Immobilized enzyme composition and application thereof in efficient preparation of rebaudioside M

Abstract

The invention relates to the technical field of industrial microorganisms and food industry, and discloses an immobilized enzyme composition and application thereof in efficient preparation of rebaudioside M. The immobilized enzyme composition provided by the invention comprises immobilized UDP-glycosyl transferase UGT and immobilized UDP-glycosyl transferase UGT76G1 according to a mass ratio of (1 to 2): (1 to 3). By adopting the immobilized enzyme composition disclosed by the invention, the rebaudioside A can be synergistically and efficiently promoted to be converted into the rebaudioside M, and the conversion rate is up to 85% or above and can be up to 97% at most; the working efficiency of the enzyme is remarkably improved, and the rebaudioside M is efficiently prepared; meanwhile, the enzyme is good in stability and can be repeatedly used for at least five times, and the catalytic efficiency is still higher than 95%. The method has great popularization and application values.

Description

Technical Field

[0001] The present invention relates to the fields of industrial microorganisms and the food industry, and relates to an immobilized enzyme composition and its application in the efficient preparation of rebaudioside M. Specifically, it relates to the use of two immobilized glycosyltransferases for the production of rebaudioside M. Background Art

[0002] The statements in this section only provide background information related to the disclosure of the present application and may not constitute prior art.

[0004] In Stevia rebaudiana, stevioside and Reb A are the most abundant steviol glycosides, accounting for 5 - 10% and 2 - 4% (w / w) of the dry weight of the leaves respectively. Reb M has attracted wide interest due to its high sweetness and low bitterness, and thus has a higher price than Reb A. However, the content of Reb M in Stevia rebaudiana is relatively low (about 0.4 - 0.5% w / w). The production of Reb M using traditional extraction methods requires a large amount of Stevia rebaudiana raw materials, and the process is cumbersome. After extraction, it requires multiple column chromatographies and desalination, decolorization, and recrystallization, and generates a large amount of wastewater during the production process, resulting in a high production cost and being not suitable for large-scale industrial production.

[0005] In recent years, the in vitro biosynthesis of Reb M has attracted extensive research. Currently, free enzymes are mainly used to catalyze the conversion of Reb A to Reb M. In the cost calculation of the catalytic synthesis of Reb M, the production cost of the enzyme accounts for the vast majority of the total cost, thus resulting in poor economy and lack of market competitiveness in the enzyme-catalyzed synthesis of Reb M from Reb A. Summary of the Invention

[0006] The purpose of the present invention is: aiming at the deficiencies of the current prior art, the present invention provides an immobilized enzyme composition and its application in the efficient preparation of rebaudioside M. UDP-glycosyltransferase and sucrose synthase are prepared using an engineered bacterium capable of overexpressing UDP-glycosyltransferase or sucrose synthase, and then the enzymes are immobilized on a fixed carrier to efficiently catalyze the conversion of Reb A to prepare Reb M by the immobilized enzymes. This method has a high conversion rate for the synthesis of rebaudioside M, is economically reliable, and is suitable for the production of rebaudioside M.

[0007] The technical solution of the present invention is as follows: An immobilized enzyme composition, comprising immobilized UDP-glycosyltransferase UGT and immobilized UDP-glycosyltransferase UGT76G1 in a mass ratio of (1 - 2) : (1 - 3).

[0008] According to a preferred embodiment, the source of the UGT gene is Panax ginseng, and the source of the UGT76G1 gene is Stevia rebaudiana.

[0009] According to a preferred embodiment, the immobilization carrier of the immobilized enzyme is at least one of LX-201A, NKA, D101, HP-20 and / or AB-8 resin.

[0010] On the other hand, the present invention provides a method for preparing an immobilized enzyme, and the immobilized enzyme is the immobilized UDP-glycosyltransferase UGT and the immobilized UDP-glycosyltransferase UGT76G1 as described above; the method includes the following steps: Step (1): Construct engineering bacteria: separately ligate the UDP-glycosyltransferase (UGT) gene and the UDP-glycosyltransferase (UGT76G1) gene to an expression vector to obtain two recombinant plasmids, and transfer the two recombinant vectors into Escherichia coli respectively to obtain engineering bacteria overexpressing UDP-glycosyltransferase (UGT) and engineering bacteria overexpressing the UDP-glycosyltransferase (UGT76G1) gene.

