Alkali-resistant glycosyl transferase and method for preparing gastrodin by enzyme method

Abstract

The invention provides alkali-resistant glycosyl transferase and a coding gene, a biological material, a preparation method and application thereof. The glycosyl transferase has a specific amino acid sequence and can be efficiently expressed in escherichia coli through a genetic engineering means. The preparation method comprises the steps of codon optimization, recombinant plasmid cloning, expression strain transformation, induced expression, purification and the like. The obtained glycosyltransferase has significant application in preparation of gastrodin by enzymatic catalysis of p-hydroxybenzyl alcohol, and enzymatic hydrolysis conditions such as substrate concentration, enzyme concentration, UDP-glucose concentration and temperature, and buffer solution type and pH value are optimized. Particularly, when a glycine-sodium hydroxide buffer solution is used at the pH value of 9.5, the enzymolysis effect is remarkable. According to the alkali-resistant glycosyl transferase as well as the preparation method and the application thereof, a powerful tool is provided for biosynthesis of natural products such as gastrodin, and the alkali-resistant glycosyl transferase has an important industrial application value. Meanwhile, the biological material disclosed by the invention provides a basis for production and application of alkali-resistant glycosyl transferase.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and biocatalysis, and specifically relates to the preparation of an alkali-resistant UDP-glycosyltransferase SlyUGT derived from Solanum lycopersicum and its application in enzymatic biosynthesis of gastrodin. Background Art

[0002] Gastrodin is the main bioactive ingredient of the precious medicinal material Gastrodia elata. It is a phenolic glycoside compound with the chemical name of 4-hydroxybenzyl alcohol-4-O-β-D-pyranoglucoside. It has good clinical efficacy on cardiovascular diseases and is also widely used in the adjuvant treatment of vertigo, neuralgia, headache, neurasthenia and epilepsy, with significant efficacy and no obvious toxic side effects. In addition, gastrodin also has anti-inflammatory, anti-anxiety, anti-alcoholic liver damage, antioxidant, anti-obesity, neuroprotective and memory-improving effects. There are currently 44 kinds of medicines and health products on the market with gastrodin as the main ingredient. So far, the production of gastrodin mainly adopts plant extraction and compound synthesis. However, wild gastrodia resources are scarce, artificial cultivation is complicated, and the content of gastrodin in plant gastrodia is extremely low (mass fraction <0.7%), resulting in high extraction cost and long time. Due to the complex structure of gastrodin, chemical synthesis of gastrodin will produce many analogues, which are very difficult to separate. Moreover, the toxic phenols, phosphates and bromides used in the production process will cause serious environmental pollution. Biological methods have the advantages of strong specificity, mild reaction conditions, and low pollution. Therefore, in recent years, reports on the preparation of gastrodin by biological methods have appeared in large numbers, becoming a research hotspot.

[0003] Glycosyltransferases can use small molecular weight compounds as acceptor substrates and uridine 5'-diphosphate (UDP) as a donor. Research reports show that gastrodin can be biosynthesized by glycosylation of p-hydroxybenzyl alcohol by UDP-glycosyltransferase. Therefore, the catalytic activity of UGT plays a decisive role in the biosynthesis of gastrodin. However, the natural glycosyltransferase for gastrodin biosynthesis is unknown. Although it has been reported that a single gene encoding a glycosyltransferase may be involved in the natural biosynthetic pathway of gastrodin, the sequence of the natural UGT used for gastrodin biosynthesis is still unclear. Therefore, it is crucial to obtain a glycosyltransferase with good enzymatic properties and the ability to efficiently biosynthesize gastrodin. Summary of the invention

[0004] To solve the above problems, we disclose an alkali-resistant glycosyltransferase and a method for preparing gastrodin by enzymatic method.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a glycosyltransferase, the amino acid sequence of which is shown in SEQ ID NO.1; the enzyme has an optimum reaction temperature of 40°C, an optimum reaction pH of 9.5, a wide pH application range and good alkali resistance.

[0007] The present invention provides a coding gene of the glycosyltransferase optimized according to the codon preference of Escherichia coli, which has a nucleotide sequence as shown in SEQ ID NO.2.

[0008] The invention provides a recombinant plasmid comprising a nucleotide sequence encoding the glycosyltransferase.

