Preparation of carrier-free immobilized sucrose isomerase and application thereof in catalyzing sucrose to produce isomaltulose

Abstract

The application discloses a kind of preparation of carrier-free immobilized sucrose isomerase and its application in catalyzing sucrose to produce isomaltulose, and belongs to the field of bioengineering technology.The specific method of the application comprises the following steps: using polyethylene glycol as a precipitant, dextran aldehyde as a crosslinking agent, realizing the immobilization of sucrose isomerase by carrier-free immobilization-crosslinked enzyme aggregate method, adding bovine serum albumin as a stabilizer and amino donor in the preparation process, and then using the immobilized sucrose isomerase to convert sucrose raw materials to produce isomaltulose.The crosslinked sucrose isomerase aggregate prepared by the method has significantly improved temperature stability, pH tolerance and storage stability, and the enzyme activity recovery rate can be maintained at more than 90% after 10 times of continuous repeated use;after continuously preparing isomaltulose in batches, the substrate conversion rate of CLSIAs-BSA can be maintained at more than 85%.The application improves the efficiency of enzymatic preparation of isomaltulose, reduces production cost and separation and purification difficulty, is suitable for large-scale continuous production, and has great significance for improving the technical level of isomaltulose production in China.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and specifically relates to the preparation of a carrier-free immobilized sucrose isomerase and its application in catalyzing the production of isomaltulose from sucrose. Background Technology

[0002] Isomaltulose is a functional disaccharide and an isomer of sucrose. It shares similar physical properties and taste with sucrose and is naturally found in sugar beets, molasses, etc. It has advantages such as low sweetness, easy absorption, non-cariogenicity, and low calories. Compared to sucrose, isomaltulose has good acid stability, extremely low hygroscopicity, and extremely high safety, making it very suitable for food applications. Furthermore, as a reducing sugar, isomaltulose can be further hydrogenated to produce novel functional sugar alcohols such as isomaltulitol.

[0003] Chemical and biological conversion methods are the main approaches for synthesizing isomaltulose. However, due to the high energy consumption and pollution associated with chemical conversion, isomaltulose production currently utilizes a biocatalytic sucrose isomerization method, where sucrose isomerase (SIase) uses sucrose as a substrate to convert it into isomaltulose. Compared to free enzymes, immobilized enzymes offer advantages such as good operational and storage stability, and easy product separation, making their industrial application more economical. Currently, SIase has been immobilized using two methods: diatomaceous earth adsorption and microencapsulation (J Biotechnol, 2012, 158(3): 137-43). The resulting immobilized enzymes have low enzyme activity recovery rates and operational stability, far below the level required for industrial applications. Later, SIase was immobilized using e-poly-L-lysine-modified mesoporous TiO2 and sponges as carriers (Food Chem, 2015, 187: 182-8; Adv Synth Catal, 2016, 358(24): 4030-4040). The enzyme activity recovery rates of the two immobilized enzymes were 93.2% and 84.5%, respectively. They can be reused multiple times and still maintain a high conversion rate. The materials are also relatively novel, but the production cost is high and they are not suitable for large-scale industrial production.

[0004] Cross-linked enzyme aggregates (CLEAs) are a carrier-free immobilization technique. First, free enzymes are precipitated using water-soluble organic solvents, neutral salt solutions, or nonionic polymers. Then, bifunctional reagents are added to crosslink the precipitate, resulting in water-insoluble enzyme aggregates. Because the non-covalent interactions during enzyme precipitation are weak, the three-dimensional structure of the protein is not destroyed, thus preserving most of the enzyme's activity. Compared to carrier-based immobilization, CLEAs offer advantages such as simple preparation, low requirements for enzyme purity, high activity per unit volume, good specificity, and wide applicability. They also exhibit excellent tolerance to organic solvents and high temperatures and pressures, demonstrating good storage and operational stability. Furthermore, CLEAs have a high enzyme loading capacity and minimal activity loss during preparation, effectively avoiding enzyme inactivation that may occur when solid carriers are introduced. However, there is currently no research utilizing CLEAs technology—a carrier-free enzyme immobilization technique—for SIase immobilization and its catalytic formation of isomaltulose. Existing methods for preparing isomaltulose suffer from problems such as the inability to recover and reuse the enzyme and high costs. Therefore, researching a carrier-free immobilized SIase that is cost-effective, easy to operate, has excellent performance, and can be industrially produced is of great practical significance. Summary of the Invention

[0005] The first objective of this invention is to provide a method for preparing carrier-free immobilized sucrose isomerase, so as to achieve immobilized enzymes with low preparation cost and easy operation.