[0011] Preferably, the UDP-glycosyltransferase gene has the nucleotide sequence shown in SEQ ID NO:1; the UDP-glycosyltransferase (UGT76G1) gene has the nucleotide sequence shown in SEQ ID NO:2.

[0012] Preferably, the expression vector is pET32(a)+, pET28(a)+, pET22(b); the Escherichia coli is BL21(DE3).

[0013] Step (2): Inoculate the engineering bacteria overexpressing UDP-glycosyltransferase (UGT) and the engineering bacteria overexpressing the UDP-glycosyltransferase (UGT76G1) gene into a fermentation medium (TB medium and LB medium) respectively, add an inducer for induced expression, and collect the bacterial cells by low-temperature centrifugation. Add Na 2 HPO 4 -NaH 2 PO 4 buffer for resuspension, ultrasonically or homogenize the bacterial solution under high pressure and then centrifuge to collect the supernatant, which is the crude enzyme solution; and purify the UDP-glycosyltransferase with a protein purification column respectively, collect the purified enzyme and the crude enzyme solution without enzyme protein.

[0014] Preferably, the starting time for adding the inducer is when OD 600 is 1.0 - 1.8. Preferably 1.4.

[0015] Preferably, the TB medium includes: peptone 20 g / L, yeast extract 24 g / L, dipotassium hydrogen phosphate 12.25 g / L, potassium dihydrogen phosphate 2.3 g / L, glycerol 4 g / L.

[0016] Preferably, the LB medium comprises: 10 g / L of peptone, 5 g / L of yeast extract, and 10 g / L of NaCl.

[0017] Preferably, the inducer is IPTG with a working concentration of 0.1 - 0.4 mM.

[0018] Preferably, the low-temperature centrifugation is specifically: at 4 - 10 °C, 3000 - 5000 rpm, for 10 - 20 min.

[0019] Preferably, Na 2 HPO 4 -NaH 2 PO 4 The concentration of the buffer solution is 0.1 - 0.3 mM, and the pH is 7 - 9.

[0020] Preferably, the protein purification column is a His-tag purification column.

[0021] Step (3): Prepare the immobilized enzyme: Mix UDP-glycosyltransferase (UGT, UGT76G1) with the treated immobilization carrier, perform immobilization in a constant-temperature shaker, and obtain the immobilized enzyme of each enzyme through filtration, washing, and drying.

[0022] Preferably, the immobilization temperature is 4 °C - 40 °C, the rotation speed is 100 - 300 rpm, and the immobilization time is 2 - 8 h.

[0023] Preferably, the treatment method of the carrier in step (3) is to soak the immobilization carrier in absolute ethanol at a concentration of 10 - 20 g / L for 8 - 24 h, remove the ethanol (or filter), then wash away all the ethanol with ultrapure water, soak it in a 2% (W / V) NaOH solution for 8 - 24 h, wash it to neutral with ultrapure water, filter, and vacuum extract the excess water. Immerse the filtered immobilization carrier in pure water, add 0.1% - 2% glutaraldehyde, and under the condition of 10 - 20 °C, with a shaker rotation speed of 100 - 300 rpm for 4 - 12 h, filter and wash to obtain the activated resin.

[0024] Preferably, the concentration of UDP-glycosyltransferase (UGT, UGT76G1) in step (3) is 0.5 - 3 mg / mL.

[0025] Preferably, the mixing ratio of UDP-glycosyltransferase (UGT, UGT76G1) to the carrier in step (3) is set such that the enzyme solution can completely immerse the immobilization carrier. Specifically, approximately 10 - 20 g of immobilization carrier corresponds to 100 - 220 ml of enzyme solution.

