Method for large-scale production of rare ginsenoside Rk3

Abstract

The invention discloses a method for large-scale production of rare ginsenoside Rk3. According to the invention, wild type beta-glucosidase derived from Sulfobus acidocaldarius is modified, so that the beta-glucosidase has efficient enzymatic activity of catalyzing the conversion of ginsenoside Rg1 into ginsenoside Rh1, and meanwhile, the heat-resistant and acid-resistant characteristics of the enzyme are retained, so that the ginsenoside Rg1 can be converted into ginsenoside Rk3 by adopting a one-pot method. The technology for large-scale production of ginsenoside Rk3 is obtained through optimization, and a foundation is laid for large-scale application of ginsenoside Rk3 in the market.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a method for large-scale production of rare ginsenoside Rk3 in a high-temperature acidic conversion system by utilizing a new beta-glucosidase mutant. Background Art

[0002] Ginsenoside Rk3 is a rare ginsenoside, but in natural ginseng, American ginseng and other Chinese medicinal materials rich in ginsenosides, the content of ginsenoside Rk3 is extremely low, only at the ppm level. The active ingredients that can really play a role in the known Araliaceae Panax plants are these rare ginsenosides with extremely low or even undetectable contents. Compared with ordinary ginsenosides, rare ginsenosides are more effective in improving or maintaining skin conditions, anti-tumor, regulating the body's immunity, etc., and have higher bioavailability. Because the content of rare ginsenosides in natural plants is very low, and it is also quite difficult to chemically synthesize them from simple initial products. Therefore, strengthening the preparation research of rare ginsenosides, further improving the yield of rare ginsenosides, and then obtaining a large amount of high-purity, highly targeted rare ginsenosides has important scientific research significance and social value. Through transformation pathway analysis, ginsenoside Rk3 is a triol group ginsenoside, which is generated by ginsenoside Rg1, Re through deglycosylation and dehydration. So far, no enzyme or reaction system has been found that can catalyze the direct production of Rk3 from ginsenoside Rg1 or Re. The conversion of ginsenoside Rg1 or Re to Rk3 is achieved step by step through intermediates in different systems.

[0003] Therefore, the process of efficiently converting prototriol ginsenoside Rg1 or Re into Rk3 is the basis for expanding the application market of ginsenoside Rk3. Summary of the invention

[0004] The object of the present invention is to overcome the defects and shortcomings of the prior art and to provide a method for large-scale production of rare ginsenoside Rk3 in a reaction system using a new β-glucosidase mutant.

[0005] The inventors conducted in-depth research to solve the above technical problems and found that:

[0006] 1. Ginsenoside Rg1 can be converted into rare ginsenoside Rk3 (second step) via rare ginsenoside Rh1 (first step).

[0007] 2. The first step of the above conversion pathway is a deglycosylation reaction, which is highly efficient under the catalysis of β-glucosidase; the second step of the above conversion pathway is a dehydration reaction, which is highly efficient under high temperature and acidic environment.

[0008] 3. In order to enable the first and second steps mentioned above to be carried out continuously in the same reaction system (one-pot method), the key is to develop a heat-resistant and acid-resistant β-glucosidase that can catalyze the deglycosylation of ginsenoside Rg1 to produce Rh1.

[0009] 4. Sulfolobus acidocaldarius is an acidophilic and thermophilic archaeon. Researchers have publicly reported the bacterium's β-glucosidase BgaS (GenBank: CP020364.1), but unfortunately, it cannot catalyze the deglycosylation reaction of ginsenoside Rg1 to produce Rh1.

[0010] 5. The inventors genetically engineered the β-glucosidase BgaS from Sulfolobus acidocaldarius to obtain the N81W / W195E variant, which has the ability to catalyze the deglycosylation of ginsenoside Rg1 to produce Rh1.

[0011] 6. Moreover, this variant is also resistant to high temperature and acid, and can maintain enzyme activity stably for a long time in a high temperature and acidic environment.

[0012] 7. The variant is applied to a reaction system for converting ginsenoside Rg1 to rare ginsenoside Rk3 in a high temperature acidic one-step method. Ginsenoside Rg1 is deglycosylated under enzyme catalysis to generate ginsenoside Rh1, which is then dehydrated under high temperature acidic conditions to generate ginsenoside Rk3. The consumption of ginsenoside Rh1 relieves the substrate inhibition of the activity of the β-glucosidase variant, thereby allowing the reaction of ginsenoside Rg1 to ginsenoside Rk3 to continue via ginsenoside Rh1.

