rpm, 180rpm, and 190rpm.

[0050] The present invention also provides an immobilized trehalose synthase prepared by the above preparation method.

[0051] The present invention also provides an application of an immobilized trehalose synthase in producing trehalose, wherein the production method of trehalose is as follows:

[0052] The maltose solution is mixed with immobilized trehalose synthase, and the reaction, centrifugal filtration, and concentration and crystallization are carried out in sequence to obtain trehalose;

[0053] Alternatively, immobilized trehalose synthase is loaded into a reaction column, and the maltose solution is circulated through the reaction column for reaction. After the reaction is completed, centrifugal filtration and concentration crystallization are sequentially performed to obtain trehalose.

[0054] In the present invention, the pH value of the maltose solution in the two reaction modes is independently 6.3-6.7, specifically 6.4, 6.5, or 6.6.

[0055] In the present invention, the maltose solution is prepared by using starch solution as raw material and converting the maltose solution through α-amylase and pullulanase catalytic reaction.

[0056] In the present invention, the reaction temperature is independently 31-35°C, specifically 32°C, 33°C, or 34°C; the reaction time is independently 24-26h, specifically 24.5h, 25h, or 25.5h.

[0057] In the present invention, the trehalose obtained by the two reaction modes are independently high-purity trehalose crystals with a purity greater than 99%.

[0058] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0059] Example 1

[0060] 1) Trehalose synthase solution is obtained by fermentation of Bacillus subtilis expressing trehalose synthase, followed by isolation, purification, and concentration. The enzyme activity is 2300 U / mL.

[0061] 2) 15 nm silica nanoparticles were dissolved in 20 times their weight of 0.05 M phosphate buffer (pH 6.5) to form a stable, translucent colloidal dispersion (sol) exhibiting a Tyndall effect. Excess buffer was removed by centrifugation at 4000 rpm for 5 minutes to obtain a silica sol with a mass concentration of 0.05 mg / mL.

[0062] 3) 5% by volume glutaraldehyde was added to the silica sol and incubated at 28°C and 150 rpm for 6 hours to activate the silica nanoparticles within the sol. The activated silica sol was then recovered by centrifugation at 4000 rpm for 5 minutes. The sol was then washed five times with phosphate buffer to remove any residual glutaraldehyde.

[0063] 4) Add 2 mL of activated silica sol (silica concentration 0.05 mg / mL) to 10 mL of trehalose synthase solution and mix thoroughly. Immobilize the trehalose synthase via covalent bonding in a shaking incubator at 150 rpm and 25°C for 30 hours. Centrifuge at 4000 rpm for 5 minutes to remove excess buffer.

[0064] 5) Add 0.15% volume of glutaraldehyde to the silica sol containing trehalose synthase (the mass concentration of silica containing trehalose synthase is 0.05 mg / mL) and react at 28°C and 150 rpm for 6 hours to perform a cross-linking reaction. The silica sol after the reaction is then recovered by centrifugation at 4000 rpm for 5 minutes. Repeat washing twice with phosphate buffer to remove residual trace glutaraldehyde. A silica sol containing immobilized trehalose synthase with a mass concentration of 0.05 mg silica / mL (immobilized trehalose synthase) is obtained. It has been determined that the activity of the silica sol containing immobilized trehalose synthase is 9020 U / mL (enzyme concentration makes the activity higher).

[0065] Example 2

[0066] The only difference between this example and example 1 is that the amount of glutaraldehyde added in step 3) is 3% of the volume of the silica sol. The activity of the silica sol containing immobilized trehalose synthase was measured to be 7960 U / mL.

[0067] In Examples 1 and 2, the silica sol containing immobilized trehalose synthase activated with 3% glutaraldehyde had an initial enzyme activity of 7960 U / mL, capable of adsorbing approximately 69.2% of trehalose synthase. The silica sol containing immobilized trehalose synthase activated with 5% glutaraldehyde had an initial enzyme activity of 9020 U / mL, capable of adsorbing approximately 78.4% of trehalose synthase, demonstrating excellent results.

[0068] Example 3

[0069] The only difference between this example and Example 1 is that the amount of glutaraldehyde added in step 5) is 0.05% of the volume of the silica sol containing trehalose synthase. The activity of the silica sol containing immobilized trehalose synthase was measured to be 9430 U / mL.

[0070] The resulting silica sol containing immobilized trehalose synthase was added to a 10% maltose solution (pH 6.5) and allowed to react for 24 hours. The reaction solution was then removed by centrifugation and washed twice with 0.05M phosphate buffer (pH 6.5). This step was repeated five times before the enzyme activity of the silica sol was measured.