[0009] The present invention provides a host cell, comprising the recombinant vector or a nucleotide sequence as shown in SEQ ID NO.2 integrated into the genome.

[0010] The invention provides a method for preparing the recombinant glycosyltransferase.

[0011] In the expression conditions of the recombinant glycosyltransferase of the present invention:

[0012] The induction temperature is 16-45°C;

[0013] Induction IPTG concentration: 0.01mmol / L-5mmol / L;

[0014] Induction OD600 value: 0.2-2.0;

[0015] Induction time: 8-72h.

[0016] In some specific embodiments, the induction temperature is 20°C;

[0017] The induction IPTG concentration was: 0.1mmol / L;

[0018] The induced OD600 value was: 1.8;

[0019] Induction time: 36h.

[0020] The present invention provides application of the glycosyltransferase in biosynthesis of gastrodin.

[0021] In the method provided by the present invention, the buffer for enzymatic hydrolysis is a citric acid-disodium hydrogen phosphate buffer or a Tris-HCl buffer or a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer or a glycine-sodium hydroxide buffer.

[0022] The citric acid-disodium hydrogen phosphate buffer solution is 10mmol / L-200mmol / L, and the pH is 5.0-8.0;

[0023] or Tris-HCl buffer with a concentration of 10mmol / L-200mmol / L and a pH of 7.0-9.0;

[0024] Or disodium hydrogen phosphate-sodium dihydrogen phosphate buffer is 10mmol / L-200mmol / L, pH is 7.0-9.5;

[0025] Or glycine-sodium hydroxide buffer is 10mmol / L-200mmol / L, pH is 8.5-10.5.

[0026] In some specific embodiments, the buffer is a glycine-sodium hydroxide buffer with a concentration of 100 mmol / L and a pH of 9.5.

[0027] In the enzymatic hydrolysis system of the present invention:

[0028] The concentration of p-hydroxybenzyl alcohol is 0.5mmol / L-5mmol / L;

[0029] The concentration of glycosyltransferase is 2mU / mL-20mU / mL;

[0030] The concentration of UDP-glucose is 0.5mmol / L-10mmol / L.

[0031] In some specific embodiments, the concentration of p-hydroxybenzyl alcohol is 2 mmol / L;

[0032] The concentration of glycosyltransferase was 2 mU / mL;

[0033] The UDP-glucose concentration was 7 mmol / L.

[0034] In the present invention, the conditions for enzymolysis are:

[0035] React at 25°C-50°C.

[0036] In some specific embodiments, the enzymatic hydrolysis condition is: reacting at 40°C.

[0037] Compared with the prior art, the present invention has the following outstanding beneficial effects:

[0038] The invention obtains a glycosyltransferase which has a wide pH applicable range and can efficiently catalyze the conversion of p-hydroxybenzyl alcohol into gastrodin, has a short enzymolysis time, a high conversion rate and a high gastrodin yield.

[0039] (1) The glycosyltransferase of the present invention has a wide pH application range and excellent alkali resistance;

[0040] (2) The glycosyltransferase of the present invention can efficiently biosynthesize gastrodin, with a gastrodin yield of 559.83 mg / L and a molar conversion rate of up to 97.82%. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The enzymatic method of the present invention is shown as a biosynthesis route of p-hydroxybenzyl alcohol to gastrodin;

[0042] Figure 2 The result diagram of the change of enzyme yield at different time of induced expression of glycosyltransferase SlyUGT described in Example 3 is shown;

[0043] Figure 3 The SDS-PAGE diagram of the purification process of the glycosyltransferase SlyUGT described in Example 4 is shown; wherein lane M is a protein Marker (purchased from Beijing Quanshijin, item number DM111-02), lane 1 is the whole cell protein after the cells containing plasmid pGEX2T are broken, lane 2 is the supernatant protein after the cells containing plasmid pGEX2T are broken, lanes 3, 5, 7 and 9 are the whole cell proteins after the cells containing plasmid pGEX2T-SlyUGT are induced to express at 20°C, 25°C, 30°C and 37°C, respectively, lanes 4, 6, 8 and 10 are the supernatant proteins after the cells containing plasmid pGEX2T-SlyUGT are induced to express at 20°C, 25°C, 30°C and 37°C, respectively, and lane 11 is the glycosyltransferase SlyUGT separated using a GST-tagged protein purification column;