[0013] The present invention comprises:

[0014] 1. A β-glucosidase variant, wherein an N81W mutation and a W195E mutation occur in the wild-type β-glucosidase derived from Sulfolobus acidocaldarius whose amino acid sequence is shown in SEQ ID No: 2, i.e., asparagine N at position 81 mutates to tryptophan W and tryptophan W at position 195 mutates to glutamate E.

[0015] The amino acid sequence of the β-glucosidase N81W / W195E variant can be shown as SEQ ID No: 3. The β-glucosidase variant not only has the enzyme activity of catalyzing the conversion of ginsenoside Rg1 to ginsenoside Rh1, but also retains the heat and acid resistance of Sulfolobus acidocaldarius.

[0016] 2. A host cell expressing the β-glucosidase variant according to item 1.

[0017] 3. Use of the β-glucosidase variant according to item 1 as a catalyst, which is used to catalyze the continuous reaction of converting ginsenoside Rg1 into ginsenoside Rk3 via ginsenoside Rh1, and the continuous reaction is carried out in one reaction system.

[0018] 4. A method for producing ginsenoside Rk3, comprising:

[0019] Step A: converting ginsenoside Rg1 into ginsenoside Rh1; and

[0020] Step B: converting ginsenoside Rh1 into ginsenoside Rk3;

[0021] Wherein, the method is a one-pot method, that is, the β-glucosidase variant described in item 1 is used as a catalyst, and step A and step B are continuously carried out in the same reaction system.

[0022] 5. The method according to item 4, wherein the reaction temperature of the reaction system is 70°C-120°C, preferably 76°C-84°C, and more preferably 80°C.

[0023] 6. The method according to item 4, wherein the reaction time is 12 hours to 60 hours, preferably 18 hours to 48 hours, more preferably 18 hours to 30 hours, and more preferably 24 hours.

[0024] 7. The method according to item 4, wherein the pH of the reaction system is 1-5, preferably 2.6-3.4, and more preferably 3.

[0025] 8. The method according to item 4, wherein the reaction system is acetate buffer, phosphate buffer or citrate buffer.

[0026] 9. The method according to item 4, wherein the initial concentration of ginsenoside Rg1 in the reaction system is 1 g / L-200 g / L, preferably 5 g / L-150 g / L, more preferably 10 g / L-100 g / L, and more preferably 20 g / L-50 g / L.

[0027] 10. The method according to item 4 further comprises the step of purifying the generated ginsenoside Rk3 by preparative high performance liquid chromatography, wherein the purity of the purified ginsenoside Rk3 is ≥93%.

[0028] 11. The method according to item 4, wherein the ginsenoside Rk3 produced by the method can be used to manufacture cosmetics, medicines, foods or health products.

[0029] Effects of the Invention

[0030] The present invention transforms the wild-type β-glucosidase from Sulfolobus acidocaldarius to have an efficient enzyme activity for catalyzing the conversion of ginsenoside Rg1 into ginsenoside Rh1, while retaining the heat and acid resistance of the enzyme, so that ginsenoside Rg1 can be converted into ginsenoside Rk3 by a one-pot method. Through optimization, a process for large-scale production of ginsenoside Rk3 is obtained, laying a foundation for the large-scale application of ginsenoside Rk3 in the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A is a graph showing the stability of the N81W-W195E variant enzyme at different temperatures.

[0032] Figure 1 B is a graph showing the stability of the N81W-W195E variant enzyme at different pH.

[0033] Figure 2 A is a graph showing the optimal temperature for the conversion of ginsenoside Rg1 to produce ginsenoside Rk3.

[0034] Figure 2 B is a graph showing the optimal pH for the conversion of ginsenoside Rg1 to produce ginsenoside Rk3.

[0035] Figure 2 C is a graph showing the optimal conversion time of ginsenoside Rg1 into ginsenoside Rk3.

[0036] Figure 3 A is the liquid chromatogram of the conversion product.

[0037] Figure 3 B is the liquid chromatogram of ginsenoside Rk3 after separation and purification. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings, but it should not be construed as limiting the present invention. The experimental methods used in the examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the examples can be obtained from commercial sources unless otherwise specified.

