UDP-glucosyltransferase mutant and its application and preparation method of stevioside rebaudioside D

Abstract

The present invention belongs to the field of biotechnology, and discloses a UDP-glucosyltransferase mutant and its application and a method for preparing stevioside rebaudioside D. The UDP-glucosyltransferase mutant provided by the present invention includes an H18P mutation and / or an R370H mutation compared to the original UDP-glucosyltransferase including an amino acid fragment as shown in a sequence such as SEQ ID NO: 1, and the above mutations endow the UDP-glucosyltransferase mutant with a higher specific catalytic activity of converting rebaudioside A to rebaudioside D, and can achieve high-efficiency and sufficient conversion of rebaudioside A to rebaudioside D, which is conducive to the realization of large-scale and high-efficiency production of rebaudioside D, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a UDP-glucosyltransferase mutant and an application thereof and a preparation method of stevioside rebaudioside D. Background Art

[0002] Stevioside, a natural sweetener in stevia, is a steviol diterpene glycoside, commonly known as steviol glycosides (SGs). Stevioside is a mixture of multiple components with high sweetness, low calories, stable properties, etc. It is easily soluble in water, methanol and ethanol. Stevioside mainly includes stevioside, rebaudioside A~O, dulcoside A, rubusoside and stevioside. Among them, stevioside and rebaudioside A have the highest content in stevioside, which are the main components of stevioside flavor, accounting for more than 80% of stevioside, with slow release ability and long duration, and taste very close to sucrose; however, high concentrations of stevioside and rebaudioside A have a bitter taste, which limits their application. Recent studies have found that the increase in the content of rebaudioside D is beneficial to improving the bitter taste of stevioside, and also has a taste close to sucrose. It is called "next generation stevioside" and has been widely used.

[0003] At present, the production process of rebaudioside D mainly includes extraction method and biocatalysis method. Among them, the extraction method refers to extracting, separating and purifying rebaudioside D from stevia; however, the content of rebaudioside D in stevia is extremely low (<0.5%), which limits the large-scale production of rebaudioside D. The biocatalysis method refers to the technology of using enzymes or microbial cells as biocatalysts to catalyze reactions to synthesize rebaudioside D. Compared with the extraction method, it has a higher rebaudioside D yield, and therefore has been more widely used in the production of rebaudioside D.

[0004] However, the UDP-glucosyltransferase used in the biocatalytic method often has the disadvantages of low enzyme activity and low conversion efficiency, which leads to high costs for its application in industrial large-scale production of rebaudioside D.

[0005] Therefore, it is urgent to upgrade and transform UDP-glucosyltransferase to improve the biocatalytic synthesis efficiency of rebaudioside D. Summary of the invention

[0006] The first object of the present invention is to provide a UDP-glucosyltransferase mutant. Compared with the original UDP-glucosyltransferase, the UDP-glucosyltransferase mutant has a higher catalytic activity of catalyzing the conversion of rebaudioside A into rebaudioside D. When applied to the preparation of rebaudioside D, the highly efficient synthesis of rebaudioside D can be well achieved, and the mutant has good application prospects.

[0007] The second object of the present invention is to provide a nucleic acid molecule.

[0008] The third object of the present invention is to provide a recombinant strain.

[0009] The fourth object of the present invention is to provide a complex enzyme preparation.

[0010] The fifth object of the present invention is to provide the use of the above-mentioned UDP-glucosyltransferase mutant, nucleic acid molecule, recombinant strain or complex enzyme preparation in the preparation of rebaudioside D.

[0011] The sixth object of the present invention is to provide a method for preparing rebaudioside D.

[0012] Specifically, the UDP-glucosyltransferase mutant provided by the present invention comprises an H18P mutation and / or an R370H mutation compared to the UDP-glucosyltransferase; wherein the UDP-glucosyltransferase comprises an amino acid fragment having a sequence as shown in SEQ ID NO: 1.

[0013] Furthermore, the UDP-glucosyltransferase mutant includes an amino acid fragment having a sequence as shown in SEQ ID NO:10.

[0014] Furthermore, the UDP-glucosyltransferase mutant includes an amino acid fragment having a sequence as shown in SEQ ID NO:11.