[0044] Figure 4 Graphs showing the qualitative determination results of the glycosyltransferase SlyUGT described in Example 5; wherein Graph a is a graph showing the determination results of the optimum reaction pH, Graph b is a graph showing the determination results of the optimum reaction temperature, Graph c is a graph showing the determination results of the temperature stability, and Graph d is a graph showing the determination results of the pH stability;

[0045] Figure 5 The graph shows the change in the yield of gastrodin at different times when the glycosyltransferase SlyUGT catalyzes p-hydroxybenzyl alcohol as described in Example 6. DETAILED DESCRIPTION

[0046] The present invention provides an alkali-resistant glycosyltransferase and a method for preparing gastrodin by enzymatic method. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired effect. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0047] The present invention provides a glycosyltransferase with a wide pH application range and alkaline resistance, the amino acid sequence of which is shown in SEQ ID NO.1 and is named SlyUGT.

[0048] The present invention also provides a DNA molecule fragment encoding the glycosyltransferase of the present invention. Due to the degeneracy of codons, there may be many nucleotide sequences that can encode the glycosyltransferase of the present invention.

[0049] In some embodiments, the present invention provides a DNA molecule that can encode the glycosyltransferase SlyUGT, and its nucleotide sequence is shown in SEQ ID NO.2.

[0050] The present invention provides a recombinant plasmid containing the DNA molecule of the glycosyltransferase of the present invention, wherein the recombinant plasmid is pGEX2T-SlyUGT.

[0051] The host bacteria for transformation and expression of the present invention are Escherichia coli expression strains, including BL21 and JM109 series strains. In a preferred embodiment, the host cell is BL21 (DE3) strain.

[0052] The induced expression of the preparation method of the glycosyltransferase of the present invention is specifically to culture the recombinant bacteria containing the SlyUGT gene in LB, collect the bacteria by centrifugation after the enzyme production stage is completed, then crush the cells with an ultra-high pressure crusher, centrifuge to obtain the supernatant, and purify it through a GST-tagged protein purification column to obtain the desired target enzyme protein.

[0053] The present invention also provides a method for preparing gastrodin by enzymatic conversion of p-hydroxybenzyl alcohol, specifically, the glycosyltransferase of the present invention can catalyze p-hydroxybenzyl alcohol to obtain gastrodin under the conditions of pH 9.5 and 40°C.

[0054] The sequences SEQ ID NO.1-4 involved in the present invention are as follows:

[0055] Sequence 1: Amino acid sequence of glycosyltransferase SlyUGT from Solanum lycopersicum

[0056] 1 MAQIPHIAIL PSPGMGHLIP LVEFAKRIFL HHHFSVSLIL PTDGPISNAQ KIFLNSLPSS

[0057] 61 MDYHLLPPVN FDDLPEDVKI ETRISLTVSR SLTSLRQVLE SIIESKKTVA LVVDLFGTDA

[0058] 121 FDVAIDLKIS PYIFFPSTAM GLSLFLHLPN LDETVSCEYR DLPDPIQIPG CTPIHGKDLL

[0059] 181 DPVQDRNDES YKWLLHHAKR YGMAEGIIVN SFKELEGGAI GALQKDEPGK PTVYPVGPLI

[0060] 241 QMDSGSKVDG SECMTWLDEQ PRGSVLYISY GSGGTLSHEQ LIEVAAGLEM SEQRFLWVVR

[0061] 301 CPNDKIANAT FFNVQDSTNP LEFLPKGFLE RTKGFGLVLP NWAPQARILS HESTGGFLTH

[0062] 361 CGWNSTLESV VHGVPLIAWP LYAEQKMNAV MLSEDIKVAL RPKVNEENGI VGRLEIAKVV

[0063] 421 KGLMEGEEGK GVRSRMRDLK DAAAKVLSED GSSTKALAEL ATKLRKKCQM IDVANH

[0064] Sequence 2: The nucleotide sequence of SlyUGT after optimizing according to the codon preference of Escherichia coli