[0039] Example 1 Construction of β-glucosidase variant library

[0040] The β-glucosidase variant library was established by error-prone PCR mutation, using low-fidelity taqDNA polymerase, and the nucleotides of the β-glucosidase derived from Sulfolobus acidocaldarius (GenBank: CP020364.1) were synthesized as templates. The primers were P1 (5′- CATATG ATGTTATCATTCCCAAAGGGTT-3′) and P2(5′- AAGCTT ATGTCTCAAAGGTTTTATTGGT-3′), a certain amount of magnesium ions and manganese ions are added to the system to increase the error rate for mutation. The specific error-prone PCR system is: 5ul 10x taq enzyme buffer, 0.5uM upstream primer P1, 0.5uM downstream primer P2, 40ng template, 1mM dTTP, dCTP, 0.2mM dATP, dGTP, 5U Taq DNA polymerase, 7.5mM MgCl2, 0.3mM MnCl2, 3% DMSO, and finally sterile water is added to 50uL. The PCR conditions are as follows:

[0041]

[0042] The amplified error-prone PCR fragment and pET-30a(+) vector were double-digested with HindⅢ and NdeⅠ, respectively. The digested products were connected at a ratio of 3:1 to 10:1 between the fragment and the vector, and finally the obtained recombinant plasmid was introduced into the competent E. coli according to the conventional E. coli heat shock method to obtain a mutant plasmid library.

[0043] Example 2 Screening of β-glucosidase variant library

[0044] (1) Initial screening of mutation library

[0045] Screening principle: Aesculin is decomposed into aesculin by β-glucosidase, so if the culture medium turns black, it is positive, and if it does not turn black, it is negative. It reacts with the divalent iron ions in the culture medium to form black compounds, so if the culture medium turns black, it is positive, and if it does not turn black, it is negative. Positive strains can produce β-glucosidase, and the enzyme activity can be preliminarily determined by the color depth.

[0046] The mutant plasmid library constructed in Example 1 was introduced into BL-21 (DE3) expression competent cells to form a recombinant bacterial library capable of expressing β-glucosidase variants, and the recombinant bacteria constructed according to the nucleotide sequence of SEQ ID No: 1 without mutation were used as control bacteria, and the recombinant bacteria were transferred to the esculin-R2A screening medium to determine whether they would produce active β-glucosidase. The culture medium color of the mutant bacteria was darker than that of the positive control bacteria culture medium as a forward mutation, and the color of the culture medium was lighter than that of the positive control bacteria culture medium as a reverse mutation, and 17 positive variants (single point mutations) were finally screened.

[0047] Esculin-R2A screening culture: 1 g of esculin, 0.5 g of yeast powder, 0.5 g of ferric citrate, 0.3 g of sodium pyruvate, 0.5 g of casein, 0.3 g of potassium dihydrogen phosphate, 0.25 g of peptone, 0.5 g of glucose, 0.1 g of magnesium sulfate, and 12 g of agar were dissolved in 1000 mL of distilled water.

[0048] (2) Rescreening of mutation libraries

[0049] 17 positive variants were used as research objects to express β-glucosidase variants efficiently. The 17 mutant bacteria obtained were transferred to LB medium. When the OD (580nm) value reached about 1, IPTG was added to induce expression, and the induction was carried out at 20°C overnight. The bacteria were collected (5000rpm, centrifuged for 10min), dissolved in a certain amount of phosphate buffer, crushed, centrifuged to obtain the supernatant, dialyzed and desalted at low temperature, and finally freeze-dried to prepare β-glucosidase variant preparations. Ginsenoside Rg1 (1g / L) and the prepared β-glucosidase variant preparation were dissolved in phosphate buffer (pH3) at a ratio of 10:1 and reacted at 80°C for 12h. HPLC was used to detect the components of the conversion product, and it was found that some of the above 17 positive variants showed catalytic activity in converting ginsenoside Rg1 to ginsenoside Rk3, but the catalytic activity was weak.