[0006] The immobilized sucrose isomerase was prepared according to the following method:

[0007] (1) Preparation of sucrose isomerase fermentation broth;

[0008] (2) Collect the enzyme solution from step (1) by centrifugation, and dilute it with phosphate buffer;

[0009] (3) Add a precipitant to step (2) and stir to precipitate at 4°C;

[0010] (4) Add crosslinking agent to the system after precipitation in step (3) and stir at 4°C for crosslinking;

[0011] (5) While performing step (3), add additives to (1) and simultaneously precipitate crosslinking;

[0012] (6) Centrifuge the cross-linked solution from step (4) at 12000 rpm for 10 min to remove the supernatant, and wash repeatedly with buffer solution until no enzyme activity is detected in the washing solution, thus obtaining cross-linked sucrose isomerase aggregates.

[0013] In one embodiment of the invention, the enzyme solution is added at a ratio of 20-40 U / mL;

[0014] In one embodiment of the invention, the precipitant comprises PEG (10-50%), ammonium sulfate (60-100%), and ethanol (60-100%), with a precipitation time of 10-120 min and a rotation speed of 100-300 rpm;

[0015] In one embodiment of the invention, the crosslinking agent comprises dextranaldehyde (Dex-CHO) and glutaraldehyde (GA), with a Dex-CHO mass concentration of 20-40 mg / mL, a GA mass fraction of 0.3-0.8%, a crosslinking time of 5-15 h, and a rotation speed of 100-300 rpm.

[0016] In one embodiment of the invention, the additive includes bovine serum albumin (BSA) and trehalose (Tre), wherein the concentration of BSA is 30-50 mg / mL and the concentration of Tre is 4-20 mg / mL.

[0017] In one embodiment of the invention, the method for preparing the crude sucrose isomerase solution includes:

[0018] Pichia pastoris SG115 was inoculated into 5-10 mL of YPD liquid medium and cultured at 28-32℃ and 200-240 rpm for 20-30 h. Then, the bacterial culture was transferred to 250 mL of BMGY medium at a 2% inoculation rate and cultured at 28-32℃ and 200-240 rpm for 16-18 h. The cells were collected by centrifugation and transferred to 250 mL of BMMY medium under aseptic conditions and cultured at 28-32℃ and 200-240 rpm for 3-7 days, during which 0.4-0.6% methanol was added twice a day. Finally, the supernatant was collected by centrifugation at 6000-8000 rpm for 5-15 min to obtain the crude sucrose isomerase solution.

[0019] A second objective of this invention is to provide the application of the aforementioned carrier-free immobilized sucrose isomerase in the catalytic production of isomaltulose from sucrose, thereby simplifying the process, increasing enzyme reusability, and reducing costs. This application utilizes cross-linked sucrose isomerase aggregates as catalysts and sucrose or similar raw materials to produce isomaltulose in multiple batches.

[0020] In one embodiment of the present invention, the amount of cross-linked sucrose isomerase aggregate added is 20-40 U / mL, and the raw materials include, but are not limited to, sucrose, sugarcane juice, apple juice, mango juice, orange juice, etc., with a concentration of 150-300 g / L.

[0021] In one embodiment of the present invention, the catalytic reaction time is 12-48 h, the reaction temperature is 25-45 °C, and the reaction pH is 5-9.

[0022] The beneficial effects of this invention are:

[0023] This invention presents a method for preparing cross-linked sucrose isomerase aggregates via carrier-free immobilization and catalyzing the production of isomaltulose from sucrose. This method offers advantages such as low cost, ease of operation, high stability, and high conversion rate. Compared to free enzymes, the cross-linked sucrose isomerase aggregates exhibit significantly improved temperature stability and pH tolerance. Even after 10 reuses, the immobilized enzyme retains over 90% of its initial activity, demonstrating excellent reusability and effectively reducing costs. The immobilized enzyme achieves a conversion rate of up to 88% in sucrose solution and up to 81% in complex environments such as sugarcane juice, far exceeding the substrate conversion rate of free enzymes. The conversion reaction can be carried out continuously, making it suitable for large-scale production, reducing product separation difficulties, and improving production efficiency. Attached Figure Description

[0024] Figure 1 The effect of temperature on the activity of free and immobilized enzymes.

