Glucosyltransferase catalyzing rebaudioside A to produce rebaudioside M and its application

Abstract

The present invention belongs to the technical field of enzyme engineering, and specifically relates to a glucosyltransferase for catalyzing rebaudioside A to produce rebaudioside M and an application thereof. The present invention screens and obtains glucosyltransferases derived from hollyhock and walnut, and finds that the glucosyltransferase derived from hollyhock is high in catalyzing rebaudioside A to synthesize rebaudioside D, and the glucosyltransferase derived from walnut is high in catalyzing rebaudioside D to synthesize rebaudioside M, and the two are applied to catalyze rebaudioside A to synthesize rebaudioside M with a high conversion rate. The glucosyltransferases derived from hollyhock and walnut proposed by the present invention are used to prepare steviol glycoside rebaudioside M, which has not been reported, and provides excellent candidate protein resources and corresponding gene sequences for the enzyme conversion process of steviol glycoside.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to a glucosyltransferase for catalyzing rebaudioside A to produce rebaudioside M and an application thereof. Background Art

[0002] Steviosides (SGs) include stevioside, rebaudioside A (RA), rebaudioside B (RB), rebaudioside C (RC), rebaudioside D (RD), rebaudioside E (RE), rebaudioside F (RF), dulcoside A (Dulcoside A), stevioside, rebaudioside M (RM) and rebaudioside I (RI). Among them, RM is formed by connecting the R1 end of the RD structure to the glucosyl group with a β-1,3 glycosidic bond. The content of RM in stevia is less than 0.1%, which is much lower than RA and stevioside. However, compared with RA and stevioside, RM has high sweetness (about 300-350 times the sweetness of sucrose), rapid sweetness, pure sweetness and no bitterness, astringency, sourness, licorice and other impurities. Its high sweetness and good taste make it a new generation of sweetener. As an alternative to sucrose, RM has shown significant application potential in the food industry. It can effectively reduce dependence on traditional sweeteners, thereby reducing the burden of these sweeteners on the environment, which covers land use, water resource consumption and greenhouse gas emissions. In this way, the use of RM helps to maintain the balance and health of the entire ecosystem to a certain extent, especially playing a positive protective role in our marine environment.

[0003] The glucosyltransferases in the RM synthesis process mainly include three enzymes, namely β-1,2-glucosyltransferase that converts RA into RD, β-1,3-glucosyltransferase that converts RD into RM, and sucrose synthase used to construct the glycosyl cycle. As a key factor affecting the efficiency of RM synthesis, the diversity of the source and type of glucosyltransferase is particularly important. In the RM synthesis process, different enzymes may have different catalytic efficiency, substrate specificity and stability. If different types of glucosyltransferases can be obtained from multiple sources, it is possible to find one or more enzymes that can catalyze the RM synthesis reaction more efficiently and specifically. However, the currently known enzyme types are relatively limited, which prompts us to continuously explore and develop new enzyme resources to further improve the synthesis efficiency of RM. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a glucosyltransferase for catalyzing rebaudioside A to produce rebaudioside M and its application. The enzyme has high activity, can transfer glucose in a directional manner to prepare RM, and has a high conversion rate.

[0005] The technical solution of the present invention is as follows:

[0006] The glucosyltransferase for catalyzing the production of rebaudioside M from rebaudioside A of the present invention comprises a glucosyltransferase derived from tetrapodioides grandiflora ( Cornus florida ) and glucosyltransferase from walnut ( Juglans regia ), the amino acid sequence of the glucosyltransferase derived from the large-flowered tetraspermum is shown in SEQ ID NO.1, and the amino acid sequence of the glucosyltransferase derived from walnut is shown in SEQ ID NO.2.

[0007] The nucleotide sequence of the glucosyltransferase derived from Tetraspermum grandiflorum is shown as SEQ ID NO.3, and the nucleotide sequence of the glucosyltransferase derived from Juglans regia is shown as SEQ ID NO.4.

[0008] The amino acid sequence of the glucosyltransferase from Tetraspermum grandiflorum is shown in SEQ ID NO.1:

[0009] MDGRHSRISVLMFPWLAHGHISPFLELAKKLTKRNFHIYFCSTPVNLRPIKEKLPEKYSLSIQLVELHLPSLPELPPHYHTTNGLPPHLMPTLKKAFDMASPNFSTIVKNLRP DLLIYDFLQQWAPSIALSRNIPAVEFLPTNAAMTSFVIHLTKNPGVEFPFPEIYLRDNYAISKFQNVLESSANGIKDTERAVECCEQSSEIVLIKTFREIEAKYLDYLSELTG KKIVPVGPLVQDPVHEYDEKPGIMEWLNNKERSSTVFVSFGSEYFLSKEEMEEIAYGLEQSMVPFIWVVRFPGGAKVSLEMALPKGFLDRVGDGGMVVEGWAPQTKILEHSSI GGFSSHCGWSSVLESMKLGVPIIAMPMHLDQPINARLVEEVGMGLEVERDMNGKLKREEVAKVIREVVVEKAGESVRQKAKELKEKLISKGEEEIDEVVQEVVQLCRKKNRH.