[0050] Example 3 β-glucosidase combined mutation

[0051] The single point mutations in the above 17 positive variants were combined, and the catalytic activity of the variants with combined mutations was measured. The results showed that the combined mutation of asparagine N at position 81 to tryptophan W and tryptophan W at position 212 to alanine W, namely the N81W-W195E variant (amino acid sequence shown in SEQ ID No: 3) had stronger catalytic activity, and its conversion rate of catalytic conversion of ginsenoside Rg1 to ginsenoside Rk3 reached 69.3%. Among them,

[0052]

[0053] The designed combination variant N81W-W195E was commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd. CATATG ATGTTATCATTCCCAAAGGGTT-3′) and P2(5′- AAGCTT ATGTCTCAAAGGTTTTATTGGT-3′) was used as the upstream and downstream primers, and the variant was prepared according to the conventional cloning and expression method, and the enzyme preparation of the variant was obtained as in Example 2. The unmutated enzyme was used as the control group, and the conversion rate was detected according to the method of Example 2. The conversion rate of the β-glucosidase variant N81W-W195E reached 69.3%, and the unmutated β-glucosidase had no conversion effect.

[0054] Example 4 Study on the enzymatic properties of β-glucosidase variants

[0055] (1) The N81W-W195E variant enzyme preparation was dissolved in a certain phosphate buffer and incubated at 60°C, 70°C, 80°C, 90°C, and 100°C for 30 min. The relative enzyme activity of the variant was measured. The stability at different temperatures was Figure 1 As shown in A. From the above results, the variant has comparable temperature tolerance to the non-mutated enzyme. After being treated at 100°C for 30 minutes, it still maintains 90% of its activity.

[0056] (2) The variant enzyme preparation was dissolved in buffer solutions of different pH values ​​at 90° C., wherein the pH values ​​were 1, 2, 3, 4, 5, and 6, and the relative enzyme activity of the variant was determined. The stability of the variant at different pH values ​​was determined. Figure 1 As shown in B. From the above results, compared with the non-mutated enzyme, the variant has improved tolerance to low pH and can maintain 93.2% of the enzyme activity when treated at pH 1 for 30 minutes.

[0057] Example 5 Optimization of Ginsenoside Rk3 Conversion Process

[0058] (1) The N81W-W195E variant enzyme preparation and ginsenoside Rg1 were mixed in a certain ratio, placed in a pH 3 buffer, and reacted at 70°C, 80°C, 90°C, and 100°C for 24 h. Three reactions were performed in parallel at each temperature condition. The conversion rate of ginsenoside Rk3 in the conversion product was detected by high performance liquid chromatography.

[0059] (2) The N81W-W195E variant enzyme preparation and ginsenoside Rg1 were mixed in a certain ratio, placed at 80°C, and reacted at pH 1, 2, 3, 4, 5, and 6 for 24 h. Three reactions were performed in parallel under each pH condition. The conversion rate of ginsenoside Rk3 in the conversion product was detected by high performance liquid chromatography.

[0060] (3) The N81W-W195E variant enzyme preparation was mixed with ginsenoside Rg1 in a certain ratio and reacted at 80°C and pH 3 for 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, and 48 h. Three reactions were performed at each time point. The conversion rate of ginsenoside Rk3 in the conversion product was detected by HPLC.

[0061] The optimal temperature, pH and time for the conversion of ginsenoside Rg1 to ginsenoside Rk3 are as follows: Figure 2 As shown. When the temperature is 80℃, the conversion rate of ginsenoside Rk3 is the highest, and when it exceeds 90℃

[0062] The conversion rate decreases ( Figure 2 A); With the increase of pH, the conversion rate of ginsenoside Rk3 gradually increased.

[0063] The highest value is at pH 3. When the pH value exceeds 3, the conversion rate decreases ( Figure 2 B); As the conversion time increases, the conversion rate of ginsenoside Rk3 increases, and after 24 hours the conversion rate tends to be stable ( Figure 2 C).

[0064] Example 5 Validation of large-scale conversion production of β-glucosidase variant Rk3

[0065] Add 35L of phosphate buffer (pH 3) to a conversion tank with an effective volume of 50L, and then add 5kg of ginsenoside Rg1 and fully dissolve it. Then take 0.5kg of the prepared β-glucosidase variant (N81W-W195E) enzyme preparation and dissolve it in a small amount of phosphate buffer, and add it to the above conversion tank, slowly stir at 80°C (speed 20rpm) to react for 24h. After the conversion is completed, separate the supernatant and precipitate, place the precipitate in a 60°C vacuum drying oven under reduced pressure and crush it. The yield was counted, and the conversion product was analyzed by HPLC, and the conversion rate reached 69%. After the conversion product was separated and purified by high-pressure preparative chromatography, ginsenoside Rk3 with a purity of ≥93% was obtained, and the yield of Rk3 reached 90% during the purification process. The conversion product ( Figure 3 A) and Rk3 after separation and purification ( Figure 3 B) The liquid chromatogram is as follows Figure 3 shown.

