Mutant enzyme for preparing rebaudioside i and application thereof
By performing site-directed mutagenesis on the UGT76G1 enzyme, a highly efficient riboside I mutant enzyme was prepared, solving the problem of low conversion efficiency of riboside A and realizing the efficient synthesis and industrial application of riboside I.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- 成都圆大生物科技有限公司
- Filing Date
- 2026-03-19
- Publication Date
- 2026-07-14
Smart Images
Figure CN121852350B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biocatalysis technology, specifically to a mutant enzyme for preparing ribobandi glycoside I and its application. Background Technology
[0002] The statements in this section provide only background information relevant to the disclosure of this application and may not constitute prior art.
[0003] Rebaudioside I is a natural, calorie-free sweetener isolated from stevia (S. rebaudiana Morita). It is a type of steviol glycoside, with an English name of Rebaudioside I (RI). Its sweetness is approximately 200-300 times that of sucrose, and its sweetness is purer. The bitter aftertaste and licorice-like finish are significantly lower than rebaudioside A (RA) and some other steviol glycosides (such as stevioside). It is characterized by high sweetness and low calories, making it suitable for diabetics or those trying to lose weight. It is stable to heat, acids, and alkalis (it does not decompose below 200℃) and can be used in the baking, beverage, and other food industries. It is not broken down by human digestive enzymes, produces almost no calories, and is excreted unchanged through the kidneys after consumption. It poses no teratogenic or carcinogenic risk, and daily intake does not require strict limits, making it a promising candidate for food applications.
[0004] However, its extremely low content in natural stevia leaves constitutes the primary limitation to its commercial development. In typical dried stevia leaves, the total content of steviol glycosides is approximately 10%-20% (dry weight), while the content of ribobadiin I is usually only 0.2%-0.6% of the dry leaf weight, far lower than ribobadiin A (usually 3%-5%) and steviol glycosides (usually 5%-10%). Even in high-glycoside varieties selected through traditional breeding methods, the increase in ribobadiin I content is very limited. This naturally low abundance results in exceptionally high costs for large-scale isolation and purification of ribobadiin I directly from plant materials, making raw material supply unstable and unable to meet market demand.
[0005] Converting the higher-content rebaudioside A into the more valuable rebaudioside I is an effective method. Conventional methods use enzymes such as β-glucosidase for glycosylation modification, but the conversion process is cumbersome and inefficient, further reducing the utilization rate of the raw materials. A highly efficient biocatalyst determines the final yield of rebaudioside I; therefore, developing high-efficiency biocatalysts is of great value. Summary of the Invention
[0006] The purpose of this invention is to address the problem of low conversion efficiency in converting rebaudioside A to the more valuable rebaudioside I, and to provide a mutant enzyme for preparing rebaudioside I and its application. A new UGT76G1 mutant was discovered, which catalyzes the conversion of rebaudioside A to rebaudioside I with an efficiency higher than 45%.
[0007] The technical solution of the present invention is as follows:
[0008] In one aspect, the present invention provides a mutant enzyme for preparing riboside I, which is a mutant of UGT76G1, and the mutant has at least 80% similarity to UGT76G1.
[0009] Preferably, the mutant has at least 90% similarity to UGT76G1.
[0010] Preferably, the mutant is obtained by the following mutation of the original UGT76G1: the leucine in the 379th amino acid sequence of UGT76G1 is mutated to valine, and the leucine in the 126th amino acid sequence is mutated to alanine.
[0011] Preferably, the mutation is caused by the site-directed mutagenesis primers shown in sequences SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.
[0012] Another aspect of the present invention provides a genetically engineered strain comprising a genetically engineered expression plasmid capable of expressing a mutant enzyme for preparing riboside I as described above, wherein the promoter of the genetically engineered expression plasmid is followed by a nucleic acid sequence encoding a mutant enzyme for preparing riboside I as described above.
[0013] Preferably, the nucleic acid sequence is as shown in SEQ ID NO.1; it can also be a sequence as shown in SEQ ID NO.1 after generating meaningless or neutral mutations.
[0014] Preferably, the genetically engineered strain is Escherichia coli, Bacillus subtilis, Escherichia coli, Aspergillus oryzae, Penicillium, Aspergillus niger, Streptomyces or yeast.