[0015] Furthermore, the UDP-glucosyltransferase mutant includes an amino acid fragment whose sequence is shown in SEQ ID NO:12.

[0016] The nucleic acid molecule provided by the present invention comprises a nucleotide fragment encoding the above-mentioned UDP-glucose transferase mutant.

[0017] The recombinant strain provided by the invention synthesizes and expresses the above-mentioned UDP-glucose transferase mutant.

[0018] Furthermore, the chassis cells of the recombinant strain are selected from one or more of Escherichia coli DH5α, Escherichia coli TOP10, Escherichia coli BL21 and Escherichia coli BL21 (DE3).

[0019] The composite enzyme preparation provided by the invention comprises the above-mentioned UDP-glucose transferase mutant.

[0020] The present invention also provides the use of the above-mentioned UDP-glucose transferase mutant, nucleic acid molecule, recombinant strain or composite enzyme preparation in the preparation of rebaudioside D.

[0021] The preparation method of rebaudioside D provided by the present invention comprises: taking the above-mentioned UDP-glucosyltransferase mutant or composite enzyme preparation and mixing it with a raw material liquid, and performing an enzyme catalytic reaction to obtain the rebaudioside D; wherein the raw material liquid comprises sucrose synthase, rebaudioside A and UDP-glucose.

[0022] Furthermore, in the raw material solution, the concentration of sucrose synthase is 10-40 mM, the concentration of rebaudioside A is 5-20 g / L, and the concentration of UDP-glucose is 5-20 mM.

[0023] Furthermore, when the reagent mixed with the raw material liquid is a complex enzyme preparation, the addition amount of the complex enzyme preparation is 600-800 mg / L based on the amount of protein in the complex enzyme preparation.

[0024] Furthermore, the temperature of the enzyme catalytic reaction is 30-40°C and the time is 24-36 hours.

[0025] Beneficial effects:

[0026] The UDP-glucosyltransferase mutant provided by the present invention has an H18P mutation and / or an R370H mutation compared to the original UDP-glucosyltransferase including an amino acid fragment as shown in SEQ ID NO: 1. The above mutations give the UDP-glucosyltransferase mutant a higher specific catalytic activity for converting rebaudioside A into rebaudioside D, and can achieve efficient and sufficient conversion of rebaudioside A to rebaudioside D, which is conducive to the large-scale and efficient production of rebaudioside D, and has good application prospects.

[0027] In some specific embodiments, a composite enzyme preparation is also provided, which is obtained by fermenting a recombinant strain capable of synthesizing and expressing the UDP-glucosyltransferase mutant and subjecting it to cell disruption treatment. The composite enzyme preparation includes not only the UDP-glucosyltransferase mutant, but also a complete enzyme system in the cells of the recombinant strain. The UDP-glucosyltransferase mutant and the enzyme system cooperate with each other to form an enzyme cascade reaction, which can effectively circulate related substances in the fermentation treatment system, so as to effectively reduce the effect of reduced substrate concentration on the activity of the UDP-glucosyltransferase mutant in catalyzing the conversion of rebaudioside A to rebaudioside D, and can further improve the conversion efficiency and conversion rate of rebaudioside A to rebaudioside D. DETAILED DESCRIPTION

[0028] The amino acid and nucleotide sequences involved in the present invention are specifically shown in Table 1.

[0029] Table 1.

[0030]

[0031] Based on a deep understanding of the catalytic mechanism of the original UDP-glucosyltransferase catalyzing the conversion of rebaudioside A to rebaudioside D and the mechanism of substrate recognition, the inventors of the present invention creatively discovered through extensive and in-depth research and a large number of experiments that on the basis of the structure of the original UDP-glucosyltransferase, specific sites and specific types of mutations were introduced, and the substrate binding region in the resulting mutant had strict recognition and binding capabilities of rebaudioside A, and the catalytic active region had catalytic activity of catalyzing glycosylation reactions to form β-1,2 glycosidic bonds, thereby giving the mutant highly specific and efficient catalytic activity of converting rebaudioside A to rebaudioside D. Based on this, the technical solution of the present invention was obtained.