[0071] It was determined that the enzyme activity was 8200 U / mL after five repeated uses, which was 87% of the initial enzyme activity. The decline was small and the product could continue to be used.

[0072] Example 4

[0073] The only difference between this embodiment of the present invention and Example 1 is that the enzyme activity of the trehalose synthase solution in step 1) is 2200 U / mL; and the amount of glutaraldehyde added in step 5) is 0.5% of the volume of the silica sol containing trehalose synthase. The activity of the silica sol containing immobilized trehalose synthase was measured to be 8140 U / mL.

[0074] After repeated use for 5 times as in Example 3, the enzyme activity was tested to be 7490 U / mL, which was 92% of the initial enzyme activity. The decrease was small and the product could continue to be used.

[0075] Example 5

[0076] The only difference between the embodiment of the present invention and embodiment 1 is that the enzyme activity of the trehalose synthase solution in step 1) is 2200 U / mL, and the activity of the silica sol containing immobilized trehalose synthase is measured to be 8690 U / mL.

[0077] After repeated use for 5 times as in Example 3, the enzyme activity was tested to be 7950 U / mL, which was 91.5% of the initial enzyme activity. The decrease was small and the product could continue to be used.

[0078] Example 6

[0079] 1) Trehalose synthase solution is obtained by fermentation of Bacillus subtilis expressing trehalose synthase, followed by isolation, purification, and concentration. The enzyme activity is 2100 U / mL.

[0080] 2) 50 nm silica nanoparticles were dissolved in 20 times their weight of 0.05 M phosphate buffer (pH 6.5) to form a stable, translucent colloidal dispersion (sol) exhibiting a Tyndall effect. Excess buffer was removed by centrifugation at 4000 rpm for 5 minutes to obtain a silica sol with a mass concentration of 0.045 mg / mL.

[0081] 3) 3% by volume glutaraldehyde was added to the silica sol and incubated at 25°C and 150 rpm for 10 hours to activate the silica nanoparticles within the sol. The activated silica sol was then recovered by centrifugation at 4000 rpm for 5 minutes. The sol was then washed five times with phosphate buffer to remove any traces of residual glutaraldehyde.

[0082] 4) Add 2 mL of activated silica sol (silica concentration 0.045 mg / mL) to 10 mL of trehalose synthase solution and mix thoroughly. Immobilize the trehalose synthase via covalent bonding in a shaking incubator at 150 rpm and 25°C for 25 hours. Remove excess buffer by centrifugation at 4000 rpm for 5 minutes.

[0083] 5) 0.1% by volume glutaraldehyde was added to the silica sol containing trehalose synthase (the silica mass concentration containing trehalose synthase was 0.045 mg / mL) and reacted at 25°C and 200 rpm for 10 hours to perform a cross-linking reaction. The silica sol was then recovered by centrifugation at 4000 rpm for 5 minutes. The silica sol was washed twice with phosphate buffer to remove residual traces of glutaraldehyde. This resulted in a silica sol containing immobilized trehalose synthase (immobilized trehalose synthase) with a mass concentration of 0.045 mg silica / mL.

[0084] Example 7

[0085] 1) Trehalose synthase solution is obtained by fermentation of Bacillus subtilis expressing trehalose synthase, followed by isolation, purification, and concentration. The enzyme activity is 2100 U / mL.

[0086] 2) 80 nm silica nanoparticles were dissolved in 20 times their weight of 0.05 M phosphate buffer (pH 6.5) to form a stable, translucent colloidal dispersion (sol) exhibiting a Tyndall effect. Excess buffer was removed by centrifugation at 4000 rpm for 5 minutes to obtain a silica sol with a mass concentration of 0.055 mg / mL.

[0087] 3) 4% glutaraldehyde was added to the silica sol, and the mixture was incubated at 26°C and 200 rpm for 8 hours to activate the silica nanoparticles in the sol. The activated silica sol was recovered by centrifugation at 4000 rpm for 5 minutes. The residual glutaraldehyde was removed by washing the silica sol with phosphate buffer five times.

[0088] 4) 2 mL of the activated silica sol (0.055 mg / mL of silica) was mixed with 10 mL of the trehalose synthase solution. Covalent binding was performed by incubating the mixture in a shaking incubator at 26°C and 200 rpm for 28 hours. The excess buffer was removed by centrifugation at 4000 rpm for 5 minutes.