[0066] Industrial Applicability

[0067] The present invention provides a β-glucosidase variant and a production method for large-scale production of ginsenoside Rk3 using the β-glucosidase variant, which is of great significance to the industrial production of ginsenoside Rk3.

[0068] Sequence information

[0069] SEQ ID No:1: Nucleotide sequence of β-glucosidase from Sulfolobus acidocaldarius 1 atgttatcat tcccaaaggg tttcaaattt ggctggtctc agtcgggatt ccagtctgaa

[0070] 61 atgggaactc caggtagcga ggatccgaac agcgattggc acgtctgggt tcatgacagg

[0071] 121 gagaacatag tctcacaggt tgtcagtgga gatttacccg aaaatggtcc agggtactgg

[0072] 181 ggggaactata agagatttca tgacgaagca gagaaaatag gactaaatgc agtgagaatt

[0073] 241 aacgtagagt ggagtagaat atttcccaga ccactaccca agcctgaaat gcaaacaggg

[0074] 301 actgataaag agaacagtcc tgtcattagc gtagacttaa atgagtctaa gctgagagaa

[0075] 361 atggacaact acgctaatca tgaagcgtta tcacattaca ggcaaatact ggaggatcta

[0076] 421 agaaacagag gatttcacat agtactgaac atgtatcatt ggactttgcc catatggttg

[0077] 481 cacgacccta tcagagtgag gagaggagac tttacaggac caacaggttg gttaaactcc

[0078] 541 aggacagttt atgagttcgc taggttctcg gcttacgtag cctggaaatt agatgatttg

[0079] <h2 style=";text-align:left;direction:ltr">601gcgagtgaat atgcaacaat gaatgaacct aacgtggttt ggggagcagg ttacgctttt<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0080] <h2 style=";text-align:left;direction:ltr"> 661cctagagcag gctttccacc taattacctt agcttcaggc tttcagaaat agctaaatgg<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0081] <h2 style=";text-align:left;direction:ltr"> 721aatataattc aggctcatgc gagggcttat gacgccatca agagcgtatc aaaaaagagt<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0082] <h2 style=";text-align:left;direction:ltr"> 781gtaggtataa tatatgcaaa cacatcatat tacccactca gaccacaaga taacgaagct<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0083] <h2 style=";text-align:left;direction:ltr"> 841gtggaaatag cagagagatt gaacagatgg agtttctttg actccattat aaagggagag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0084] <h2 style=";text-align:left;direction:ltr"> 901ataactagtg agggacaaaa tgtcagagag gacttaagga acaggttaga ctggattggc<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0085] <h2 style=";text-align:left;direction:ltr"> 961gtaaactatt acacgaggac tgtggtaaca aaagctgaga gtggttattt aacccttccg<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0086] <h2 style=";text-align:left;direction:ltr"> 1021ggttatggag atcgttgtga aaggaactca ttgagtttag ctaacctccc taccagtgat<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0087] <h2 style=";text-align:left;direction:ltr"> 1081ttcggttggg agttctttcc tgagggtcta tatgatgtac ttttgaagta ttggaatagg<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0088] <h2 style=";text-align:left;direction:ltr"> 1141tatgggttac cattatacgt aatggagaac ggtatcgctg atgacgctga ctaccaaaga<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0089] 1201ccgtattact tagtatcaca tatctaccag gtgcacaggg ctttaaacga gggagtagat

[0090] 1261gtaagaggtt atcttcattg gtctttggca gataattatg agtggtcgtc aggtttttca

[0091] 1321atgaggttcg gtctacttaa ggtagattat ctaacaaaga gattgtactg gagaccttct

[0092] 1381gcattagttt acagggat tactaggagt aacggtattc ctgaggagct ggaacatcta

[0093] 1441aacagagtac caccaataaa acctttgaga catSEQ ID No: 2Amino acid sequence of β-glucosidase from Sulfolobusacidocaldarius 1mlsfpkgfkf gwsqsgfqse mgtpgsedpnsdwhvwvhdr enivsqvvsg dlpengpgyw