[0010] The amino acid sequence of the walnut-derived glucosyltransferase is shown in SEQ ID NO.2:

[0011] MLITNPTTKISSANTRDRLSSYLLQTTTERERERELLMEAKKNTISILMVPWLAHHISPLELAKKLTNRNFHIYMCSTPVNLSSIKKNVTEKYSQSIKLVEIHLPSLPNLPPHYHTTN GLPHHLISTLKTAFEMSTPNFSKILQTLNPDLVIYDFNLPWAADCASSVNIPAVQFLTFSAAVVALGIHMYDKPGEMFPFPEIYLHEYEMLTIKKALEYLPGNKFPFDEALRRSRDIILV KTCRDFEGKYMDYLSSLVSKKVVPVGALVQESIDQDGHEEIMQWLDKKETSSTVFVSFGSEYFLSKEKIHAVAQGLELSKVNFILVIRFPQGERISTQDALPEGFLERIGERGMILEGWA PQAKILQHSSIGGFVSHCGWNSLMESMKFGVPIIAMPMQVDQPMNARLVEYIGIGGMEAMRDEKGNLQSEEIAKVIRKVVVDEMGKAVRKNARELSEKMNAKGDEKIDGVVEELVALCNNK.

[0012] The nucleotide sequence of the glucosyltransferase from Tetraspermum grandiflorum is shown in SEQ ID NO.3:

[0013]

[0014] The nucleotide sequence of the walnut-derived glucosyltransferase is shown in SEQ ID NO.4:

[0015]

[0016] The present invention provides the use of a glucosyltransferase having an amino acid sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2 in catalyzing rebaudioside A to produce rebaudioside M.

[0017] The glucosyltransferase with the amino acid sequence shown in SEQ ID NO.1 is β-1,2-UDP-glucosyltransferase, and the glucosyltransferase with the amino acid sequence shown in SEQ ID NO.2 is β-1,3-UDP-glucosyltransferase. The β-1,2-UDP-glucosyltransferase uses RA as a substrate and prepares RD through a cycle constructed by sucrose synthase; the β-1,3-UDP-glucosyltransferase uses RD as a substrate and prepares RM through a cycle constructed by sucrose synthase.

[0018] The method for catalyzing rebaudioside A to produce rebaudioside M comprises the following steps:

[0019] (1) preparing enzyme solutions of glucosyltransferases having an amino acid sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2 respectively;

[0020] (2) RA is used as a substrate, sucrose and sucrose synthase are added, and then the enzyme solution prepared in step (1) is added to carry out a catalytic reaction to obtain RM. The catalytic reaction conditions are: pH 6-7 and temperature 37-38° C. The addition amounts of RA, sucrose, sucrose synthase and enzyme solution can be adjusted according to actual needs.

[0021] In step (1), the method for preparing the enzyme solution comprises the following steps:

[0022] a. constructing an expression vector carrying a glucosyltransferase gene, wherein the expression vector is a pET vector, preferably pET-28a(+);

[0023] b. transforming the expression vector into a host cell to obtain a recombinant strain, wherein the host cell comprises Escherichia coli, preferably E. coli BL21 (DE3) or E. coli Rosetta (DE3), more preferably E. coli BL21 (DE3);

[0024] c. Induce the recombinant strain to express at 16-25°C for 16-36h with isopropyl-β-d-thiogalactoside (IPTG) as the inducer, collect the bacteria, and then break them by low-temperature ultrasonication to obtain the supernatant, which is the enzyme solution.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention screened and obtained glucosyltransferases from hollyhock and walnut, and found that the glucosyltransferase from hollyhock catalyzed the synthesis of RA into RD, and the glucosyltransferase from walnut catalyzed the synthesis of RD into RM, and the enzyme activity was high, and the conversion rate of the two in catalyzing the synthesis of RA into RM was high. There is no report that the glucosyltransferases from hollyhock and walnut are used to prepare steviol glycoside RM. The present invention provides excellent candidate protein resources and corresponding gene sequences for the enzyme conversion process of steviol glycoside, improves the application potential of glucosyltransferase in food, medicine and chemical industries, and has important significance for the industrial production of glucosyltransferase. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the HPLC chart of the glucosyltransferase activity from Tetraspermum grandiflorum;