[0089] 5) 0.5% glutaraldehyde was added to the silica sol containing the trehalose synthase (0.055 mg / mL of silica containing the trehalose synthase), and the mixture was incubated at 26°C and 180 rpm for 8 hours to perform the cross-linking reaction. The reacted silica sol was recovered by centrifugation at 4000 rpm for 5 minutes. The residual glutaraldehyde was removed by washing the silica sol with phosphate buffer twice. The silica sol containing the immobilized trehalose synthase (0.055 mg of silica / mL) was obtained.

[0090] Comparative Example 1

[0091] The only difference between this comparative example and Example 5 is that the silica nanoparticles have a particle size of 1000 nm, and the silica nanoparticles remain in a particulate state after mixing with the phosphate buffer, and do not form a sol.

[0092] In this example, the volume of the 1000 nm silica nanoparticles does not change much after dissolution, and the surface area of the particulate state is too small. The enzyme activity of the immobilized trehalose synthase is 2530 U / mL, and only about 23% of the enzyme is immobilized on the nanoparticles. Therefore, silica nanoparticles of this size are not suitable as a carrier for immobilizing trehalose synthase.

[0093] Comparative Example 2

[0094] The only difference between this comparative example and Example 1 is that the enzyme activity of the trehalose synthase solution in step 1) is 2500 U / mL, and the activation reaction in step 3) is not performed. The silica sol obtained in step 2) is directly mixed with the trehalose synthase solution and reacted.

[0095] It was determined that the activity of the final sol was 5620 U / mL. In this example, the silica sol without the activation reaction step poorly adsorbed the trehalose synthase, and only about 45% of the enzyme was immobilized on the nanoparticles.

[0096] Comparative Example 3

[0097] The only difference between this comparative example and Example 1 is that the enzyme activity of the trehalose synthase solution in step 1) is 2500 U / mL; and step 5) the cross-linking reaction is not performed.

[0098] The activity of the final sol was measured to be 9720 U / mL. After repeated use for 5 times as in Example 3, the enzyme activity was 5880 U / mL, which was only 60% of the initial enzyme activity. The decrease was large and the sol could not be used further.

[0099] Application Example 1

[0100] Trehalose is produced using a silica sol containing immobilized trehalose synthase. The specific steps are as follows:

[0101] 1) Maltose solution is made from starch solution through the catalytic conversion of α-amylase and pullulanase.

[0102] 2) The silica sol containing immobilized trehalose synthase prepared according to the conditions of Example 5 was added to the maltose solution at a pH of 6.5 and a temperature of 33° C. The reaction was carried out for 24 hours to obtain a trehalose solution with a reducing sugar content of ≤1.5%.

[0103] 3) After the trehalose solution has reacted completely, the solution is centrifuged at 4000 rpm for 5 minutes to recover the silica sol containing the immobilized trehalose synthase.

[0104] 4) After centrifugal filtration, the trehalose solution is concentrated, crystallized and dried to obtain high-purity trehalose crystals with a purity of 99%.

[0105] Trehalose synthase is reusable. After conversion, the silica sol containing the immobilized trehalose synthase must be recovered by centrifugation. The weight of the sol decreases by 2-4% each time it is recovered. After repeating the production process five times, new silica sol containing the immobilized trehalose synthase must be added.

[0106] Application Example 2

[0107] The specific steps for producing trehalose using trehalose synthase immobilized enzyme column are as follows:

[0108] 1) Maltose solution is made from starch solution through the catalytic conversion of α-amylase and pullulanase.

[0109] 2) The silica sol containing immobilized trehalose synthase, prepared according to the conditions of Example 5, was loaded into a reaction column. A sieve plate with numerous small holes was installed at the bottom of the column. The silica sol could not pass through the holes, while the reaction solution could freely enter and exit. This resulted in a trehalose synthase-immobilized enzyme column that could be reused for trehalose production.

[0110] 3) The maltose solution is circulated in the immobilized enzyme column of trehalose synthetase at pH 6.5 and 33℃, and the reaction is carried out for 24 hours to obtain a trehalose solution with a reducing sugar content of ≤1.5%.

[0111] 4) The trehalose solution is filtered by centrifugation, concentrated, crystallized and dried to obtain high-purity trehalose crystals with a purity of 99%.

[0112] The trehalose synthetase can be repeatedly used, and the immobilized enzyme column of trehalose synthetase can be used for 10 times to obtain qualified trehalose solution. After the immobilized enzyme column of trehalose synthetase is used for 10 times, the initial enzyme activity is maintained at more than 90%, and the repeatability is good. The conditions are all optimal.

