A mutant enzyme of rebaudioside i l379a and its use

Abstract

The present application relates to the technical field of biological catalysis, and discloses an L379A mutant enzyme for preparing rebaudioside I and application thereof, wherein the enzyme is obtained by making the following mutation to UGT76G1: the leucine in the 379th amino acid sequence is mutated into alanine; the enzyme can be applied to the preparation of RI with higher utilization value through in-vitro conversion of RA, and the conversion rate is higher than 50%, and the catalytic conversion enzyme activity is significantly improved by 7 times compared with the original enzyme. The UGT76G1 mutant discovered in the present application has the following advantages: 1) filling the blank: the special enzyme catalyst for realizing the efficient in-vitro synthesis of rebaudioside I (RI) is provided for the first time; 2) efficiency leap: the catalytic conversion rate is greatly improved from the original about 7% to more than 50% (more than 7 times); 3) stable and reliable: the catalytic performance is stable in the cross-scale reaction of 10 mL to 5 L, and excellent industrial application potential is exhibited; and the present application is suitable for popularization and application.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, specifically to an L379A 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 the current process of converting rebaudioside A to rebaudioside I, which has higher utilization value. This invention provides an L379A 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 50%.

[0007] The technical solution of the present invention is as follows:

[0008] In one aspect, the present invention provides an L379A 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: L379A: the leucine in the 379th amino acid sequence of UGT76G1 is mutated to alanine.

[0011] Preferably, the mutation is caused by the site-directed mutagenesis primers shown in sequences SEQ ID NO.2 and SEQ ID NO.3.

[0012] In another aspect, the present invention provides an isolated nucleic acid molecule encoding an enzyme for preparing ribobandibidine I as described above.

[0013] Preferably, the nucleic acid sequence of the isolated nucleic acid molecule is as shown in SEQ ID NO.1, or a meaningless mutation or neutral mutation is generated from the sequence shown in SEQ ID NO.1.

[0014] Another aspect of the present invention provides an expression vector comprising an isolated nucleic acid molecule as described above.

[0015] Another aspect of the present invention provides a host cell comprising the expression vector as described above.

[0016] Preferably, the host cell is Escherichia coli, Bacillus subtilis, Escherichia coli, Aspergillus oryzae, Penicillium, Aspergillus niger, Streptomyces or yeast.

[0017] Another aspect of the present invention provides the use of enzymes, isolated nucleic acid molecules, expression vectors, or host cells for preparing ribobadiidine I as described above in catalyzing the conversion of ribobadiidine A to ribobadiidine 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 L379A mutant, in which the leucine of the 379th amino acid sequence of UGT76G1 is mutated to alanine.

[0021] Preferably, the mutation is caused by the site-directed mutagenesis primers shown in sequences SEQ ID NO.2 and SEQ ID NO.3.

[0022] According to a preferred embodiment, the biocatalyst is selected from pure protein molecules, expression systems containing the expression vectors as described above, or lysates or whole-cell suspensions of host cells as described above.

[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 50%-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. An L379A mutant enzyme for preparing riboside I and its application. This invention fills the technical gap in in vitro culture of RI and lays the foundation for the industrial application of RI.

[0027] 2. An L379A mutant enzyme for preparing ribobandiside I and its application. The core beneficial effect of this invention lies in the fundamental modification of the catalytic performance of the UGT76G1 enzyme. The modified mutant exhibits a revolutionary improvement in the conversion efficiency of ribobandiside A to ribobandiside I: the original conversion rate of less than 10% (approximately 7%) is stably increased to no less than 50%, a relative improvement of more than 7 times. This significant performance leap not only proves the effectiveness of the mutant site design but also makes high-efficiency, targeted synthesis of RI possible. Furthermore, verification from small-scale (10 mL) to pilot-scale (5 L) shows that this highly efficient catalytic performance has excellent stability for large-scale production, fully meeting the core requirements of enzyme catalysts for industrial production. Attached Figure Description

[0028] Figure 1 A schematic diagram of the gene of a UDP-glycosyltransferase UGT76G1 constructed on the pETDuet plasmid;

[0029] Figure 2A schematic diagram showing the results of polyacrylamide gel electrophoresis of the enzyme solution.

[0030] Figure 3 A schematic diagram showing the results of gel electrophoresis of the enzyme solution. Detailed Implementation

[0031] 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.

[0032] 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.

[0033] The gene sequence of UDP-glycosyltransferase UGT76G1 was obtained from NCBI and synthesized artificially by a biotechnology company.

[0034] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0035] Example 1: Preparation of the UGT76G1 mutant

[0036] The gene for UDP-glycosyltransferase UGT76G1 was constructed in the pETDuet plasmid (purchased from EMD Biosciences (Novagen)) vector (e.g.) Figure 1Subsequently, 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.

[0037] Table 1

[0038]

[0039] 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).

[0040] The sequence of the UGT76G1 mutant is shown in SEQ ID NO.1.

[0041] Enzyme solution collection:

[0042] Stock solution preparation: Inoculated strains were cultured overnight;

[0043] 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... 600Once 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.