[0065] 1 ATGGCGCAGA TTCCGCATAT TGCGATTCTG CCGAGCCCGG GCATGGGCCA TCTGATTCCG

[0066] 61 CTGGTGGAAT TTGCGAAACG CATTTTTCTG CATCACCATT TTAGCGTGAG CCTGATTCTG

[0067] 121 CCGACCGATG GCCCGATTAG CAACGCGCAG AAAATTTTTC TGAACAGCCT GCCGAGCAGC

[0068] 181 ATGGATTATC ATCTGCTGCC GCCGGTGAAC TTTGATGATC TGCCGGAAGATGTGAAAATT

[0069] 241 GAAACCCGCA TTAGCCTGAC CGTGAGCCGC AGCCTGACGA GCCTGCGCCA AGTGCTGGAA

[0070] 301 AGCATTATTG AAAGCAAAA AACCGTGGCG CTGGTGGTGG ATCTGTTTGG CACCGATGCG

[0071] 361 TTTGATGTGG CGATCGACCT GAAAATTAGC CCGTATATTT TTTTTCCGAG CACCGCGATG

[0072] 421 GGCCTGAGCC TGTTTCTGCA TCTGCCGAAC CTGGATGAAA CCGTGAGCTG CGAATATCGC

[0073] 481 GATCTGCCGG ACCCGATTCAGATTCCGGGC TGCACCCCGA TTCATGGCAA GGATCTGTTA

[0074] 541 GATCCGGTGC AAGATCGCAA CGATGAAAGC TATAAATGGC TGCTGCATCA TGCGAAACGC

[0075] 601 TATGGCATGG CGGAAGGCAT TATTGTGAAC AGCTTTAAAG AACTGGAAGG CGGCGCGATT

[0076] 661 GGCGCGCTGC AGAAAGATGA ACCGGGCAAA CCGACCGTGT ATCCGGTGGG CCCGCTGATT

[0077] 721 CAGATGGATA GCGGCAGCAA AGTGGATGGC AGCGAATGCA TGACCTGGCT GGATGAACAG

[0078] 781 CCGCGCGGCA GCGTGCTGTA TATTAGCTAT GGCAGCGGCG GCACCCTGAG CCATGAACAG

[0079] 841 CTGATTGAAG TGGCGGCGGG CCTGGAAATG AGCGAACAGC GCTTTCTGTG GGTGGTGCGC

[0080] 901 TGCCCGAACG ATAAAATTGC GAACGCGACC TTTTTTAACG TGCAAGATAG CACCAACCCG

[0081] 961 CTGGAATTTC TGCCGAAAGG CTTTCTGGAA CGCACCAAAG GCTTTGGCCT GGTGCTGCCG

[0082] 1021 AACTGGGCGC CGCAAGCGCG CATTCTGAGC CACGAAAGCA CGGGCGGCTTTCTGACCCAT

[0083] 1081 TGCGGCTGGA ACAGCACCCT GGAAAGCGTG GTGCATGGCG TGCCGCTGATTGCGTGGCCG

[0084] 1141 CTGTATGCGG AACAGAAAAT GAACGCGGTG ATGTTAAGTG AAGATATTAAAGTGGCGCTG

[0085] 1201 CGCCCGAAAG TGAACGAAGA AAACGGCATT GTGGGCCGCC TGGAAATTGCGAAAGTGGTG

[0086] 1261 AAAGGCCTGA TGGAAGGCGAAGAAGGCAAA GGCGTGCGCAGCCGCATGCG CGATCTGAAA

[0087] 1321 GATGCGGCCG CGAAAGTTCT GAGCGAAGAC GGCAGCAGCACCAAAGCGCT GGCGGAACTG

[0088] 1381 GCGACCAAAC TGCGCAAAAAATGTCAGATG ATTGACGTGG CCAACCACTA A

[0089] Sequence 3:5'-CGCGGATCCATGGCGCAGATTCCGCATATTGCG-3'

[0090] Sequence 4: 5'-CCGGAATTCTTAGTGGTTGGCCACGTCCAATCATCTG-3'

[0091] In order to further understand the present invention, the present invention is described in detail below in conjunction with embodiments.

[0092] Example 1

[0093] Construction of recombinant plasmid pGEX2T-SlyUGT.

[0094] Primers were designed according to the SlyUGT gene after codon optimization in Escherichia coli and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The primer sequences are as follows:

[0095] SlyUGT-F:CGC GGATCC ATGGCGCAGATTCCGCATATTGCG, the underline indicates the BamH I site (SEQ ID NO. 3).