[0094] 61gnykrfhdea ekiglnavri nvewsrifpr plpkpemqtg tdkenspvis vdlnesklre

[0095] 121mdnyanheal shyrqiledl rnrgfhivln myhwtlpiwl hdpirvrrgd ftgptgwlns

[0096] 181rtvyefarfs ayvawklddl aseyatmnep nvvwgagyaf pragfppnyl sfrlseiakw

[0097] 241niiqaharay daiksvskks vgiiyantsy yplrpqdnea veiaerlnrw sffdsiikge

[0098] 301itsegqnvre dlrnrldwig vnyytrtvvt kaesgyltlp gygdrcerns lslanlptsd

[0099] 361fgweffpegl ydvllkywnr yglplyvmen giaddadyqr pyylvshiyq vhralnegvd

[0100] 421vrgylhwsla dnyewssgfs mrfgllkvdy ltkrlywrps alvyreitrs ngipeelehl

[0101] 481nrvppikplr h

[0102] Amino acid sequence of SEQ ID No: 3 combinatorial mutant (N81W-W195E):

[0103] 1mlsfpkgfkf gwsqsgfqse mgtpgsedpn sdwhvwvhdr enivsqvvsg dlpengpgyw

[0104] 61gnykrfhdea ekiglnavri wvewsrifpr plpkpemqtg tdkenspvis vdlnesklre

[0105] 121mdnyanheal shyrqiledl rnrgfhivln myhwtlpiwl hdpirvrrgd ftgptgwlns

[0106] 181rtvyefarfs ayvaeklddl aseyatmnep nvvwgagyaf pragfppnyl sfrlseiakw

[0107] 241niiqaharay daiksvskks vgiiyantsy yplrpqdnea veiaerlnrw sffdsiikge

[0108] 301itsegqnvre dlrnrldwig vnyytrtvvt kaesgyltlp gygdrcerns lslanlptsd

[0109] 361fgweffpegl ydvllkywnr yglplyvmen giaddadyqr pyylvshiyq vhralnegvd

[0110] 421vrgylhwsla dnyewssgfs mrfgllkvdy ltkrlywrps alvyreitrs ngipeelehl

[0111] 481nrvppikplr h。

Claims

1. A β-glucosidase variant, wherein In the wild-type β-glucosidase derived from Sulfolobus acidocaldarius whose amino acid sequence is shown in SEQ ID No: 2, N81W mutation and W195E mutation occur, that is, asparagine N at position 81 mutates to tryptophan W and tryptophan W at position 195 mutates to glutamate E. Preferably, the amino acid sequence of the β-glucosidase N81W / W195E variant can be, for example, as shown in SEQ ID No:

3.

2. A host cell expressing the β-glucosidase variant according to claim 1.

3. Use of the β-glucosidase variant according to claim 1 as a catalyst, which is used to catalyze the continuous reaction of converting ginsenoside Rg1 into ginsenoside Rk3 via ginsenoside Rh1, and the continuous reaction is carried out in one reaction system.

4. A method for producing ginsenoside Rk3, comprising: Step A: converting ginsenoside Rg1 into ginsenoside Rh1; and Step B: converting ginsenoside Rh1 into ginsenoside Rk3; The method is a one-pot method, that is, the β-glucosidase variant according to claim 1 is used as a catalyst, and step A and step B are continuously performed in the same reaction system.

5. The method according to claim 4, wherein: The reaction temperature of the reaction system is 80°C.

6. The method according to claim 4, wherein: The reaction time is 12 to 60 hours, preferably 18 to 48 hours, more preferably 18 to 30 hours, and still more preferably 24 hours.

7. The method according to claim 4, wherein: The pH of the reaction system is 1-5, preferably 2.6-3.4, more preferably 3.

8. The method according to claim 4, wherein: The reaction system is acetate buffer, phosphate buffer or citrate buffer.

9. The method according to claim 4, wherein: The initial concentration of ginsenoside Rg1 in the reaction system is 1 g / L-200 g / L, preferably 5 g / L-150 g / L, more preferably 10 g / L-100 g / L, and more preferably 20 g / L-50 g / L.

10. The method according to claim 4, further comprising the step of purifying the generated ginsenoside Rk3 by preparative high performance liquid chromatography, wherein the purity of the purified ginsenoside Rk3 is ≥93%.

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