[0028] Figure 2 This is the HPLC chart of the glucosyltransferase activity from walnut;

[0029] Figure 3 This is the enzyme solution catalytic HPLC chart of glucosyltransferase from Tetraspermum grandiflorum and Juglans regia. DETAILED DESCRIPTION

[0030] The embodiments of the present invention are only used as further explanations of the present invention and cannot be used as limiting the content or scope of the present invention. For molecular biology experimental methods not specifically described in the present embodiments, reference can be made to the Molecular Cloning Experiment Guide.

[0031] The culture medium involved in the embodiment is as follows:

[0032] LB medium: tryptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L;

[0033] TB medium: tryptone 12 g / L, yeast powder 24 g / L, glycerol 5 g / L, KH2PO4 2.31 g / L, K2HPO4·3H2O 16.43 g / L.

[0034] Example 1 Construction of Glucosyltransferase Strain

[0035] Expression vectors carrying glucosyltransferase genes were constructed, wherein the glucosyltransferases were glucosyltransferases derived from Cornus florida, Nyssa sinensis Oliv., Camellia japonica L., Actinidia chinensis, Juglans regia L., Solanum bulbocastanum, Solanum dulcamara L., and Nicotiana attenuata.

[0036] The expression vector was transformed into the host cell E. coli BL21 (DE3), and then spread on LB plates containing 50 mg / L kanamycin sulfate and cultured overnight at 37°C. Single colonies were picked from the plates and verified by PCR using universal primers. Positive clones were retained to obtain recombinant strains.

[0037] The recombinant strain was inoculated into LB liquid culture medium at a 1% inoculation rate to prepare seed solution, and the seed solution was inoculated into TB liquid culture medium at a 1% inoculation rate. After culture and fermentation for 24 h on a shaking induction platform at 25°C (the inducer was 1 mM IPTG), the fermentation broth was centrifuged at 4°C and 12000 rpm for 15 min, the bacterial precipitate was taken, and the supernatant was taken after low-temperature ultrasonic disruption to obtain the enzyme solution.

[0038] Wherein, the nucleotide sequence of the forward primer is: 5'-TAATACGACTCACTATAGGG-3', as shown in SEQ ID NO.5;

[0039] The nucleotide sequence of the reverse primer is: 5'-TGCTAGTTATTGCTCAGCGG-3', as shown in SEQ ID NO.6.

[0040] PCR reaction system: 1 μL each of 10 μM forward primer and reverse primer, 25 μL of 2×PCR mix, 1 μL of template, and make up to 50 μL with double distilled water.

[0041] PCR reaction program: pre-denaturation at 94°C for 4 min; followed by 25 cycles (94°C for 10 s, 55°C for 5 s, 72°C for 7 min 50 s); extension at 72°C for 10 min; and finally incubation at 4°C.

[0042] The PCR products were detected by 1% agarose gel electrophoresis.

[0043] Example 2 Detection of Glucosyltransferase Activity

[0044] The enzyme activity of the glucosyltransferase enzyme solution from Rhizoma Coptidis, blue fruit tree, camellia, kiwi fruit and walnut prepared in Example 1 was determined by RA-RD catalytic reaction. The enzyme activity determination method was as follows: 1 mL 1.2 mM RA, 1 mL UDPG, 1 mL enzyme solution, at 37°C, 200 rpm, after 1 hour of reaction, HPLC determination was performed. The results are shown in Table 1, wherein the HPLC chart of the glucosyltransferase activity from Rhizoma Coptidis is as follows: Figure 1 .

[0045] The enzyme activity of the glucosyltransferase obtained in Example 1 from Castanea sphaerocarpa, Elaeagnus oleraceus, Nicotiana tabacum, Cyperus rotundus, and Juglans regia was determined by RD-RM catalytic reaction. The enzyme activity determination method was as follows: 1 mL 1.2 mM RD, 1 mL UDPG, 1 mL enzyme solution, at 37° C., 200 rpm, after reacting for 1 h, HPLC determination was performed. The results are shown in Table 2. The HPLC chart of the glucosyltransferase activity from Juglans regia is as follows: Figure 2 .