[0096] SlyUGT-R:CCG GAATTC TTAGTGGTTGGCCACGTCAATCATCTG, the underline indicates the EcoR I site (SEQ ID NO. 4).

[0097] The SlyUGT gene optimized according to the codon of Escherichia coli was used as a template for PCR amplification. The amplification conditions were 94°C, 3 min; 30 cycles (94°C, 5s; 57°C, 15s; 72°C, 43s); 72°C, 10 min; reaction stop, 4°C insulation. The PCR amplification product was purified by a PCR product purification kit to obtain the DNA molecule of the glycosyltransferase SlyUGT.

[0098] The obtained DNA molecule of glycosyltransferase SlyUGT and pGEX2T were double-digested with BamH I and EcoR I, respectively, and recovered with gel recovery kits, respectively, and ligated at 16° C. overnight after concentration. The ligation products were transformed into Escherichia coli DH5α competent cells, positive clones were screened, and sequence analysis was performed; clones with correct sequences were selected to extract plasmids, and recombinant plasmid pGEX2T-SlyUGT containing the DNA molecule of alkali-resistant glycosyltransferase SlyUGT was obtained, and its nucleotide sequence is shown in ISEQ ID N0.2.

[0099] Example 2

[0100] Glycosyltransferase SlyUGT enzyme activity assay.

[0101] With p-hydroxybenzyl alcohol (pHBA) as the glycosyl acceptor and UDP-glucose as the glycosyl donor, the biosynthesized gastrodin was detected by HPLC. The reaction volume was 100 μL, including 1 mM pHBA, 50 mM glycine buffer (pH 9.5) and 1 mM UDP-glucose, 10 μL enzyme solution was added after preheating, and the reaction was carried out at 40°C for 1 hour, and then 4 μL 10% trifluoroacetic acid aqueous solution and 900 μL methanol were added to terminate the reaction. The yield of gastrodin was then detected by high performance liquid chromatography (HPLC).

[0102] One unit of enzyme activity (U) was defined as the amount of enzyme required to release 1 μmol of gastrodin per minute at 40°C and pH 9.5.

[0103] Example 3

[0104] The expression of glycosyltransferase SlyUGT is induced.

[0105] The recombinant plasmid pGEX2T-SlyUGT was transformed into competent cells of Escherichia coli strain BL21 (DE3) by heat shock transformation method and plated on LB plates (LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 5g / L, agar 15g / L) after culturing at 37°C overnight, pick the transformant into 50mL LB medium (50mg / mL ampicillin) and culture at 37°C with shaking at 180rpm until OD600 is 1.8, add isopropyl β-D-thiogalactopyranoside (IPTG) inducer with a final concentration of 0.1mM, and culture at 30°C for 12h, 18h, 24h, 36h, 42h, and 48h, respectively. Centrifuge the culture solution at 12,000g for 8min at 4°C in a high-speed refrigerated centrifuge to collect the bacteria, add 50mL of 1×PBS buffer for resuspending and washing, and centrifuge at 12,000g for 8min at 4°C in a high-speed refrigerated centrifuge to collect the washed bacteria. Add 50 mL of 1× PBS buffer to the washed cells, resuspend, and disrupt them three times with a French high-pressure cell disruptor. Centrifuge at 12,000 g for 8 min at 4°C in a high-speed refrigerated centrifuge, collect the crude enzyme solution in the supernatant, and measure its enzyme activity. Figure 2 As shown. Figure 2 The results show that the glycosyltransferase SlyUGT of the present invention obtains the maximum enzyme yield when the expression is induced for 36 hours, and the maximum enzyme yield is 29.48 mU / mL.

[0106] Example 4

[0107] Purification of glycosyltransferase SlyUGT

[0108] Since the recombinant plasmid pGEX2T-SlyUGT contains a GST tag, it can be purified through a GST tag protein purification column to obtain purified recombinant enzyme. Specific operation process:

[0109] A. Preparation of crude enzyme solution

[0110] (1) Take the crude enzyme solution and filter it through a 0.22 μm microporous membrane.

[0111] B. Treatment of columns

[0112] (1) Wash the column with 5 mL of pure water to remove ethanol.

[0113] (2) Use 5 mL of equilibration buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) to equilibrate the filler in the same buffer system as the target protein, and repeat 2-3 times.