[0026] Preferably, the immobilization carrier is at least one of LX-201A, NKA, D101, HP-20, and / or AB-8 resin.

[0027] On the other hand, the present invention provides an application of the immobilized enzyme composition in the preparation of Rebaudioside M, and the immobilized enzyme composition is as described above.

[0028] On the other hand, the present invention provides a method for efficiently preparing Reb M by using an immobilized enzyme composition, comprising the following steps: Add Reb A with a final concentration of 1-40 g / L to a container, add uridine diphosphate disodium equivalent to 10%-50% of the molar amount of Reb A, as well as sufficient sucrose and sucrose synthase, and then add the immobilized UDP-glycosyltransferase (UGT, UGT76G1) with a final concentration of 50-150 g / L respectively. The solvent is water, the pH value is 7-9, and the catalytic reaction is carried out at 20-40 °C for 12-48 h to prepare Reb M.

[0029] Preferably, the concentration of Rebaudioside A is 30 g / L. The concentration of uridine diphosphate disodium is 3 g / L. The concentration of sucrose is 120 g / L. The pH value of the reaction solution after mixing is 8.0.

[0030] Preferably, the addition ratio of the immobilized UDP-glycosyltransferase (UGT) and the immobilized UDP-glycosyltransferase (UGT76G1) is (1-2):(1-3). More preferably, it is 1:3 or 2:3.

[0031] It should be understood that the two glycosyltransferases described in the present invention can be used in the form of whole cells of engineering bacteria, or in the form of unpurified crude enzymes, or in the form of partially purified or completely purified enzymes.

[0032] Preferably, the working concentrations of the immobilized enzymes UGT and UGT76G1 are 20 g / L and 60 g / L respectively.

[0033] Preferably, the catalytic reaction temperature is 25-35 °C, more preferably 35 °C; the reaction time is preferably 24-48 h, more preferably 48 h.

[0034] According to the above method, high-purity Rebaudioside M with a purity of ≥95% (w / w) is finally obtained after drying under vacuum conditions.

[0035] Compared with the existing technology, the beneficial effects of the present invention are: 1. By using the immobilized enzyme composition of the present application, it can synergistically and efficiently promote the conversion of Rebaudioside A into Rebaudioside M, with a conversion rate as high as 95%, and up to 97% at the highest; the working efficiency of the enzyme is improved, and the efficient preparation of Rebaudioside M is realized; 2. Through the immobilized enzyme technology, the present invention significantly improves the stability and durability of UDP-glycosyltransferase, enabling it to maintain activity for a long time, achieve continuous catalytic reactions, and the catalytic conversion rate in continuous reactions is not less than 95%, which is conducive to continuous production; 3. Reduction of production costs: Compared with traditional methods, the immobilized enzyme of the present invention can be reused, and even after being reused more than 5 times, the conversion rate of rebaudioside M can still reach more than 95%. The method of the present invention is simple and easy to implement. It reduces the process flow, lowers costs, improves production efficiency, and is more suitable for large-scale production. Specific embodiments

[0036] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or instruments not specified by the manufacturer can be obtained through regular commercial products. To better illustrate the present invention, numerous specific details are given in the following specific embodiments. Those skilled in the art should understand that the present invention can still be implemented without some specific details. In other embodiments, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Without special instructions, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0038] The characteristics and performance of the present invention will be further described in detail below in conjunction with the embodiments.

[0039] Example 1 Preparation of UDP-glycosyltransferase (UGT, UGT76G1) and sucrose synthase (SUS1) Step (1): Preparation of engineering bacteria overexpressing UDP-glycosyltransferase (UGT), engineering bacteria overexpressing the UDP-glycosyltransferase (UGT76G1) gene, and engineering bacteria overexpressing the sucrose synthase (SUS1) gene.