[0046] The specific method parameters of HPLC determination are as follows: add 200 μL of chromatographic acetonitrile, shake and mix, let stand for 10 minutes, centrifuge at 12000 rpm for 10 minutes at room temperature, and analyze the supernatant after passing through a 0.22 μm organic membrane. HPLC uses a C18 reverse-phase bonded silica gel separation column (4.6 mm×250 mm, 5 μm), 25% acetonitrile as the mobile phase, a flow rate of 1 mL / min, a column temperature of 40°C, a UV detector PDA, a PDA detector wavelength of 210 nm, and an injection volume of 10 μL.

[0047] Table 1 Results of enzyme activity determination of RA-RD catalytic reaction

[0048] Sample name Retention time (min) Peak area Four-orange-flowered holly 5.735 82819 Blueberry Tree 5.734 9858 Camellia 5.741 7724 kiwi 5.754 9815 walnut 5.742 6959

[0049] Table 2 Results of enzyme activity determination of RD-RM catalytic reaction

[0050] Sample name Retention time (min) Peak area Chestnut 7.799 39018 Ou Baiying 7.756 40278 tobacco 7.765 44516 Tetraphyllum grandiflorum 7.767 56501 walnut 7.733 207717

[0051] It can be seen from Tables 1 and 2 that in the RA-RD catalytic reaction, the enzymatic activity of the glucosyltransferase from the four o'clock flower is significantly higher than that of enzymes from other sources, and in the RD-RM catalytic reaction, the enzymatic activity of the glucosyltransferase from walnut is significantly higher than that of enzymes from other sources. Therefore, the glucosyltransferases from the four o'clock flower and walnut can be used for the directional transfer of glucosyl groups and catalyze RA to prepare stevioside RM.

[0052] Example 3 Conversion of Glucosyltransferase

[0053] The catalytic reaction was carried out using the enzyme solution of glucosyltransferase from Rhizoma Coptidis and Juglans regia prepared in Example 1: water was used as the solvent, 20 g / L RA, 300 g / L sucrose, 50 g / L sucrose synthase, 100 g / L Rhizoma Coptidis glucosyltransferase solution, and 150 g / L Juglans regia glucosyltransferase solution were added, and the reaction was catalyzed at 37°C and pH 7 for 24 h.

[0054] After the reaction was completed, HPLC was performed to detect the product formation. The results were as follows Figure 3 The glucosyltransferases from the four-flowered odonata and walnut can transfer glucose in a targeted manner to catalyze RA to prepare RM. After 24 hours of reaction, 10 g / L RM was produced.

Claims

1. A glucosyltransferase that catalyzes the production of rebaudioside M from rebaudioside A, characterized in that: The glucosyltransferases include glucosyltransferases derived from Tetrapoda macrophylla and glucosyltransferases derived from Juglans regia. The amino acid sequence of the glucosyltransferases derived from Tetrapoda macrophylla is shown in SEQ ID NO.1, and the amino acid sequence of the glucosyltransferases derived from Juglans regia is shown in SEQ ID NO.

2.

2. The glucosyltransferase for catalyzing the production of rebaudioside M from rebaudioside A according to claim 1, characterized in that: The nucleotide sequence of the glucosyltransferase derived from Tetraspermum grandiflorum is shown as SEQ ID NO.3, and the nucleotide sequence of the glucosyltransferase derived from Juglans regia is shown as SEQ ID NO.

4.

3. An application of the glucosyltransferase for catalyzing the production of rebaudioside M from rebaudioside A according to claim 1, characterized in that: The glucosyltransferases with the amino acid sequence shown in SEQ ID NO.1 and the amino acid sequence shown in SEQ ID NO.2 are used to catalyze rebaudioside A to produce rebaudioside M.

4. The use according to claim 3, characterized in that: The method for catalyzing rebaudioside A to produce rebaudioside M comprises the following steps: (1) preparing enzyme solutions of glucosyltransferase having an amino acid sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2 respectively; (2) Using rebaudioside A as a substrate, adding sucrose and sucrose synthase, and then adding the enzyme solution prepared in step (1) to carry out a catalytic reaction to obtain rebaudioside M.

5. The use according to claim 4, characterized in that: The preparation method of the enzyme solution comprises the following steps: a. Constructing an expression vector carrying a glucosyltransferase gene; b. Transforming the expression vector into host cells to obtain a recombinant strain; c. After inducing the expression of the recombinant strain, the bacteria are collected, and the supernatant is obtained after low-temperature ultrasonic disruption, which is the enzyme solution.

6. The use according to claim 5, characterized in that: The expression vector is a pET vector, and the host cell includes Escherichia coli.

7. The use according to claim 5, characterized in that: The induction agent used for inducing expression was isopropyl-β-d-thiogalactoside.

8. The use according to claim 4, characterized in that: The catalytic reaction conditions in step (2) are: pH 6-7, temperature 37-38°C.

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