Momordica charantia lectin and its use

Abstract

The application discloses a momordica grosvenori glycosyltransferase mutant R192A and a construction method and application thereof, and belongs to the technical field of genetic engineering and enzyme engineering. The amino acid sequence of the mutant R192A is shown as SEQ ID NO. 1. In the application, the amino acid at the 192th position of the amino acid sequence of a wild-type momordica grosvenori glycosyltransferase SgUGT94-289-3 is subjected to point mutation, and arginine is replaced by alanine, so that the momordica grosvenori glycosyltransferase mutant R192A is obtained. Research proves that the activity and biosynthesis efficiency of the mutant in catalyzing the synthesis of momordica grosvenori glycoside V from a substrate momordica grosvenori glycoside IV A are significantly improved, the relative yield of the product momordica grosvenori glycoside V is 5 times higher than that of the wild type, the application can meet the application in industrial production, and has important application value.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, and more specifically to the mogroside glycosyltransferase mutant R192A, its construction method, and its application. Background Technology

[0002] Mogrosides are the main active ingredient of the traditional Chinese medicine monk fruit (Luo Han Guo), and are also non-sugar, purely natural sweeteners. Pharmacological studies have shown that mogrosides have antitussive, expectorant, lung-moistening, liver-protecting, anti-inflammatory, anti-cancer, and hypoglycemic effects. Furthermore, mogrosides have become one of the few novel sweeteners with therapeutic functions discovered from traditional Chinese medicine. Mogrosides are a class of cucurbitane-type triterpenoid saponins with mogroside as the aglycone, formed by the linkage of various glucose groups. The sweetness of mogrosides is related to the number and linkage of sugar residues. When the total number of glucose residues linked at positions 3 and 24 of the aglycone mogroside is 4–6 glucose residues, high-intensity sweetness mogrosides IV, IVA, V, VI, and symmenidine I are formed.

[0003] Current research has found that the mogroside glycosyltransferase SgUGT94-289-3 has problems such as substrate heterogeneity and low in vitro catalytic activity, which hinder its application in industrial production.

[0004] In summary, how to provide a glycosyltransferase with high enzyme activity, high substrate specificity, and the ability to meet the requirements of industrial production of mogrosides is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides the mogroside glycosyltransferase mutant R192A, its construction method and application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A mogroside glycosyltransferase mutant R192A has the amino acid sequence shown in SEQ ID NO.1.

[0008] MDAAQQGDTTTILMLPWLGYGHLSAFLELAKSLSRRNFHIYFCSTSVNLDAIKPKLPSSFSDSIQFVELHLPSSPEFPPHLHTTNGLPPTLMPALHQAFSMAAQHFESILQTLA PHLLIYDSLQPWAPRVASSLKIPAINFNTTGVFVISQGLHPIHYPHSKFPFSEFVLHNHWKAMYSTADGASTERTRKAGEAFLYCLHASCSVILINSFRELEGKYMDYLSVLLN KKVVPVGPLVYEPNQDGEDEGYSSIKNWLDKKEPSSTVFVSFGSEYFPSKEEMEEIAHGLEASEVNFIWVVRFPQGDNTSGIEDALPKGFLERAGERGMVVKGWAPQAKILKHW SEQ ID NO.1.

[0009] Conservative substitutions, additions or deletions of one or more amino acids, amino-terminal truncation, and carboxyl-terminal truncation of the mogroside glycosyltransferase mutant R192A are also included within the scope of protection of this invention.

[0010] Furthermore, the amino acid sequence encoded by the monk fruit glycosyltransferase SgUGT94-289-3 was modified by making a point mutation at position 192, replacing arginine with alanine.

[0011] The amino acid sequence encoded by the mogroside glycosyltransferase SgUGT94-289-3 is shown in SEQ ID NO.2.

[0012] MDAAQQGDTTTILMLPWLGYGHLSAFLELAKSLSRRNFHIYFCSTSVNLDAIKPKLPSSFSDSIQFVELHLPSSPEFPPHLHTTNGLPPTLMPALHQAFSMAAQHFESILQTLA PHLLIYDSLQPWAPRVASSLKIPAINFNTTGVFVISQGLHPIHYPHSKFPFSEFVLHNHWKAMYSTADGASTERTRKRGEAFLYCLHASCSVILINSFRELEGKYMDYLSVLLN KKVVPVGPLVYEPNQDGEDEGYSSIKNWLDKKEPSSTVFVSFGSEYFPSKEEMEEIAHGLEASEVNFIWVVRFPQGDNTSGIEDALPKGFLERAGERGMVVKGWAPQAKILKHW SEQ ID NO.2.

[0013] The gene encoding the above-mentioned mogroside glycosyltransferase mutant R192A has the nucleotide sequence shown in SEQ ID NO.3.

[0014]

[0015] Expression vectors containing the above-mentioned genes.

[0016] Furthermore, the original vector was the pET28a plasmid.

[0017] Microorganisms that have been transformed with the above expression vectors.