[0040] Using pET 32a(+) as the vector and the UGT, UGT76G1, and SUS1 genes as the target fragments, after digestion with enzymes, purification, and ligation, the pET 32a(+)-UGT / UGT76G1 / SUS1 expression plasmid was constructed. Then, the expression plasmid was transferred into Escherichia coli BL21(DE3) to obtain engineering bacteria overexpressing UDP-glycosyltransferase (UGT, UGT76G1) and sucrose synthase (SUS1) respectively. The source of the SUS1 gene is Arabidopsis thaliana. The sucrose synthase (SUS1) gene has the nucleotide sequence shown in SEQ ID NO:3.

[0041] Step (2): Expression and fermentation of the engineering bacteria of UDP-glycosyltransferase (UGT, UGT76G1) and sucrose synthase (SUS1).

[0042] The single colonies of the engineering bacteria of UDP-glycosyltransferase (UGT, UGT76G1) and sucrose synthase (SUS1) were respectively inoculated into LB medium and cultured overnight at 37°C and 200 rpm, and then inoculated into TB medium at an inoculation amount of 2% with a medium volume of 3 L. When the OD reached about 1.2 at 37°C and 300 rpm, IPTG with a final concentration of 0.2 mM was added, and then the temperature was adjusted to 20°C and cultured for 24 - 36 h. The whole process ensured that the dissolved oxygen in the medium was ≥20% by aeration and adjusting the rotation speed. 600 After the fermentation was completed, centrifugation was carried out at 4°C and 4200 rpm for 15 - 20 min, the supernatant was discarded, and the cell precipitate was collected. The cells were washed twice with a NaHPO₄-NaH₂PO₄ buffer with pH = 8.0 and a concentration of 0.1 Mm. After removing the washing buffer, a NaHPO₄-NaH₂PO₄ buffer 10 times the weight of the bacterial strain was added, and cell disruption was carried out using a high-pressure homogenizer at 4°C under the conditions of 800 bar for 3 times; then centrifugation was carried out to remove the cell debris, and the supernatant was collected to obtain the crude enzyme solutions containing UDP-glycosyltransferase (UGT, UGT76G1) and sucrose synthase (SUS1) respectively.

[0043] Separation and purification of glycosyltransferase: 2 HPO 4 -NaH 2 PO 4 -NaH 2 HPO 4 -NaH 2 PO 4 After removing the washing buffer, a NaHPO₄-NaH₂PO₄ buffer 10 times the weight of the bacterial strain was added, and cell disruption was carried out using a high-pressure homogenizer at 4°C under the conditions of 800 bar for 3 times; then centrifugation was carried out to remove the cell debris, and the supernatant was collected to obtain the crude enzyme solutions containing UDP-glycosyltransferase (UGT, UGT76G1) and sucrose synthase (SUS1) respectively.

[0044] Separation and purification of glycosyltransferase: Ni-NTA affinity chromatography was used to separate and purify the obtained crude enzyme solution. After loading, washing, and elution, the eluate was collected, ultrafiltered, and then desalted to remove imidazole to obtain the pure enzyme solutions of UDP-glycosyltransferase (UGT, UGT76G1) and sucrose synthase (SUS1) for immobilization; the buffer used for washing was the washing buffer, and the buffer used for elution was the elution buffer. An appropriate amount of buffer was used to remove loose miscellaneous proteins, and finally a small amount of elution buffer was used to obtain the target protein.

[0045] The protein concentration and purity were measured using Nanodrop 2000, and then the protein was desalted and secondarily purified using a protein purification instrument with desalting buffer as the mobile phase. Finally, protein gel electrophoresis was used to determine the protein size and purity, and after verification, it was stored at -80 °C.

[0046] Example 2 Immobilization of Glycosyltransferase and Sucrose Synthase 1. Pretreatment of the Carrier After the LX-201A resin (all resins used in this experiment were wet resins) was pretreated and transformed, the LX-201A resin was soaked in absolute ethanol at a concentration of 10 g / L for 20 h, filtered, and then all ethanol was washed away with ultrapure water. Then it was soaked in 2% (W / V) NaOH solution for 22 h, and then washed with ultrapure water until neutral, and the excess water was removed by vacuum pumping. The filtered resin was soaked in pure water, 1% glutaraldehyde was added, and the reaction was carried out at 18 °C and 210 rpm for 4 h. It was washed with deionized water multiple times until neutral and soaked in deionized water for standby.