[0015] Another aspect of the present invention provides an isolated nucleic acid molecule encoding an enzyme for preparing ribobandibidine I as described above, wherein the sequence encoding the ribobandibidine contains a meaningless mutation.
[0016] Another aspect of the present invention provides an expression vector comprising an isolated nucleic acid molecule as described above.
[0017] Another aspect of the present invention provides the use of the enzyme, expression vector, or genetically engineered strain for preparing rebaudioside I as described above in catalyzing the conversion of rebaudioside A to rebaudioside I.
[0018] According to a preferred embodiment, the catalysis is in vitro catalysis or in vivo catalysis.
[0019] Another aspect of the present invention provides a method for preparing riboflavin I, comprising the following steps:
[0020] A starting composition containing ribobadiidine A is contacted with a biocatalyst capable of converting ribobadiidine A into ribobadiidine I to prepare ribobadiidine I; the biocatalyst is a mutant of UGT76G1, specifically: the leucine in the 379th amino acid sequence of UGT76G1 is mutated to valine, and the leucine in the 126th amino acid sequence is mutated to alanine.
[0021] According to a preferred embodiment, the biocatalyst is selected from high-purity substances, cell lysates, or whole-cell suspensions.
[0022] According to a preferred embodiment, the cells in the cell lysate or whole-cell suspension are microbial cells. For example, the microbial cells are Escherichia coli.
[0023] According to a preferred embodiment, the starting composition containing ribobadiol A is a plant extract or a pure substance. For example, it is a stevia extract.
[0024] According to a preferred embodiment, the content of the prepared ribobadiin I is 45%-60% of the content of ribobadiin A in the starting composition containing ribobadiin A.
[0025] Compared with existing technologies, the advantages of this invention are:
[0026] 1. Pioneering a New Path: It provides a dedicated enzyme catalyst that enables efficient in vitro synthesis of RI, successfully filling a gap in this technological field and serving as an important cornerstone for the industrial application of RI.
[0027] 2. A significant leap in catalytic efficiency: Under optimized reaction conditions, the mutant significantly enhanced the conversion efficiency of RA to RI. The conversion rate increased dramatically from approximately 7% at the baseline level to the range of 45%-60%, an absolute increase of over 38 percentage points and a relative increase of 6-9 times. This improvement is significant both statistically and in industrial applications.
[0028] 3. Verification of process scalability: Through systematic process validation, the excellent stability and scalability of this mutant were demonstrated. In reaction systems of 10 mL (laboratory scale), 100 mL (process development scale), and 5 L (preliminary scale-up scale), its conversion efficiency for RA remained stable, and the final RI yield consistently remained above 50%, effectively overcoming the common scale-up challenges in biocatalysis and providing solid data support and process assurance for its industrial continuous production. Attached Figure Description
[0029] Figure 1 A schematic diagram of the gene of a UDP-glycosyltransferase UGT76G1 constructed on the pETDuet plasmid;
[0030] Figure 2 A schematic diagram showing the results of polyacrylamide gel electrophoresis of the enzyme solution.
[0031] Figure 3 A schematic diagram showing the results of gel electrophoresis of the enzyme solution. Detailed Implementation
[0032] The specific embodiments listed in this invention are merely examples, and the 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 this invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of this invention should be covered within its scope. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments whose manufacturers are not specified are commercially available conventional products. To better illustrate this invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of this invention.
[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.
[0034] The gene sequence of UDP-glycosyltransferase UGT76G1 was obtained from NCBI and synthesized artificially by a biotechnology company.
[0035] The features and performance of the present invention will be further described in detail below with reference to embodiments.
[0036] Example 1: Preparation of the UGT76G1 mutant
[0037] The gene for UDP-glycosyltransferase UGT76G1 was constructed in the pETDuet plasmid (purchased from EMD Biosciences (Novagen)) vector (e.g.) Figure 1 Subsequently, the mutant plasmid was constructed using the Novizan MutExpress II Fast Mutagenesis Kit V2. Primers for site-directed mutagenesis were designed using the Novizan website. The designed primers are shown in Table 1 below. The target plasmid was amplified using the designed primers via Phanta Max Super-Fidelity DNA Polymerase technology. The amplified products and the original UGT76G1 gene were constructed into the pET32a plasmid vector, respectively, and then transformed into single E. coli DE3 (BL21) competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd.). 70 μL of each culture was spread onto LB agar plates containing Ampicillin (Amp, 50 mg / L) using glass beads and incubated overnight at 37°C.