[0113] Comparative Example 1

[0114] The trehalose is produced by using the trehalose synthetase solution, and the specific steps are as follows:

[0115] 1) The trehalose synthetase is prepared by fermenting the integrated trehalose synthetase of Bacillus subtilis, isolating, purifying and concentrating, and the enzyme activity is 2400 U / mL.

[0116] 2) The maltose solution is prepared by using starch solution as raw material and through catalytic reaction of α-amylase and pullulanase.

[0117] 3) The maltose solution is added with the trehalose synthetase solution at pH 6.5 and 33℃, and the reaction is carried out for 24 hours to obtain a trehalose solution with a reducing sugar content of ≤1.5%.

[0118] 4) After the reaction of the trehalose solution is completed, the trehalose synthetase is inactivated by heating to 85℃ and maintaining for 30 minutes.

[0119] 5) The trehalose solution is filtered by centrifugation, concentrated, crystallized and dried to obtain high-purity trehalose crystals with a purity of 99%.

[0120] In this example, the trehalose synthetase cannot be repeatedly used, and the trehalose synthetase needs to be inactivated at high temperature after conversion. The use amount of the trehalose synthetase is large in large-scale production, and the production cost is high.

[0121] Each of the embodiments in the specification is described in a progressive manner, and each embodiment focuses on the difference from other embodiments. The same or similar parts of each embodiment can be referred to each other.

[0122] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited

[0123] The present invention is not limited to the embodiments shown herein, but is intended to be consistent with the principles and novelties disclosed herein.

[0124] The widest range of consistent characteristics.

Claims

1. A method for preparing immobilized trehalose synthase, characterized in that: The steps include: 1) preparing silica nanoparticles into silica sol; 2) mixing the silica sol with glutaraldehyde and incubating the mixture to obtain an activated silica sol; 3) mixing the activated silica sol with a trehalose synthase solution to perform a trehalose synthase immobilization reaction to obtain a silica sol containing trehalose synthase; 4) Mixing the silica sol containing trehalose synthase with glutaraldehyde and performing a cross-linking reaction to obtain immobilized trehalose synthase.

2. The method for preparing immobilized trehalose synthase according to claim 1, wherein: The particle size of the silicon dioxide nanoparticles is 1 to 100 nm; The mass concentration of silicon dioxide in the silicon dioxide sol is 0.045-0.056 g / mL.

3. The method for preparing immobilized trehalose synthase according to claim 2, wherein: The volume ratio of the silica sol to glutaraldehyde in step 2) is 100:3-5; The incubation temperature in step 2) is 25-28° C., the incubation time is 6-10 h, and the incubation is carried out under stirring at a stirring rate of 150-200 rpm.

4. The method for preparing immobilized trehalose synthase according to any one of claims 1 to 3, characterized in that: The volume ratio of the activated silica sol to the trehalose synthase solution in step 3) is 1-2:5-10; The mass concentration of silicon dioxide in the activated silicon dioxide sol is 0.045 to 0.056 g / mL; The immobilization reaction temperature is 25-28° C., the time is 24-30 hours, and the immobilization reaction is carried out under stirring conditions at a stirring rate of 150-200 rpm.

5. The method for preparing immobilized trehalose synthase according to claim 4, characterized in that: The volume ratio of the silica sol containing trehalose synthase to glutaraldehyde in step 4) is 100:0.05-0.5; The mass concentration of the silica sol containing trehalose synthase is 0.045-0.056 g / mL; The cross-linking reaction in step 4) is carried out at a temperature of 25 to 28° C. for 6 to 10 hours, and is carried out under stirring at a stirring rate of 150 to 200 rpm.

6. The immobilized trehalose synthase prepared by the preparation method according to any one of claims 1 to 8.

7. Use of the immobilized trehalose synthase according to claim 6 in the production of trehalose, characterized in that: The production method of trehalose is: The maltose solution is mixed with immobilized trehalose synthase, and the reaction, centrifugal filtration, and concentration and crystallization are carried out in sequence to obtain trehalose; Alternatively, immobilized trehalose synthase is loaded into a reaction column, and the maltose solution is circulated through the reaction column for reaction. After the reaction is completed, centrifugal filtration and concentration crystallization are sequentially performed to obtain trehalose.

8. Use of the immobilized trehalose synthase according to claim 7 in the production of trehalose, characterized in that: The pH value of the maltose solution is independently 6.3 to 6.7; The reaction temperature is independently 31 to 35° C., and the reaction time is independently 24 to 26 hours.