[0032] In the present invention, the UDP-glucosyltransferase has a typical structural feature of glycosyltransferases of the GT-B fold family, specifically including an amino acid fragment as shown in SEQ ID NO: 1 or a variant sequence having at least 90% homology with the sequence shown in SEQ ID NO: 1. The variant sequence is obtained by substituting, deleting or adding one or more amino acids of the amino acid fragment as shown in SEQ ID NO: 1, and the substitution, deletion or addition does not affect the biological activity of the UDP-glucosyltransferase.

[0033] In the present invention, the amino acid residue positions are numbered with reference to the amino acid sequence shown in SEQ ID NO: 1, and the UDP-glucosyltransferase mutant comprises an H18P mutation and / or an R370H mutation compared to the original UDP-glucosyltransferase.

[0034] In the present invention, the H18P mutation is defined as a mutation of the amino acid residue at position 18 of the original UDP-glucosyltransferase from histidine to proline.

[0035] In the present invention, the R370H mutation is defined as a mutation of the amino acid residue at position 370 of the original UDP-glucosyltransferase from arginine to histidine.

[0036] In some specific embodiments, the UDP-glucosyltransferase mutant has an H18P mutation compared to the original UDP-glucosyltransferase; in this case, the UDP-glucosyltransferase mutant specifically includes an amino acid fragment as shown in SEQ ID NO: 10 or a variant sequence having at least 90% homology with the sequence shown in SEQ ID NO: 10. The variant sequence is obtained by substituting, deleting or adding one or more amino acids of the amino acid fragment as shown in SEQ ID NO: 10, and the substitution, deletion or addition does not affect the biological activity of the UDP-glucosyltransferase mutant.

[0037] In some specific embodiments, the UDP-glucosyltransferase mutant has a R370H mutation compared to the original UDP-glucosyltransferase; in this case, the UDP-glucosyltransferase mutant specifically includes an amino acid fragment as shown in SEQ ID NO: 11 or a variant sequence having at least 90% homology with the sequence shown in SEQ ID NO: 11.

[0038] In some specific embodiments, the UDP-glucosyltransferase mutant has an H18P mutation and an R370H mutation compared to the original UDP-glucosyltransferase; in this case, the UDP-glucosyltransferase mutant specifically includes an amino acid fragment as shown in SEQ ID NO: 12 or a variant sequence having at least 90% homology with the sequence shown in SEQ ID NO: 12.

[0039] Based on the purpose of obtaining the above UDP-glucosyltransferase mutant, the present invention also provides a nucleic acid molecule comprising a nucleotide fragment encoding the above UDP-glucosyltransferase mutant.

[0040] In the present invention, the nucleic acid molecule is limited to being able to guide the synthesis of a UDP-glucosyltransferase mutant of a corresponding structure. Those skilled in the art can design the nucleotide sequence of the nucleic acid molecule according to the structure of the desired UDP-glucosyltransferase mutant and the host cell used, and the present invention does not impose any particular limitation thereto.

[0041] In some specific embodiments, when the desired UDP-glucosyltransferase mutant comprises an amino acid fragment having a sequence as shown in SEQ ID NO:10, and the host cell expressing the UDP-glucosyltransferase mutant is Escherichia coli, the nucleic acid molecule preferably comprises a nucleotide fragment having a sequence as shown in SEQ ID NO:7.

[0042] In some specific embodiments, when the desired UDP-glucosyltransferase mutant comprises an amino acid fragment having a sequence as shown in SEQ ID NO:11, and the host cell expressing the UDP-glucosyltransferase mutant is Escherichia coli, the nucleic acid molecule preferably comprises a nucleotide fragment having a sequence as shown in SEQ ID NO:8.

[0043] In some specific embodiments, when the desired UDP-glucosyltransferase mutant comprises an amino acid fragment having a sequence as shown in SEQ ID NO:12, and the host cell expressing the UDP-glucosyltransferase mutant is Escherichia coli, the nucleic acid molecule preferably comprises a nucleotide fragment having a sequence as shown in SEQ ID NO:9.

[0044] In the present invention, in addition to the coding gene of the above-mentioned UDP-glucosyltransferase mutant, the nucleic acid molecule preferably also includes nucleotide sequences that realize different functions, which may specifically be but are not limited to one or more of a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, a homologous recombination site, an NdeⅠ restriction site and an XhoⅠ restriction site.