[0025] Figure 2 The effect of pH on the activities of free and immobilized enzymes.

[0026] Figure 3 To ensure the reusability of immobilized enzymes.

[0027] Figure 4 To produce isomaltulose for immobilized enzymes. Detailed Implementation

[0028] (I) Determination of sucrose isomerase activity

[0029] Determination of free enzyme: 400 μL of sucrose solution (w / v, 25%) and 100 μL of crude enzyme solution were thoroughly mixed in 10 mM, pH 6.0 phosphate buffer. The mixture was incubated in a 35°C water bath for 10 min, and then immediately transferred to a boiling water bath for 5 min to terminate the reaction. The absorbance of the reaction solution was measured at 540 nm.

[0030] Assay of immobilized enzyme: Take the immobilized enzyme and add 0.1 g of 400 μL of sucrose solution (w / v, 25%) and 100 μL of 10 mM, pH 6.0 phosphate buffer. Mix thoroughly and incubate at 35°C for 10 min. Immediately transfer to a boiling water bath for 5 min to terminate the reaction. Measure the absorbance of the reaction solution at 540 nm.

[0031] Enzyme activity definition: Under the above reaction conditions, the amount of enzyme required to generate 1 μmol of isomaltulose per minute is defined as one enzyme activity unit (U).

[0032] (II) Determination of sucrose conversion rate by HPLC method

[0033] HPLC conditions: Agilent HPLC 1260 high performance liquid chromatograph, Agilent column (4.6 mm × 250 mm, 5 μm), evaporative light scattering detector (ELSD, Agilent 1260 Infinity, China), mobile phase acetonitrile:water = 80:20, flow rate 0.8 mL / min, column temperature 30 °C, injection volume 10 μL.

[0034] (III) Culture Medium

[0035] YPD test tubes: 1% yeast extract, 2% peptone, 2% glucose (solid culture medium with 1.5% agar powder) autoclaved at 115℃ for 20 min.

[0036] BMGY medium: 5g peptone, 2.5g yeast extract, 5mL glycerol, total 200mL (autoclave at 121℃ for 20min), then add (10×YNB 25mL, biotin 500μL, phosphate buffer solution (1M, pH=6.0), 25mL).

[0037] BMMY medium: 5g peptone, 2.5g yeast extract, 200mL total (autoclave at 121℃ for 20min), then add (10×YNB 25mL, biotin 500μL, phosphate buffer solution (1M, pH=6.0) 25mL).

[0038] Example 1: Process for immobilizing sucrose isomerase using a carrier-free immobilization method

[0039] (1) Preparation of sucrose isomerase: Pichia pastoris SG115 was inoculated into 5 mL of YPD liquid medium and cultured at 30℃ and 220 rpm for 24 h; then, the above bacterial solution was transferred to 250 mL of BMGY medium with an inoculation amount of 2% and cultured at 30℃ and 220 rpm for 18 h; the bacterial cells were collected by centrifugation and transferred to 250 mL of BMMY medium under aseptic conditions and cultured at 30℃ and 220 rpm for 5 days, during which 0.5% methanol was added twice a day; finally, the supernatant was collected by centrifugation at 6000 rpm for 10 min to obtain crude sucrose isomerase solution.

[0040] (2) Process of immobilizing sucrose isomerase using the cross-linking enzyme aggregate method: 1 mL of crude enzyme solution (20 mg / mL) was added to PEG solution to achieve a final concentration of 50%, and the mixture was stirred at 4℃ and 220 rpm for 60 min. Then, dextran aldehyde (35 mg / mL) was added, and the cross-linking reaction was carried out at 4℃ and 220 rpm for 8 h. The concentrations of additives BSA and Tre were 45 mg / mL and 12 mg / mL, respectively. After centrifugation and removal of the supernatant, the enzymes were repeatedly washed with buffer to obtain three immobilized enzymes: CLSIAs (without additives), CLSIAs-BSA (with BSA), and CLSIAs-Tre (with trehalose). The enzyme activity recovery rates were 18.5%, 30.2%, and 20.7%, respectively.

[0041] Example 2: Temperature stability of free enzymes and immobilized enzymes

[0042] (1) Enzyme activity assay of free and immobilized enzymes at 45℃: Equal concentrations of free and immobilized enzyme solutions were prepared using phosphate buffer (10 mM, pH 6.0), and each enzyme preparation was incubated at 45℃ for 30 min. The enzyme activities of the four enzyme preparations—free SIase, CLSIAs, CLSIAs-BSA, and CLSIAs-Tre—were 41.3%, 60.2%, 66.3%, and 64.7%, respectively.