[0114] C. Loading and Elution

[0115] (1) Add the crude enzyme solution to a balanced gravity column. Keep the sample for at least 2 minutes to ensure that the sample and filler are in full contact. Collect the effluent and repeat the loading 2-3 times.

[0116] (2) Wash with 10 mL of washing solution to remove non-specifically adsorbed impurities and collect the washing solution.

[0117] (3) Elute with 10 mL of eluent at a low flow rate and collect the eluate.

[0118] (4) Wash the medium with 8 mL of purified water and remove the eluent in the medium.

[0119] (5) Wash the filler with 3 mL of equilibration solution and 5 mL of deionized water.

[0120] (6) Equilibrate the filler with 5 mL of 20% ethanol and store the filler in 20% ethanol at 4-8°C.

[0121] The purified recombinant enzyme SlyUGT was obtained through this process, and its purity was identified by SDS-PAGE method. Figure 3 shown.

[0122] Depend on Figure 3 The results showed that the SlyUGT gene was highly expressed in the dormitory bacteria BL21 (DE3), and after the target band was purified by GST tag, the recombinant glycosyltransferase SlyUGT in its eluate had a high purity, reaching the electrophoresis purity level.

[0123] Example 5

[0124] Qualitative determination of the glycosyltransferase SlyUGT.

[0125] 5.1 Optimal reaction pH

[0126] The enzyme activity was measured at 40°C in different pH ranges (citric acid-disodium hydrogen phosphate buffer: pH 5.0-8.0; disodium hydrogen phosphate-sodium dihydrogen phosphate buffer: pH 6.0-8.0; Tris-HCl buffer: pH 7.0-9.5; glycine-NaOH buffer: pH 8.5-10.5). Figure 4 As shown in A.

[0127] Depend on Figure 4 A The results show that the optimal reaction pH of the recombinant glycosyltransferase SlyUGT of the present invention is 9.5, and has a wide pH range of use.

[0128] 5.2 Determination of the optimal reaction temperature

[0129] The enzyme activity was measured at 5°C intervals in the range of 25-50°C in a glycine-NaOH buffer solution at pH 9.5. The results are shown in Table 1. Figure 4 As shown in B.

[0130] Depend on Figure 4 B. The results show that the optimal reaction temperature of the recombinant glycosyltransferase SlyUGT of the present invention is 40°C.

[0131] 5.3 Temperature stability

[0132] At pH 9.5, the enzyme was incubated at 35°C, 40°C and 45°C, and the enzyme activity was measured every 30 minutes, with the enzyme activity without incubation being 100%. The results are as follows: Figure 4 As shown in C.

[0133] Depend on Figure 4 C The results show that the recombinant glycosyltransferase SlyUGT of the present invention has almost no loss of enzyme activity at 35°C, still has more than 40% residual enzyme activity after being incubated at 40°C for 3 hours, and only has about 8% residual enzyme activity after being incubated at 45°C for 240 minutes.

[0134] 5.4 pH stability

[0135] The purified recombinant enzyme SlyUGT was treated at 35°C for 4 h at different pH values ​​(citrate-sodium hydrogen phosphate buffer: pH 5.0-8.0; sodium hydrogen phosphate-sodium dihydrogen phosphate buffer: pH 6.0-8.0; Tris-HCl buffer: pH 7.0-9.5; glycine-NaOH buffer: pH 8.5-10.5). The results were compared with those of the enzyme without incubation. Figure 4As shown in D.

[0136] Depend on Figure 4 D The results show that the recombinant glycosyltransferase SlyUGT of the present invention can still have 70% residual enzyme activity under the condition of pH 5.5-10.5, and has good alkali resistance.

[0137] Example 6

[0138] Glycosyltransferase SlyUGT catalyzes the conversion of p-hydroxybenzyl alcohol to gastrodin.

[0139] The concentration of p-hydroxybenzyl alcohol (pHBA) was 2 mmol / L, the concentration of UDP-glucose was 7 mmol / L, the conversion conditions were 40°C, 50 mmol / L pH 9.5 glycine-sodium hydroxide buffer, 14 mU / mL recombinant SlyUGT, and samples were taken at different time points (0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h) and detected by HPLC. The results are shown in Figure 5 shown.