[0045] Based on the purpose of obtaining the above UDP-glucosyltransferase mutant, the present invention also provides a recombinant strain that can synthesize and express the above UDP-glucosyltransferase mutant.

[0046] In the present invention, the recombinant strain is limited to the UDP-glucosyltransferase mutant capable of synthesizing the corresponding structure. Those skilled in the art can use the existing conventionally used genetically engineered strains as chassis cells according to the structure of the desired UDP-glucosyltransferase mutant, and use genetic engineering technology to introduce the coding gene of the corresponding UDP-glucosyltransferase mutant to construct a recombinant strain, and the present invention does not impose any particular limitation on it.

[0047] In some specific embodiments, specific examples of chassis cells of the recombinant strain include but are not limited to: one or more of Escherichia coli DH5α, Escherichia coli TOP10, Escherichia coli BL21 and Escherichia coli BL21 (DE3).

[0048] Based on the excellent biocatalytic synthesis efficiency of the above-mentioned UDP-glucosyltransferase mutant, the inventor creatively discovered in the process of purifying the protein expressed by the recombinant strain to obtain the pure enzyme preparation of the UDP-glucosyltransferase mutant: directly using the whole cell fragmentation liquid of the recombinant strain and sucrose synthase to carry out the enzyme catalytic reaction of rebaudioside A can achieve better synthesis efficiency and conversion rate of rebaudioside D. Therefore, the present invention further provides a composite enzyme preparation, which includes the above-mentioned UDP-glucosyltransferase mutant.

[0049] In the present invention, the preparation of the composite enzyme preparation specifically includes: fermenting the above-mentioned recombinant strain and then performing cell disruption treatment to obtain the composite enzyme preparation.

[0050] In the invention, the cell disruption process is a technical means commonly used in the existing biological field, which is limited to achieving sufficient release of intracellular enzyme proteins. The present invention does not impose any special limitation on it. Specific examples include but are not limited to: one or more of mechanical disruption, ultrasonic disruption, osmotic pressure mutation and freeze-thaw method.

[0051] Based on the excellent biocatalytic synthesis efficiency of the above-mentioned UDP-glucosyltransferase mutant, the present invention also provides the use of the above-mentioned UDP-glucosyltransferase mutant, recombinant strain or composite enzyme preparation in the preparation of rebaudioside D.

[0052] Based on the application potential of the above-mentioned UDP-glucosyltransferase mutant in the preparation of rebaudioside D, the present invention also provides a method for preparing rebaudioside D. The preparation method specifically comprises: taking the above-mentioned UDP-glucosyltransferase mutant or the composite enzyme preparation and mixing it with a raw material liquid, performing an enzyme catalytic reaction, and obtaining the rebaudioside D; wherein the raw material liquid comprises sucrose synthase, rebaudioside A and UDP-glucose.

[0053] In the present invention, in the raw material solution, the concentration of the sucrose synthase is preferably 10-40 mM, such as 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM or any value therebetween; the concentration of the rebaudioside A is preferably 5-20 g / L, such as 5 g / L, 8 g / L, 10 g / L, 15 g / L, 20 g / L or any value therebetween; the concentration of the UDP-glucose is preferably 5-20 mM, such as 5 mM, 7.5 mM, 15 mM, 20 mM or any value therebetween.

[0054] In the present invention, when the reagent mixed with the raw material solution is a UDP-glucosyltransferase mutant, the added amount of the UDP-glucosyltransferase mutant is preferably 600-800 mg / L, such as 600 mg / L, 610 mg / L, 630 mg / L, 680 mg / L, 700 mg / L, 750 mg / L, 800 mg / L or any value therebetween.

[0055] In the present invention, when the reagent mixed with the raw material liquid is a complex enzyme preparation, the added amount of the complex enzyme preparation is preferably 600-800 mg / L, such as 600 mg / L, 625 mg / L, 650 mg / L, 675 mg / L, 700 mg / L, 750 mg / L, 800 mg / L or any value therebetween, based on the amount of protein in the complex enzyme preparation.