[0047] 2. Immobilization of Enzymes The protein concentrations in the enzyme solutions of UDP-glycosyltransferase (UGT, UGT76G1) and sucrose synthase (SUS1) were adjusted to 1 mg / mL respectively, the reaction system was 100 mL, 10 g of LX-201A resin was added, and the immobilization was carried out at 4 °C and 210 rpm for 4 h. After immobilization, the enzyme solution was filtered with filter paper, and the change in the enzyme solution concentration before and after immobilization was measured to calculate the immobilization efficiency. The resin was washed with about 10 times the volume of the resin adsorption solution of deionized water to remove the free enzymes that were not immobilized. The immobilized enzyme was air-dried at 20 °C, and the water content was controlled at 10% - 12%, and it was left standing at 4 °C for standby.

[0048] Example 3 Preparation of Rebaudioside M Using Immobilized Glycosyltransferase and Sucrose Synthase 1. Optimization of the Addition Ratio of Immobilized Glycosyltransferase The optimal addition ratio of the immobilized enzyme composition was determined by measuring the content of rebaudioside M produced from the rebaudioside A substrate and calculating the conversion rate of catalyzing rebaudioside A to rebaudioside M.

[0049] Calculate the required amount of sucrose: Rebaudioside A and UDPG (uridine diphosphate glucose) react under the action of UDP-glycosyltransferase (UGT, UGT76G1) to produce rebaudioside M and uridine diphosphate; UDPG is produced by the reaction of sucrose and sucrose synthase SUS1. Calculate the required amount of sucrose based on the demand for UDPG: 3 g of rebaudioside A is approximately 0.0031 mol; 1 mol of rebaudioside A is converted to rebaudioside M, which requires 2 mol of glucose molecules, and 1 sucrose molecule contains 1 glucose and 1 fructose. Therefore, to completely convert 3 g of rebaudioside A to rebaudioside M, 0.0062 mol of sucrose is required, which is 2.122 g. To enable the rapid production of UDPG, 12 g of sucrose is added.

[0050] (1) In a 100 mL enzyme activity assay system solution, weigh 2 g of immobilized enzyme (UGT:UGT76G1 = 1:2) UGT, 4 g of UGT76G1, 3 g of Reb A, 0.3 g of UDP, 12 g of sucrose, 4 g of immobilized sucrose synthase SUS1, with the buffer being citric acid-phosphate (pH 8.0), and react at 35 °C for 48 h. Starting from 24 h, sample every 12 h, take the suspension, measure the content of the product rebaudioside M by HPLC, and calculate the conversion rate of catalyzing rebaudioside A to rebaudioside M.

[0051] (2) In a 100 mL enzyme activity assay system solution, weigh 2 g of immobilized enzyme (UGT: UGT76G1 = 1:3) UGT, 6 g of UGT76G1, 3 g of Reb A, 0.3 g of UDP, 12 g of sucrose, 4 g of immobilized sucrose synthase SUS1, with the buffer being citric acid-phosphate (pH 8.0), and react at 35 °C for 48 h. Starting from 24 h, sample every 12 h, take the suspension, measure the content of the product rebaudioside M by HPLC, and calculate the conversion rate of catalyzing rebaudioside A to rebaudioside M.

[0052] (3) In a 100 mL enzyme activity assay system solution, weigh 4 g of immobilized enzyme (UGT: UGT76G1 = 2:2) UGT, 4 g of UGT76G1, 3 g of Reb A, 0.3 g of UDP, 12 g of sucrose, 4 g of immobilized sucrose synthase SUS1, with the buffer being citric acid-phosphate (pH 8.0), and react at 35 °C for 48 h. Starting from 24 h, sample every 12 h, take the suspension, measure the content of the product rebaudioside M by HPLC, and calculate the conversion rate of catalyzing rebaudioside A to rebaudioside M.