[0140] Depend on Figure 5 The results show that the recombinant glycosyltransferase SlyUGT (14 mU / mL) of the present invention can achieve the maximum yield of p-hydroxybenzyl alcohol (pHBA) to gastrodin at 6 h, with the maximum yield reaching 559.83 mg / L and the molar conversion rate being 97.82%.

[0141] The above examples illustrate that the present invention has obtained a glycosyltransferase with a wide pH application range and high efficiency in catalyzing the conversion of p-hydroxybenzyl alcohol to gastrodin, with short enzymolysis time, high conversion rate, and high gastrodin yield. Experiments show that the glycosyltransferase provided by the present invention can catalyze p-hydroxybenzyl alcohol to gastrodin, with a maximum gastrodin yield of 559.83 mg / L and a molar conversion rate of 97.82%.

[0142] The above are only a few preferred embodiments of the present invention, which are described in a more specific and detailed manner, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An alkali-resistant glycosyltransferase, characterized in that The amino acid sequence of the alkali-resistant glycosyltransferase is shown in SEQ ID NO.

1.

2. A biomaterial, characterized in that: Contains any one or more of the following substances 1)-3): 1) Gene: the gene encodes the amino acid sequence of the alkali-resistant glycosyltransferase according to claim 1; 2) Recombinant plasmid: containing the gene described in 1); 3) Host cell: containing the gene described in 1) or the recombinant plasmid described in 2).

3. A gene encoding the amino acid sequence of the alkali-resistant glycosyltransferase according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. The method for preparing the alkali-resistant glycosyltransferase according to claim 1, characterized in that: The following steps are involved: S1 performs codon optimization based on the amino acid sequence to synthesize a nucleic acid sequence suitable for expression in Escherichia coli; S2 clones the above nucleic acid sequence into a recombinant plasmid; S3 transforms the recombinant plasmid into an E. coli expression strain for IPTG-induced expression; S4: washing and crushing the fermented bacteria, collecting the supernatant by centrifugation to obtain a crude enzyme solution; S5 purifies the crude enzyme solution through a purification column to obtain glycosyltransferase.

5. The method for preparing glycosyltransferase according to claim 4, characterized in that: The recombinant plasmid is pGEX2T-SlyUGT, which contains a GST tag; The expression strains include BL21 and JM109 series strains, preferably BL21 (DE3) strains.

6. The method for preparing glycosyltransferase according to claim 5, characterized in that: In the induced expression, The induction IPTG concentration is: 0.01mmol / L-5mmol / L, preferably 0.1mmol / L; The induction OD600 value is: 0.2-2.0, preferably 1.8; The induction temperature is 16-45°C, preferably 20°C; The induction time is: 18-66h, preferably 36h.

7. Use of the glycosyltransferase according to claim 1 in enzymatically catalyzing the conversion of p-hydroxybenzyl alcohol to gastrodin.

8. The use according to claim 7, characterized in that In the enzymatic hydrolysis system: The concentration of p-hydroxybenzyl alcohol is 0.5mmol / L-5mmol / L, preferably 2mmol / L; The concentration of glycosyltransferase is 2mU / mL-20mU / mL, preferably 2mU / mL; UDP-glucose concentration is 0.5 mmol / L-10 mmol / L, preferably 7 mmol / L; The temperature of enzymatic hydrolysis is 25°C-50°C, preferably 40°C.

9. The use according to claim 7, characterized in that: In the enzymatic hydrolysis system: The enzymatic hydrolysis buffer is selected from one of citric acid-disodium hydrogen phosphate buffer, Tris-HCl buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer or glycine-sodium hydroxide buffer; The citric acid-disodium hydrogen phosphate buffer solution is 10 mmol / L-200 mmol / L, and the pH is 5.0-8.0; The Tris-HCl buffer has a concentration of 10 mmol / L-200 mmol / L and a pH of 7.0-9.0; The sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer has a concentration of 10 mmol / L-200 mmol / L and a pH of 7.0-9.5; The glycine-sodium hydroxide buffer solution has a concentration of 10 mmol / L-200 mmol / L and a pH of 8.5-10.

5.

10. The use according to claim 9, characterized in that In the enzymatic hydrolysis system, the buffer is a glycine-sodium hydroxide buffer with a concentration of 100 mmol / L and a pH of 9.5.

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