[0056] In the present invention, the conditions of the enzyme-catalyzed reaction specifically include a temperature of preferably 30-40°C, such as 30°C, 32°C, 33°C, 35°C, 37°C, 39°C, 40°C or any value therebetween; and a time of preferably 4-48h, such as 4h, 8h, 12h, 16h, 36h, 40h, 48h or any value therebetween.

[0057] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0058] Example 1

[0059] This example is used to illustrate the construction method of the recombinant strain, which specifically includes:

[0060] (1) Based on the original amino acid sequence of UDP-glucosyltransferase (as shown in SEQ ID NO: 1), Escherichia coli codon optimization was performed, and a biological company was commissioned to synthesize the coding gene of UDP-glucosyltransferase with the sequence shown in SEQ ID NO: 2.

[0061] (2) The UDP-glucosyltransferase coding gene was subjected to PCR site-directed mutagenesis using the mutation primers shown in Table 2 to obtain the coding gene of the UDP-glucosyltransferase H18P mutant (as shown in SEQ ID NO:7), the coding gene of the UDP-glucosyltransferase R370H mutant (as shown in SEQ ID NO:8), and the coding gene of the UDP-glucosyltransferase H18P / R370H mutant (as shown in SEQ ID NO:9).

[0062] Table 2.

[0063]

[0064] The PCR reaction system includes: 2 μL DNA template, 0.5 μL forward primer, 0.5 μL reverse primer, 4 μL 5× reaction buffer (Nearshore Biotechnology, catalog number NR005), 1 μL NovoRec Plus recombinase (Nearshore Biotechnology, catalog number NR005), ddH 2 Make up to 20 μL with HO.

[0065] The PCR reaction program included: 95°C for 3 min; 55°C for 42 s, 70°C for 3 min, 29 cycles; and insulation at 4°C.

[0066] (3) BamHⅠ and HindIIIⅠ restriction sites were added to both ends of the above-mentioned UDP-glucosyltransferase coding gene, UDP-glucosyltransferase H18P mutant coding gene, UDP-glucosyltransferase R370H mutant coding gene, and UDP-glucosyltransferase H18P / R370H mutant coding gene, respectively, and then synthesized, and then digested with BamHⅠ and HindIIIⅠ and recombined into pET24a vector to obtain the corresponding recombinant plasmid.

[0067] (4) The recombinant plasmids obtained above were transformed into E. coli TOP10 competent cells and spread onto LB solid medium (Solebo, Catalog No. L1015) containing 100 μg / mL ampicillin. The cells were cultured at 37°C overnight. Some cells from each single colony were taken for PCR identification and sequencing. The single colony with correct amplification was inoculated into LB liquid medium (Solebo, Catalog No. L1015) containing 100 μg / mL ampicillin to extract a large amount of recombinant plasmid.

[0068] (5) The recombinant plasmids obtained above were transformed into E. coli BL21 (DE3) competent cells, and spread on LB solid culture medium containing 100 μg / mL ampicillin, and cultured overnight at 37°C. Some cells from each single colony were taken for PCR identification and sequencing to obtain recombinant strains WT, UGT-H18P, UGT-R370H and UGT-H18P / R370H. The specific amino acid sequences of UDP-glucosyltransferase (mutant) that can be synthesized and expressed by each recombinant strain are shown in Table 3.

[0069] Table 3.

[0070]

[0071] Example 2

[0072] This example is used to illustrate the preparation method of the complex enzyme preparation, which specifically includes:

[0073] (1) According to the inoculation amount of 5%, the recombinant strains WT, UGT-H18P, UGT-R370H and UGT-H18P / R370H provided in Example 1 were inoculated into LB liquid culture medium respectively, and cultured at 37°C and 180 rpm for 16 h. Then, IPTG with a final concentration of 0.5 mM was added, and cultured at 37°C and 180 rpm for 18 h to obtain the corresponding fermentation broth.

[0074] (2) The fermentation broths were centrifuged at 220 rpm to collect the bacterial cells; the bacterial cells were mixed with Tris-HCl buffer (pH=7.0) at a mass volume ratio of 1:10 to obtain a bacterial suspension; the bacterial suspension was subjected to cell disruption by an ultrasonic disruptor to obtain the corresponding cell disruption solution, i.e., composite enzyme preparations 1 to 4. The UDP-glucosyltransferase (mutant) included in each composite enzyme preparation is specifically shown in Table 4.