[0043] (2) Enzyme activity assay of free and immobilized enzymes at 55℃: Equal concentrations of free and immobilized enzyme solutions were prepared using phosphate buffer (10 mM, pH 6.0), and each enzyme preparation was incubated at 55℃ for 30 min. The enzyme activities of the four enzyme preparations—free SIase, CLSIAs, CLSIAs-BSA, and CLSIAs-Tre—were 10.8%, 30.5%, 44.2%, and 31.6%, respectively.

[0044] Example 3: pH tolerance of free and immobilized enzymes

[0045] (1) Enzyme activity assay of free and immobilized enzymes at pH 4.0: Free and immobilized enzyme solutions of equal concentration were prepared using phosphate buffer (10 mM, pH 4.0), and each enzyme preparation was incubated at 25°C for 2 hours. The enzyme activities of the four enzyme preparations—free SIase, CLSIAs, CLSIAs-BSA, and CLSIAs-Tre—were 7.3%, 61.5%, 65.9%, and 65.3%, respectively.

[0046] (2) Enzyme activity assay of free and immobilized enzymes at pH 9.0: Equal concentrations of free and immobilized enzyme solutions were prepared using phosphate buffer (10 mM, pH 9.0), and each enzyme preparation was incubated at 25°C for 2 hours. The enzyme activities of the four enzyme preparations—free SIase, CLSIAs, CLSIAs-BSA, and CLSIAs-Tre—were 18.4%, 25.8%, 38.5%, and 32.6%, respectively.

[0047] Example 4: Other conditions were the same as in Example 1, except for the amount of PEG precipitant added.

[0048] (1) Preparation of sucrose isomerase: Pichia pastoris SG115 was inoculated into 5 mL of YPD liquid medium and cultured at 30℃ and 220 rpm for 24 h; then, the above bacterial culture was transferred to 250 mL of BMGY medium with an inoculation amount of 2% and cultured at 30℃ and 220 rpm for 18 h; the bacterial cells were collected by centrifugation and transferred to 250 mL of BMMY medium under aseptic conditions and cultured at 30℃ and 220 rpm for 5 days, during which 0.5% methanol was added twice a day; finally, the supernatant was collected by centrifugation at 6000 rpm for 10 min to obtain crude sucrose isomerase solution.

[0049] (2) Process of immobilizing sucrose isomerase using the cross-linking enzyme aggregate method: 1 mL of crude enzyme solution (20 mg / mL) was added to PEG solution to achieve a final concentration of 10%, and the mixture was stirred at 4℃ and 220 rpm for 60 min. Then, dextran aldehyde (35 mg / mL) was added, and the cross-linking reaction was carried out at 4℃ and 220 rpm for 8 h. The concentrations of additives BSA and Tre were 45 mg / mL and 12 mg / mL, respectively. After centrifugation and removal of the supernatant, the enzymes were repeatedly washed with buffer to obtain three immobilized enzymes: CLSIAs (without additives), CLSIAs-BSA (with BSA), and CLSIAs-Tre (with trehalose). The enzyme activity recovery rates were 12.1%, 23.4%, and 14.4%, respectively.

[0050] Example 5: Other conditions are the same as in Example 1, except that the amount of dextranaldehyde crosslinking agent added is changed.

[0051] (1) Preparation of sucrose isomerase: Pichia pastoris SG115 was inoculated into 5 mL of YPD liquid medium and cultured at 30℃ and 220 rpm for 24 h; then, the above bacterial culture was transferred to 250 mL of BMGY medium with an inoculation amount of 2% and cultured at 30℃ and 220 rpm for 18 h; the bacterial cells were collected by centrifugation and transferred to 250 mL of BMMY medium under aseptic conditions and cultured at 30℃ and 220 rpm for 5 days, during which 0.5% methanol was added twice a day; finally, the supernatant was collected by centrifugation at 6000 rpm for 10 min to obtain crude sucrose isomerase solution.