[0044] 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.

[0045] Example 2: Enzyme activity assay for preparing riboflavin I from the UGT76G1 mutant:

[0046] In a 10 ml reaction system, 0.3 g of RA and 0.9 g of sucrose (excess) were weighed out. Following a dosage of 20 mg of enzyme protein, the enzyme solution prepared in Example 1, 20 mg of sucrose synthase SUS1, and buffer (citric acid-phosphate (pH 8.0)) were added. The reaction was carried out at 35 °C 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, the unmutated UDP-glycosyltransferase UGT76G1 enzyme solution was used for catalysis, with other conditions the same as the experimental group. The results are shown in Table 2 below. The table shows that the enzyme activity obtained after site mutation for the preparation of rebaudioside I was higher than that of the original enzyme.

[0047] Table 2

[0048]

[0049] Example 3: Preparation of Rebaudioside I from UGT76G1 mutant

[0050] In a 100ml reaction system, 3g of RA and 9g of sucrose (excess) were weighed out, and the enzyme solution prepared in Example 1 and 0.2g of sucrose synthase SUS1 were added according to the dosage of 0.2g of enzyme protein. 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.

[0051] Table 3

[0052]

[0053] Example 4: Preparation of Rebaudioside I from UGT76G1 mutant

[0054] In a 100ml reaction system, 3g of RA and 4.5g of sucrose (excess) were weighed out. The enzyme solution prepared in Example 1 and 0.3g of sucrose synthase SUS1 were added according to the dosage of 0.3g of enzyme protein. 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.

[0055] Table 4

[0056]

[0057] Example 5: Preparation of Rebaudioside I from UGT76G1 mutant

[0058] In a 100ml reaction system, 3g of RA and 12g of sucrose (excess) were weighed out, and then the enzyme solution prepared in Example 1 and 0.1g of sucrose synthase SUS1 were added according to the dosage of 0.1g of enzyme protein. 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.

[0059] Table 5

[0060]

[0061] Example 6: Preparation of Rebaudioside I from UGT76G1 mutant

[0062] In a 100ml reaction system, 3g of RA and 6g of sucrose (excess) were weighed out, and the enzyme solution prepared in Example 1 and 0.15g of sucrose synthase SUS1 were added according to the dosage of 0.15g of enzyme protein. 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.

[0063] Table 6

[0064]

[0065] Example 7: Preparation of Rebaudioside I from UGT76G1 mutant

[0066] The scale-up experiment was conducted by scaling up the materials according to the proportions in Example 2.

[0067] 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 and 16.7 g of sucrose synthase SUS1 were added according to the dosage of 16.7 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 7 below. The mutated enzyme catalyzed the conversion of rebaudioside A to rebaudioside I with higher efficiency than the original enzyme.

[0068] Table 7

[0069]

[0070] Example 8: Preparation of Rebaudioside I from UGT76G1 mutant

[0071] The scale-up experiment was conducted by scaling up the materials according to the proportions in Example 2.

[0072] 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 and 20 g of sucrose synthase SUS1 were added according to the dosage of 20 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 8 below. The mutated enzyme has a higher efficiency in catalyzing the conversion of RA to RI than the original enzyme.

[0073] Table 8

[0074]

[0075] Example 9: Preparation of Rebaudioside I from UGT76G1 mutant

[0076] The scale-up experiment was conducted by scaling up the materials according to the proportions in Example 2.

[0077] 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 has a higher efficiency in catalyzing the conversion of RA to RI than the original enzyme.

[0078] Table 9

[0079]

[0080] In summary, the L379A 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.

[0081] The sequence of the UGT76G1 mutant (L379A) ​​(SEQ ID NO.1):

[0082]

[0083] 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. An L379A mutant enzyme for preparing riboflavin I, characterized in that, It is a mutant of UGT76G1, obtained by the following mutations in UGT76G1: The leucine at position 379 of UGT76G1 is mutated to alanine; The nucleic acid sequence encoding the L379A mutant enzyme is shown in SEQ ID NO.

1.

2. The application of the L379A mutant enzyme for preparing rebaudioside I as described in claim 1 in the catalytic conversion of rebaudioside A to rebaudioside I.

3. An isolated nucleic acid molecule, characterized in that, The enzyme for preparing ribobandi glycoside I as described in claim 1 is encoded.

4. An expression carrier, characterized in that, It includes an isolated nucleic acid molecule as described in claim 3.

5. A host cell, characterized in that, It includes an expression vector as described in claim 4.

6. A host cell according to claim 5, characterized in that, The cells are one of Bacillus subtilis, Escherichia coli, Aspergillus oryzae, Penicillium, Aspergillus niger, Streptomyces, or yeast.

7. 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 is the L379A mutant of UGT76G1. The specific mutation of the L379A mutant is that the leucine in the 379th amino acid sequence of UGT76G1 is mutated to alanine. The nucleic acid sequence encoding the L379A mutant enzyme is shown in SEQ ID NO.1.

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