[0075] Table 4.

[0076]

[0077] Example 3

[0078] This example is used to illustrate the application of the composite enzyme preparation provided in the above examples in the production of rebaudioside D, specifically including:

[0079] Each composite enzyme preparation was mixed with the raw material solution at an addition amount of 750 mg / L (based on the amount of protein in the composite enzyme preparation), and the enzyme catalyzed reaction was carried out at 37°C for 36 hours. Samples were taken every 4 hours for HPLC detection of the concentration of rebaudioside D, and the rebaudioside D conversion rate (%) was calculated according to the following formula. The results are shown in Table 5.

[0080] Conversion rate = (molar amount of rebaudioside produced / molar amount of rebaudioside A input) × 100%

[0081] The raw material solution includes 10 g / L rebaudioside A, 10 mM UDP-glucose, 20 mM sucrose synthase (Shanghai Biotech, product number D799786) and 10 mM Tris-HCl buffer (pH=7.0).

[0082] The conditions for HPLC detection include: the chromatographic column is an Agilent Zorbax SB-C18 chromatographic column (4.6mm×250mm, 5μm); the mobile phase is acetonitrile-sodium phosphate buffer (pH=2.60, 27:73v / v); the flow rate is 1.0mL / min; the column temperature is 40℃; the injection volume is 10μL; and the detection wavelength is 210nm.

[0083] Table 5.

[0084]

[0085] From the test results shown in Table 5, it can be seen that compared with the original UDP-glucosyltransferase, the UDP-glucosyltransferase mutant introduced with the H18P mutation and / or the R370H mutation has a better biocatalytic synthesis efficiency, so as to achieve the efficient preparation of rebaudioside D; and the preparation of the composite enzyme provided by the present invention does not require complicated protein purification operations, has the advantages of low production cost, high production efficiency, etc., and has excellent potential for industrial application.

[0086] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A UDP-glucosyltransferase mutant, characterized in that: The amino acid sequence of the UDP-glucosyltransferase mutant is shown in SEQ ID NO:

10.

2. A UDP-glucosyltransferase mutant, characterized in that: The amino acid sequence of the UDP-glucosyltransferase mutant is shown in SEQ ID NO:

11.

3. A UDP-glucosyltransferase mutant, characterized in that: The amino acid sequence of the UDP-glucosyltransferase mutant is shown in SEQ ID NO:

12.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the UDP-glucosyltransferase mutant according to any one of claims 1 to 3.

5. A recombinant strain, characterized in that: The recombinant strain synthesizes and expresses the UDP-glucosyltransferase mutant according to any one of claims 1 to 3.

6. A composite enzyme preparation, characterized in that: The composite enzyme preparation comprises the UDP-glucosyltransferase mutant according to any one of claims 1 to 3.

7. Use of the UDP-glucosyltransferase mutant according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant strain according to claim 5 or the complex enzyme preparation according to claim 6 in the preparation of rebaudioside D.

8. A method for preparing rebaudioside D, characterized in that: The preparation method comprises: mixing the UDP-glucosyltransferase mutant described in any one of claims 1 to 3 or the complex enzyme preparation described in claim 6 with a raw material solution, and performing an enzyme-catalyzed reaction to obtain the rebaudioside D; wherein the raw material solution comprises sucrose synthase, rebaudioside A and UDP-glucose.

9. The method for preparing rebaudioside D according to claim 8, characterized in that: In the raw material solution, the concentration of sucrose synthase is 10-40 mM, the concentration of rebaudioside A is 5-20 g / L, and the concentration of UDP-glucose is 5-20 mM.

10. The method for preparing rebaudioside D according to claim 8, characterized in that: When the reagent mixed with the raw material liquid is a complex enzyme agent, the added amount of the complex enzyme agent is 600-800 mg / L based on the amount of protein in the complex enzyme agent.

11. The method for preparing rebaudioside D according to claim 8, characterized in that: The temperature of the enzyme catalytic reaction is 30-40° C. and the time is 4-48 hours.

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