[0052] (2) Process of immobilizing sucrose isomerase using the cross-linking enzyme aggregate method: 1 mL of crude enzyme solution (20 mg / mL) was added to PEG solution to achieve a final concentration of 50%, and the mixture was stirred at 4℃ and 220 rpm for 60 min. Then, dextran aldehyde (20 mg / mL) was added, and the cross-linking reaction was carried out at 4℃ and 220 rpm for 8 h. The concentrations of additives BSA and Tre were 45 mg / mL and 12 mg / mL, respectively. After centrifugation and removal of the supernatant, the enzymes were repeatedly washed with buffer to obtain three immobilized enzymes: CLSIAs (without additives), CLSIAs-BSA (with BSA), and CLSIAs-Tre (with trehalose). The enzyme activity recovery rates were 13.7%, 22.5%, and 15.3%, respectively.

[0053] Example 6: Other conditions are the same as in Example 1, except the type of crosslinking agent is changed.

[0054] (1) Preparation of sucrose isomerase: Pichia pastoris SG115 was inoculated into 5 mL of YPD liquid medium and cultured at 30℃ and 220 rpm for 24 h; then, the above bacterial culture was transferred to 250 mL of BMGY medium with an inoculation amount of 2% and cultured at 30℃ and 220 rpm for 18 h; the bacterial cells were collected by centrifugation and transferred to 250 mL of BMMY medium under aseptic conditions and cultured at 30℃ and 220 rpm for 5 days, during which 0.5% methanol was added twice a day; finally, the supernatant was collected by centrifugation at 6000 rpm for 10 min to obtain crude sucrose isomerase solution.

[0055] (2) Process of immobilizing sucrose isomerase using the cross-linking enzyme aggregate method: 1 mL of crude enzyme solution (20 mg / mL) was added to PEG solution to achieve a final concentration of 50%, and the mixture was stirred at 4℃ and 220 rpm for 60 min. Glutaraldehyde (0.5% by mass) was then added, and the cross-linking reaction was carried out at 4℃ and 220 rpm for 8 h. The concentrations of additives BSA and Tre were 45 mg / mL and 12 mg / mL, respectively. After centrifugation and removal of the supernatant, the enzymes were repeatedly washed with buffer to obtain three immobilized enzymes: CLSIAs (without additives), CLSIAs-BSA (with BSA), and CLSIAs-Tre (with trehalose). The enzyme activity recovery rates were 10.7%, 19.5%, and 14.3%, respectively.

[0056] Example 7: Reusability of cross-linked sucrose isomerase aggregates

[0057] (1) Process of immobilizing sucrose isomerase using the cross-linking enzyme aggregate method: 1 mL of crude enzyme solution (20 mg / mL) was added to PEG solution to achieve a final concentration of 40%, and the mixture was stirred at 4℃ and 220 rpm for 60 min. Then, dextran aldehyde (35 mg / mL) was added, and the cross-linking reaction was carried out at 4℃ and 220 rpm for 8 h. The concentrations of additives BSA and Tre were 45 mg / mL and 12 mg / mL, respectively. After centrifugation and removal of the supernatant, the enzymes were repeatedly washed with buffer to obtain three immobilized enzymes: CLSIAs (without additives), CLSIAs-BSA (with BSA), and CLSIAs-Tre (with trehalose). The enzyme activity recovery rates were 19.5%, 31.2%, and 21.7%, respectively.

[0058] (2) Reusability of cross-linked sucrose isomerase aggregates: equal amounts of immobilized enzymes were taken to determine their activity. After the reaction, the enzymes were centrifuged at 12,000 rpm and washed three times with 10 mM phosphate buffer (pH 6.0). Fresh substrate solution was introduced for one cycle. After 10 cycles, the remaining enzyme activities of CLSIAs, CLSIAs-BSA and CLSIAs-Tre were 61.5%, 91.7% and 62.8%, respectively.

[0059] Example 8: Process for preparing isomaltulose from cross-linked sucrose isomerase aggregates

[0060] (1) Using a 25% sucrose solution as raw material, 20 U / mL of immobilized enzyme was added, and the reaction was carried out at 35℃ for 24 h. The immobilized enzyme and the reaction solution were separated by filtration. HPLC analysis showed that the sucrose conversion rate before and after the reaction was 85.4%, which was basically consistent with that of the free enzyme (85.3%).

[0061] (2) Using freshly squeezed sugarcane juice as raw material, 20 U / mL of immobilized enzyme was added, and the reaction was carried out at 35℃ for 24 h. The immobilized enzyme and the reaction solution were separated by filtration. HPLC analysis showed that the sucrose conversion rate before and after the reaction was 75.2%, which was 6.8% higher than that of the free enzyme (70.1%).