[0053] (4) In a 100 mL enzyme activity assay system solution, weigh out 4 g of immobilized enzyme (UGT: UGT76G1 = 2:3), 6 g of UGT76G1, 3 g of Reb A, 0.3 g of UDP, 12 g of sucrose, and 4 g of immobilized sucrose synthase SUS1 respectively. The buffer is citric acid - phosphate (pH 8.0). React at 35 °C for 48 h. Starting from 24 h, take samples every 12 h. Take the suspension and measure the content of the product rebaudioside M by HPLC, and calculate the conversion rate of catalyzing rebaudioside A to form rebaudioside M.

[0054] The conversion rates of catalyzing rebaudioside A to form rebaudioside M with different proportions of immobilized enzyme addition amounts are shown in Table 1.

[0055] Table 1 Effects of different proportions of immobilized enzyme addition amounts on the conversion rate of rebaudioside M

[0056] 2. Optimization of pH In a 100 mL enzyme activity assay system solution, put the immobilized enzyme into the reactor according to the ratio of UGT:UGT76G1 of 1:3 (2 g of UGT, 6 g of UGT76G1). Adjust the pH of each reactor to 6, 7, 8, 9 respectively (citric acid - phosphate buffer for pH 6.0 - 8.0, boric acid buffer for pH 9.0). Add 3 g of RebA, 0.3 g of uridine diphosphate disodium, 12 g of sucrose, and 4 g of immobilized sucrose synthase SUS1. React at 35 °C for 48 h. Starting from 24 h, take samples every 12 h. Take the supernatant solution and measure the content of the product rebaudioside M by HPLC, and calculate the conversion rate of catalyzing rebaudioside A to form rebaudioside M. The conversion rates of catalyzing rebaudioside A to form rebaudioside M at different pH values are shown in Table 2.

[0057] Table 2 Effects of different pH values on the conversion rate of rebaudioside M

[0058] Example 4 Scale - up experiment to test the effect of immobilized enzyme in preparing rebaudioside M In this example, prepare the immobilized enzyme according to the method of Example 2. Put the immobilized enzyme into a 1 L reaction system according to the ratio of UGT: UGT76G1 of 1:3 (20 g of UGT, 60 g of UGT76G1). Add 30 g of RebA, 3 g of uridine diphosphate disodium, 40 g of immobilized sucrose synthase SUS1, and 120 g of sucrose. React at 35 °C and pH 8.0 for 48 h. Take the suspension and measure the content of the product rebaudioside M by HPLC. At this time, almost all rebaudioside A is converted into rebaudioside M, and the conversion rate is as high as 97.59%. It is close to the small - scale test.

[0059] Example 5 Amplification Experiment to Examine the Role of Immobilized Enzyme in Preparing Rebaudioside M In this example, the immobilized enzyme was prepared according to the method of Example 2. The immobilized enzyme was added to a 5 L reaction system at a ratio of UGT:UGT76G1 of 1:3 (100 g of UGT and 300 g of UGT76G1). 150 g of RebA, 15 g of uridine diphosphate disodium, 200 g of immobilized sucrose synthase SUS1, and 600 g of sucrose were added. The reaction was carried out at 35 °C and pH 8.0 for 48 h. The suspension was taken, and the content of the product Rebaudioside M was determined by HPLC. At this time, the conversion rate of Rebaudioside M was 96.43%, which was close to that of the small-scale experiment.

[0060] Example 6 Amplification Experiment to Examine the Role of Immobilized Enzyme in Preparing Rebaudioside M In this example, the immobilized enzyme was prepared according to the method of Example 2. The immobilized enzyme was added to a 50 L reaction system at a ratio of UGT:UGT76G1 of 1:3 (1000 g of UGT and 3000 g of UGT76G1). 1500 g of RebA, 150 g of uridine diphosphate disodium, 2000 g of immobilized sucrose synthase SUS1, and 6000 g of sucrose were added. The reaction was carried out at 35 °C and pH 8.0 for 48 h. The suspension was taken, and the content of the product Rebaudioside M was determined by HPLC. At this time, the conversion rate of Rebaudioside M was 95.85%, which was close to that of the small-scale experiment.