[0018] Furthermore, the microorganism is Escherichia coli Rosetta (DE3).

[0019] The application of the aforementioned mogroside glycosyltransferase mutant R192A in the synthesis of mogroside.

[0020] Furthermore, the mogroside is mogroside V.

[0021] The method for constructing the above-mentioned mogroside glycosyltransferase mutant R192A includes the following steps:

[0022] (1) Using primers containing point mutation sites, the mutant gene of SgUGT94-289-3 glycosyltransferase from Luo Han Guo was amplified by PCR using the gene as a template.

[0023] The nucleotide sequence of the primer is as follows:

[0024] R192A-F: GCTTCTACCGAAAGAACCCGCAAAGCGGGAGAAGCGTT TCTGTATTGCTTGC, SEQ IDNO.4;

[0025] R192A-R: GCAAGCAATACAGAAACGCTTCTCCCCGCTTTGCGGGTTC TTTCGGTAGAAGC, SEQ IDNO.5;

[0026] The nucleotide sequence of the gene for the mogroside glycosyltransferase SgUGT94-289-3 is shown in SEQ ID NO. 6;

[0027]

[0028] (2) The mutant gene was ligated into the pET28a vector to obtain a recombinant plasmid;

[0029] (3) Expression and purification of the mogroside glycosyltransferase mutant R192A.

[0030] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows: The present invention performs a point mutation at position 192 of the amino acid sequence of the wild-type mogroside IVA SgUGT94-289-3, replacing arginine with alanine, thereby obtaining the mogroside IVA mutant R192A. Studies have shown that the mutant significantly improves the activity and biosynthetic efficiency of catalyzing the synthesis of mogroside V from the substrate mogroside IVA, and the relative yield of product mogroside V is increased by 5 times compared with the wild type, which can meet the application requirements in industrial production and has important application value. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0032] Figure 1 The results of protein gel electrophoresis in Example 1 of this invention;

[0033] Figure 2 The chemical reaction formula of the catalytic system in Example 2 of this invention;

[0034] Figure 3 The test results are from the ACQUITY-UPLC-PDA system in Embodiment 3 of this invention.

[0035] Figure 4 The results show the ability of the mogroside V produced by the mogroside V mutant R192A in Example 3 of this invention. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] The reagents required for this invention are conventional experimental reagents, purchased from commercially available channels; the experimental methods not mentioned are conventional experimental methods, and will not be described in detail here.

[0038] Example 1

[0039] Obtaining mogroside glycosyltransferase SgUGT94-289-3 and mogroside glycosyltransferase mutant R192A

[0040] (1) Obtaining the gene encoding the R192A mutant of mogroside glycosyltransferase

[0041] Using primers containing point mutation sites, the mutant gene of mogroside glycosyltransferase SgUGT94-289-3 was amplified by PCR using the gene (nucleotide sequence shown in SEQ ID NO.6).

[0042] The nucleotide sequences of primers R192A-F and R192A-R (codons in bold indicate mutants) are as follows:

[0043] R192A-F: GCTTCTACCGAAAGAACCCGCAAAGCGGGAGAAGCGTT TCTGTATTGCTTGC, SEQ IDNO.4;

[0044] R192A-R: GCAAGCAATACAGAAACGCTTCTCCCCGCTTTGCGGGTTC TTTCGGTAGAAGC, SEQ IDNO.5;

[0045] The PCR reaction system is shown in Table 1.

[0046] Table 1 PCR reaction system

[0047]

[0048]

[0049] The PCR reaction conditions are set as shown in Table 2.

[0050] Table 2 PCR amplification program

[0051]

[0052] The gene encoding the mogroside glycosyltransferase mutant R192A was obtained by the above PCR, and its nucleotide sequence is shown in SEQ ID NO.3.

[0053] (2) Obtaining recombinant plasmids

[0054] The PCR products were sequenced and identified, and then the recombinant plasmid pET28a-R192A was prepared. At the same time, the recombinant plasmid pET28a-WT of wild-type mogroside glycosyltransferase SgUGT94-289-3 was prepared.

[0055] (3) Expression and purification of mogroside SgUGT94-289-3 and mogroside mutant R192A

[0056] (31) The recombinant plasmids pET28a-WT and pET28a-R192A were transformed into Escherichia coli Rosetta (DE3) to obtain transgenic Escherichia coli containing the recombinant plasmids, which were named recombinant bacteria pET28a-WT and recombinant bacteria pET28a-R192A.

[0057] (32) Single clones of recombinant bacteria pET28a-WT and recombinant bacteria pET28a-R192A were inoculated into 10 ml of LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain culture solution 1.

[0058] (33) Inoculate the culture solution 1 into 1L LB liquid medium and incubate at 37℃ with shaking at 220rpm until the OD of the system reaches the specified value. 600 The concentration was 0.6–0.8; then IPTG was added to a concentration of 0.5 mM in the system, and the mixture was induced and cultured at 18°C ​​and 180 rpm for 20 h to obtain culture solution 2.