[0062] Example 9: Other conditions are the same as in Example 8, except for the amount of enzyme added.

[0063] (1) Using a 25% sucrose solution as raw material, 40 U / mL of immobilized enzyme was added, and the reaction was carried out at 35℃ for 24 h. The immobilized enzyme and the reaction solution were separated by filtration. HPLC analysis showed that the sucrose conversion rate before and after the reaction was 87.4%, which was basically consistent with that of the free enzyme (87.3%).

[0064] (2) Using freshly squeezed sugarcane juice as raw material, 40 U / mL of immobilized enzyme was added, and the reaction was carried out at 35℃ for 24 h. The immobilized enzyme and the reaction solution were separated by filtration. HPLC analysis showed that the sucrose conversion rate before and after the reaction was 80.2%, which was 6.8% higher than that of the free enzyme (75.1%).

[0065] Example 10: Other conditions were the same as in Example 8, except the reaction temperature was changed.

[0066] (1) Using a 25% sucrose solution as raw material, 40 U / mL of immobilized enzyme was added, and the reaction was carried out at 25℃ for 24 h. The immobilized enzyme and the reaction solution were separated by filtration. HPLC analysis showed that the sucrose conversion rate before and after the reaction was 86.4%, which was basically consistent with that of the free enzyme (86.3%).

[0067] (2) Using freshly squeezed sugarcane juice as raw material, 40 U / mL of immobilized enzyme was added, and the reaction was carried out at 35℃ for 24 h. The immobilized enzyme and the reaction solution were separated by filtration. HPLC analysis showed that the sucrose conversion rate before and after the reaction was 76.2%, which was 7.2% higher than that of the free enzyme (71.1%).

[0068] Example 11: Other conditions are the same as in Example 8, except that the reaction raw materials are changed.

[0069] Freshly squeezed mango juice was used as the raw material, and 40 U / mL of immobilized enzyme was added. The mixture was reacted at 35°C for 24 h, and the immobilized enzyme and reaction solution were separated by filtration. HPLC analysis showed that the sucrose conversion rate before and after the reaction was 79.2%, which was 6.9% higher than that of the free enzyme (74.1%).

[0070] Example 12: Continuous preparation of isomaltulose

[0071] Using a 25% sucrose solution as a starting material, 20 U / mL of immobilized enzyme was added and reacted at 35°C for 24 h. The immobilized enzyme and reaction solution were separated by filtration. Fresh substrate solution was then added to the immobilized enzyme to continue the reaction, and this process was repeated 5 times. The substrate conversion rate after the reaction was analyzed by HPLC, and the substrate conversion rate after 5 cycles was 85.6%.

[0072] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A cross-linked sucrose isomerase aggregate, characterized in that, The cross-linked sucrose isomerase aggregates were prepared according to the following method: (1) Preparation of crude sucrose isomerase solution; (2) Add precipitant and additive to the crude sucrose isomerase solution from step (1) and stir to precipitate; (3) Add a crosslinking agent to the system after precipitation in step (2) and stir to crosslink; (4) Centrifuge the solution after cross-linking in step (3) to remove the supernatant, and wash repeatedly with buffer solution until no enzyme activity is detected in the washing solution, thus obtaining cross-linked sucrose isomerase aggregates; The precipitant is polyethylene glycol, and the final concentration of polyethylene glycol in the crude sucrose isomerase solution is 40%-50%; the crosslinking agent is dextran, and the final concentration of dextran in the system after precipitation is 35 mg / mL; the additive is bovine serum albumin. The crosslinking process is carried out at 3-5℃ and 100-300 rpm for 5-15 hours. The precipitation was carried out at 3-5℃ and 100-300 rpm for 10-120 min.

2. The cross-linked sucrose isomerase aggregate as described in claim 1, characterized in that, The crude sucrose isomerase solution was added at a ratio of 20-40 U / mL.

3. The application of the cross-linked sucrose isomerase aggregate according to claim 1 in the catalytic production of isomaltulose, characterized in that, Cross-linked sucrose isomerase aggregates were used as catalysts to prepare isomaltulose from raw materials. The substrate conversion rate was determined by HPLC and was found to be over 85%. The raw materials were sucrose, sugarcane juice, or mango juice.

4. The application as described in claim 3, characterized in that, The amount of the cross-linked sucrose isomerase aggregate added is 20-40 U / mL; the catalytic reaction time is 12-48 h, the reaction temperature is 25-45 °C, and the reaction pH is 5-9.

Images