[0061] Example 7 Recycling of Immobilized Enzyme In this example, Rebaudioside A was catalyzed to produce Rebaudioside M in a 1 L reaction system according to the method of Example 4. Through the first reaction by this method, the conversion rate of Rebaudioside M was as high as 97.59%. The immobilized enzyme was collected by filtration and added to a new reaction for reuse 10 times. The conversion rate of catalyzing Rebaudioside A to produce Rebaudioside M could still reach 95.5% in the 5th time, and the conversion rate of Rebaudioside M was 95.09% in the 6th time. Therefore, the immobilized enzyme of the present invention can be reused at least 5 times in the reaction of catalyzing Rebaudioside A to produce Rebaudioside M, and the conversion rate of Rebaudioside M is at least 95%.

[0062] The above-described embodiments only represent the specific implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application.

[0063] SEQ ID NO:1: SEQ ID NO:2: SEQ ID NO:3:

Claims

1. An immobilized enzyme composition, characterized in that It includes immobilized UDP-glycosyltransferase UGT and immobilized UDP-glycosyltransferase UGT76G1 in a mass ratio of (1-2):(1-3).

2. An immobilized enzyme composition according to claim 1, characterized in that: The immobilized enzyme's immobilized carrier is at least one of LX-201A, NKA, D101, HP-20 and / or AB-8 resins.

3. An immobilized enzyme composition according to claim 1, characterized in that: The gene source of the UDP-glycosyltransferase UGT is ginseng, and the gene source of the UDP-glycosyltransferase UGT76G1 is stevia.

4. A method for preparing an immobilized enzyme, characterized in that: The immobilized enzyme is the UGT and UGT76G1 as described in any one of claims 1 to 3, comprising the following steps: Step S1: 10-20 g of the activated immobilized carrier is added to an enzyme solution with a concentration of 0.5-3 mg / mL for adsorption, cross-linked at 4-40° C. and 100-300 rpm for 2-8 h, then filtered and washed with deionized water to obtain an immobilized enzyme.

5. The method for preparing an immobilized enzyme according to claim 4, characterized in that: The activation method of the fixed carrier is: soak the fixed carrier in anhydrous ethanol at a concentration of 10-20g / L for 8-24h, then filter, wash with water, soak in a NaOH solution for 8-24h, filter, wash with water, add 0.1%-2% glutaraldehyde, shake at 100-300rpm for 2-8h at 10-20°C, filter, wash, and obtain the activated fixed carrier.

6. Use of an immobilized enzyme composition in the preparation of rebaudioside M, characterized in that: The immobilized enzyme composition is as described in any one of claims 1-3.

7. A method for efficiently preparing rebaudioside M using an immobilized enzyme composition, characterized in that: The steps include: Reb A with a final concentration of 1-40 mM is added to the container, uridine diphosphate disodium equivalent to 10%-50% of the molar number of Reb A, sufficient sucrose is added, and then sucrose synthase, immobilized UDP-glycosyltransferase UGT and immobilized UDP-glycosyltransferase UGT76G1 with a final concentration of 50-150 g / L are added respectively, the solvent is water, the pH value is 7-9, and the catalytic reaction is carried out at 20-40° C. for 12-48 hours to prepare Reb M.

8. The method for efficiently preparing rebaudioside M using an immobilized enzyme composition according to claim 7, characterized in that: The addition ratio of the immobilized UDP-glycosyltransferase UGT and the immobilized UDP-glycosyltransferase UGT76G1 is (1-2): (1-3).

9. The method for efficiently preparing rebaudioside M using an immobilized enzyme composition according to claim 8, characterized in that: The addition ratio of the immobilized UDP-glycosyltransferase UGT and the immobilized UDP-glycosyltransferase UGT76G1 is 1:

3.

10. The method for efficiently preparing rebaudioside M using an immobilized enzyme composition according to claim 7, characterized in that: The temperature of the catalytic reaction is 35° C., the time of the enzyme catalytic reaction is 48 h, and the pH value is 8.0.

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