[0059] (34) Take 2 liters of culture solution, centrifuge at 8000 rpm for 10 min, and collect the bacterial cells.

[0060] (35) Add lysis buffer (solutes and concentrations of 500 mM NaCl, 5% glycerol and 10 mM imidazole, solvent of pH 8.0, 50 mM Tris-HCl buffer) to the bacterial cells collected in step (34) to resuspend the bacterial cells and obtain bacterial cell lysis buffer. The bacterial cell lysis buffer should be placed on ice.

[0061] (36) The bacterial lysate obtained in step (35) was ultrasonically disrupted (ultrasonic parameters: 20 Hz, 20 min), and then centrifuged at 4 °C and 18000 g for 30 min. The supernatant was collected. The supernatant is the crude enzyme solution of mogroside SgUGT94-289-3 and the crude enzyme solution of mogroside mutant R192A.

[0062] (37) Take the supernatant collected in step (36) and purify it by nickel column affinity chromatography to obtain purified mogroside glycosyltransferase SgUGT94-289-3 and mogroside glycosyltransferase mutant R192A protein. The protein gel electrophoresis results are as follows: Figure 1 .

[0063] The amino acid sequence of the mogroside glycosyltransferase SgUGT94-289-3 is shown in SEQ ID NO.2;

[0064] The amino acid sequence of the mogroside glycosyltransferase mutant R192A is shown in SEQ ID NO.1.

[0065] Example 2

[0066] Establishing an in vitro catalytic system

[0067] The reaction system consisted of 100 μL of UDP-glucose, 10 μg of mogroside SgUGT94-289-3 or mogroside mutant R192A protein, mogroside IVA, and 50 mM PBS (pH 6.5) buffer. The concentration of UDP-glucose in the reaction system was 8 mM, and the concentration of mogroside IVA was 0.1 mM.

[0068] The catalytic reaction was incubated at 45°C for 12 hours. The reaction was terminated by adding 50 μL of methanol, then vortexed to mix, centrifuged at 13000g for 10 min, and the supernatant was collected.

[0069] The chemical reaction formula of the catalytic system is as follows: Figure 2 .

[0070] Example 3

[0071] Catalytic product analysis

[0072] The supernatant collected in Example 2 was analyzed using the ACQUITY-UPLC-PDA system.

[0073] The yield of mogroside V produced by mogroside V was calculated using peak area and mogroside mutant R192A, and the relative yield was further calculated.

[0074] Relative yield = [mutant product amount / wild-type product amount] × 100%.

[0075] The UPLC reaction parameters were as follows: the mobile phase consisted of 0.1% formic acid aqueous solution (solution A) and acetonitrile (solution B); the flow rate was 0.25 ml / min; gradient elution was performed using increasing solution B and decreasing solution A, specifically: 0–10 min, 23% solution B; 10–20 min, 23–100% solution B; 20–30 min, 100% solution B. The detection results are shown below. Figure 3 .

[0076] After 12 hours of catalytic reaction, the relative yield of mogroside V produced by the mogroside mutant R192A was 5 times that of the wild-type mogroside V produced by the mogroside SgUGT94-289-3, compared to that produced by the mogroside SgUGT94-289-3. (See results below.) Figure 4 .

[0077] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0078] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A mogrosiderin glycosyltransferase mutant R192A, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. The mogroside glycosyltransferase mutant R192A as described in claim 1, characterized in that, The method was obtained by making a point mutation at position 192 of the amino acid sequence encoded by the mogroside glycosyltransferase SgUGT94-289-3, replacing arginine with alanine; The amino acid sequence encoded by the mogroside glycosyltransferase SgUGT94-289-3 is shown in SEQ ID NO.

2.

3. The gene encoding the mogroside glycosyltransferase mutant R192A as described in claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

3.

4. An expression vector comprising the gene as described in claim 3.

5. Microorganisms transformed with the expression vector as described in claim 4.

6. The application of the mogroside mutant R192A according to claim 1 in the synthesis of mogrosides, characterized in that, Catalytic synthesis of mogroside V from substrate mogroside IVA.

7. The method for constructing the mogroside glycosyltransferase mutant R192A according to claim 1, characterized in that, Includes the following steps: (1) Using primers containing point mutation sites, the mutant gene of SgUGT94-289-3 glycosyltransferase from Luo Han Guo was amplified by PCR using the gene as a template. The nucleotide sequence of the primer is as follows: R192A-F: GCTTCTACCGAAAGAACCCGCAAAGCGGGAGAAGCGTTTCTGTATTGCTTGC, SEQ ID NO.4; R192A-R: GCAAGCAATACAGAAACGCTTCTCCCGCTTTGCGGGTTCTTTCGGTAGAAGC, SEQ ID NO.5; The nucleotide sequence of the gene for the mogroside glycosyltransferase SgUGT94-289-3 is shown in SEQ ID NO. 6; (2) The mutant gene was ligated into the pET28a vector to obtain a recombinant plasmid; (3) Expression and purification of the mogroside glycosyltransferase mutant R192A.

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