[0038] Table 1
[0039]
[0040] Recombinant product identification: Five single colonies cultured in LB solid plate medium were randomly selected and added to 1.5 mL or 2 mL centrifuge tubes containing 600 μL of LB liquid medium containing Amp antibiotic. The culture was incubated at 37℃ and 220 rpm for 5-6 h. When the bacterial solution became turbid, 100 μL was aliquoted and sent for sequencing. The remaining bacterial solution was stored at 4℃. The results of each group of mutant sequences were verified. If the mutant was identified correctly, the mutant strain was preserved using 60% glycerol (LB medium formula: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl; 15 g / L agar powder needs to be added to the solid medium).
[0041] The sequence of the UGT76G1 mutant is shown in SEQ ID NO.1.
[0042] Enzyme solution collection:
[0043] Stock solution preparation: Inoculated strains were cultured overnight;
[0044] Inoculate the stock solution into TB medium (2% v / v); (TB medium formulation: peptone 20 g / L, yeast extract 24 g / L, glycerol 4 g / L, dipotassium hydrogen phosphate 12.25 g / L, potassium dihydrogen phosphate 2.3 g / L). Incubate for approximately 4 h until OD... 600 Once the pH reaches 0.7-0.9, add IPTG (0.1 mmol / L) and express for 48 h (180 rpm, 20℃). Collect the bacterial cells; centrifuge at 4℃, 4000 rpm, 10 min to collect the cells; wash the cells once with approximately 100 mL of pure water and once with PBS; add PBS buffer (1:10 with bacterial cells) and mix well, sonicate (15%, sonicate for 4 seconds, pause for 3 seconds, 5 min), and centrifuge twice (10000 rpm, 8 min); the supernatant is the protein solution containing the target enzyme, i.e., the enzyme solution, with an enzyme protein content of 5-10 mg / mL. PBS buffer formulation: NaCl (sodium chloride): 8 g / L, KCl (potassium chloride): 0.2 g / L, Na₂HPO₄ (disodium hydrogen phosphate): 1.44 g / L, KH₂PO₄ (potassium dihydrogen phosphate): 0.24 g / L, adjust pH to 7.4.
[0045] The obtained enzyme solution was subjected to polyacrylamide gel electrophoresis and gel electrophoresis tests to detect the content of target genes and target proteins. The results showed that... Figure 2-3 By comparing the test results with the standard spectrum, it can be seen from the graph that the content of both the target protein and the target DNA is relatively high.
[0046] Example 2: Enzyme activity assay for preparing riboflavin I from the UGT76G1 mutant:
[0047] In a 10 ml reaction system, weigh out 0.3 g RA and 0.9 g sucrose (excess). Add the enzyme solution prepared in Example 1, 20 mg of sucrose synthase SUS1, and buffer (citric acid) according to the dosage of 20 mg of enzyme protein. Phosphoric acid (pH 8.0) was reacted at 35℃ for 16 h (the reaction was incomplete, to observe enzyme activity). The resulting suspension was collected, and the contents of rebaudioside I and rebaudioside A were determined by HPLC. In the control group experiment, unmutated UDP was used. The glycosyltransferase UGT76G1 enzyme solution was used for catalysis, and other conditions were the same as in the experimental group. The results are shown in Table 2 below. As can be seen from the table, the enzyme activity obtained after site mutation for the preparation of rebaudioside I was higher than that of the original enzyme.
[0048] Table 2
[0049]
[0050] Example 3: Preparation of Rebaudioside I from UGT76G1 mutant
[0051] In a 100ml reaction system, 3g of RA and 9g of sucrose (excess) were weighed out, and the enzyme solution prepared in Example 1 was added at a dosage of 0.2g of enzyme protein. 0.2g of sucrose synthase SUS1 was added, and the buffer was citrate-phosphate (pH 8.0). The reaction was carried out at 35℃ for more than 24 hours to ensure complete reaction. The suspension after reaction was taken, and the contents of rebaudioside I and rebaudioside A were determined by HPLC (the control group was catalyzed by unmutated UDP-glycosyltransferase UGT76G1 enzyme solution under the same conditions). The results are shown in Table 3 below. The mutated enzyme catalyzes the conversion of rebaudioside A to rebaudioside I with higher efficiency than the original enzyme.
[0052] Table 3
[0053]
[0054] Example 4: Preparation of Rebaudioside I from UGT76G1 mutant
[0055] In a 100ml reaction system, 3g of RA and 4.5g of sucrose (excess) were weighed out, and the enzyme solution prepared in Example 1 was added at a dosage of 0.3g of enzyme protein. 0.3g of sucrose synthase SUS1 was added, and the buffer was citrate-phosphate (pH 8.0). The reaction was carried out at 35℃ for more than 24 hours to ensure complete reaction. The suspension after reaction was taken, and the contents of rebaudioside I and rebaudioside A were determined by HPLC (the control group was catalyzed by unmutated UDP-glycosyltransferase UGT76G1 enzyme solution under the same conditions). The results are shown in Table 4 below. The mutated enzyme catalyzes the conversion of rebaudioside A to rebaudioside I with higher efficiency than the original enzyme.
[0056] Table 4
[0057]
[0058] Example 5: Preparation of Rebaudioside I from UGT76G1 mutant
[0059] In a 100ml reaction system, 3g of RA and 12g of sucrose (excess) were weighed out, and the enzyme solution prepared in Example 1 was added at a dosage of 0.1g of enzyme protein. 0.1g of sucrose synthase SUS1 was added, and the buffer was citrate-phosphate (pH 8.0). The reaction was carried out at 35℃ for more than 24 hours to ensure complete reaction. The suspension after reaction was taken, and the contents of rebaudioside I and rebaudioside A were determined by HPLC (the control group was catalyzed by unmutated UDP-glycosyltransferase UGT76G1 enzyme solution under the same conditions). The results are shown in Table 5 below. The mutated enzyme catalyzes the conversion of rebaudioside A to rebaudioside I with higher efficiency than the original enzyme.
[0060] Table 5
[0061]
[0062] Example 6: Preparation of Rebaudioside I from UGT76G1 mutant
[0063] In a 100ml reaction system, 3g of RA and 6g of sucrose (excess) were weighed out, and the enzyme solution prepared in Example 1 was added at a dosage of 0.15g of enzyme protein. 0.15g of sucrose synthase SUS1 was added, and the buffer was citrate-phosphate (pH 8.0). The reaction was carried out at 35℃ for more than 24 hours to ensure complete reaction. The suspension after reaction was taken, and the contents of rebaudioside I and rebaudioside A were determined by HPLC (the control group was catalyzed by unmutated UDP-glycosyltransferase UGT76G1 enzyme solution under the same conditions). The results are shown in Table 6 below. The mutated enzyme catalyzes the conversion of rebaudioside A to rebaudioside I with higher efficiency than the original enzyme.
[0064] Table 6
[0065]
[0066] Example 7: Preparation of Rebaudioside I from UGT76G1 mutant
[0067] The scale-up experiment was conducted by scaling up the materials according to the proportions in Example 2.
[0068] A scale-up reaction with a RA concentration of 50 g / L was carried out in a 5 L reaction system. 250 g of RA and 750 g of sucrose (excess) were weighed out, and the enzyme solution prepared in Example 1 was added at a dosage of 16.7 g of enzyme protein. 16.7 g of sucrose synthase SUS1 was added, and the buffer was citrate-phosphate (pH 8.0). The reaction was carried out at 35 °C for more than 24 h to ensure complete reaction. The suspension after reaction was taken, and the contents of rebaudioside I and rebaudioside A were determined by HPLC (the control group was catalyzed by unmutated UDP-glycosyltransferase UGT76G1 enzyme solution under the same conditions). The results are shown in Table 7 below. The mutated enzyme catalyzes the conversion of rebaudioside A to rebaudioside I with higher efficiency than the original enzyme.
[0069] Table 7
[0070]
[0071] Example 8: Preparation of Rebaudioside I from UGT76G1 mutant
[0072] The scale-up experiment was conducted by scaling up the materials according to the proportions in Example 2.
[0073] A scale-up reaction with a RA concentration of 60 g / L was carried out in a 5 L reaction system. 300 g of RA and 900 g of sucrose (excess) were weighed out, and the enzyme solution prepared in Example 1 was added at a dosage of 20 g of enzyme protein. 20 g of sucrose synthase SUS1 was added, and the buffer was citrate-phosphate (pH 8.0). The reaction was carried out at 35 °C for more than 24 h to ensure complete reaction. The suspension after reaction was taken, and the contents of rebaudioside I and rebaudioside A were determined by HPLC (the control group was catalyzed by unmutated UDP-glycosyltransferase UGT76G1 enzyme solution under the same conditions). The results are shown in Table 8 below. The mutated enzyme catalyzed the conversion of rebaudioside A to rebaudioside I with higher efficiency than the original enzyme.
[0074] Table 8
[0075]
[0076] Example 9: Preparation of Rebaudioside I from UGT76G1 mutant
[0077] The scale-up experiment was conducted by scaling up the materials according to the proportions in Example 2.
[0078] A scale-up reaction with a RA concentration of 70 g / L was carried out in a 5 L reaction system. 350 g of RA and 1050 g of sucrose (excess) were weighed out, and the enzyme solution prepared in Example 1 and 23.3 g of sucrose synthase SUS1 were added according to the dosage of 23.3 g of enzyme protein. The buffer was citrate-phosphate (pH 8.0). The reaction was carried out at 35 °C for more than 24 h to ensure complete reaction. The suspension after reaction was taken, and the contents of rebaudioside I and rebaudioside A were determined by HPLC (the control group was catalyzed by unmutated UDP-glycosyltransferase UGT76G1 enzyme solution under the same conditions). The results are shown in Table 9 below. The mutated enzyme catalyzes the conversion of rebaudioside A to rebaudioside I with higher efficiency than the original enzyme.
[0079] Table 9
[0080]
[0081] In summary, the L379V mutant enzyme of this application significantly improved the conversion rate of rebaudioside A to rebaudioside I in different reaction systems compared with the original unmutated UGT76G1. The conversion rate in the scale-up experiment decreased slightly, but still remained at about 50%, which is a very outstanding effect.
[0082] The sequence of the UGT76G1 mutant (L379V+L126A) (SEQ ID NO.1):
[0083]
[0084] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.
Claims
1. A mutant enzyme for preparing riboflavin I, characterized in that, The mutant is a UGT76G1 mutant, with mutation points L379V and L126A. The nucleotide sequence of the UGT76G1 mutant is shown in SEQ ID NO.
1.
2. The application of the mutant enzyme for preparing rebaudioside I as described in claim 1 in catalyzing the conversion of rebaudioside A to rebaudioside I.
3. The application according to claim 2, characterized in that, The catalysis is either in vitro microbial catalysis or in vivo microbial catalysis.
4. A method for preparing riboflavin I, characterized in that, Includes the following steps: A starting composition containing rebaudioside A is contacted with a biocatalyst capable of converting rebaudioside A into rebaudioside I to prepare rebaudioside I; The biocatalyst contains a mutant of UGT76G1, specifically: the leucine at amino acid 379 of the UGT76G1 sequence is mutated to valine, and the leucine at amino acid 126 is mutated to alanine. The nucleotide sequence of the mutant UGT76G1 is shown in SEQ ID NO.
1.
5. The method for preparing ribobandi glycoside I according to claim 4, characterized in that, The biocatalyst is in the form of a high-purity substance, cell lysate, or whole-cell suspension.
6. The method for preparing ribobandi glycoside I according to claim 5, characterized in that, The cells in the cell lysate or whole cell suspension are microbial cells.
7. The method for preparing ribobandi glycoside I according to claim 6, characterized in that, The microbial cells are one of Bacillus subtilis, Escherichia coli, Aspergillus oryzae, Penicillium, Aspergillus niger, Streptomyces, or yeast.
8. The method for preparing ribobandi glycoside I according to claim 4, characterized in that, The starting composition containing ribobadiol A is a plant extract or a pure substance.
9. The method for preparing ribobandi glycoside I according to claim 4, characterized in that, The content of the prepared riboside I is 45%-60% of the content of riboside A in the starting composition containing riboside A.