Beta-glucosidase mutant, nucleic acid molecule and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a beta-glucosidase mutant, a nucleic acid molecule and application of the beta-glucosidase mutant. The invention provides a beta-glucosidase mutant. The amino acid sequence of the beta-glucosidase mutant is as shown in SEQ ID NO. 1. Compared with wild type beta-glucosidase, the beta-glucosidase mutant has mutation sites of V268A, E290V and I450T, and the result of the embodiment shows that the enzyme activity of the beta-glucosidase mutant is 544U / L, and compared with the enzyme activity of 148U / L of the beta-glucosidase of the wild type strain, the enzyme activity of the beta-glucosidase mutant is improved by 267.57%.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a beta-glucosidase mutant, a nucleic acid molecule and applications thereof. Background Art

[0002] Ginsenosides are one of the most important secondary metabolites of ginseng. At present, 180 ginsenosides have been isolated from ginseng roots, among which ginsenoside Rg3 exhibits multiple pharmacological activities, such as anti-inflammatory, anti-aging, anti-cancer, and regulating the body's immunity. According to the spatial arrangement of the hydroxyl groups on the chiral carbon of the Rg3 aglycone, there are optical isomers 20(S)-Rg3 and 20(R)-Rg3. 20(S)-Rg3 is more soluble in water than 20(R)-Rg3 and has higher bioavailability. The main ginsenosides such as Rb1, Rb2, Rd, etc. can be converted into a mixture of ginsenosides 20(S)-Rg3 and 20(R)-Rg3 by acid treatment or heating, but it is complicated and time-consuming to separate each isomer from the racemic mixture; 20(S)-Rg3 can also be obtained by chemical synthesis of betulintriol, but the synthesis steps are complicated and the overall yield is low.

[0003] At present, enzymes produced by microorganisms can be used as catalysts to catalyze specific substrates to prepare ginsenoside Rg3. For example, β-glucosidase can catalyze the conversion of ginsenoside Rb1 into ginsenoside Rg3. However, how to improve the enzymatic activity of β-glucosidase is still a problem to be solved in this field. Summary of the invention

[0004] The object of the present invention is to provide a β-glucosidase mutant, a nucleic acid molecule and applications thereof. The enzyme activity of the β-glucosidase mutant of the present invention is significantly improved.

[0005] The present invention provides a beta-glucosidase mutant, and the amino acid sequence of the beta-glucosidase mutant is shown in SEQ ID NO.1.

[0006] The present invention also provides a nucleic acid molecule encoding the β-glucosidase mutant described in the above scheme.

[0007] As a preferred embodiment, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.2.

[0008] The present invention also provides a recombinant expression vector, which comprises the nucleic acid molecule described in the above scheme.

[0009] As a preferred embodiment, the expression vector includes pET28a.

[0010] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the recombinant expression vector described in the above scheme.

[0011] As a preferred embodiment, the basic bacteria of the recombinant bacteria include competent Escherichia coli.

[0012] The present invention also provides a method for preparing the β-glucosidase mutant described in the above scheme, comprising the following steps: culturing the recombinant bacteria described in the above scheme, inducing the recombinant bacteria to express, and obtaining the β-glucosidase mutant.

[0013] The present invention also provides the use of the nucleic acid molecule, the recombinant expression vector or the recombinant bacteria in the above scheme in preparing the β-glucosidase mutant.

[0014] The present invention also provides the use of the β-glucosidase mutant or the nucleic acid molecule or the recombinant expression vector or the recombinant bacteria described in the above scheme in the production of ginsenoside Rg3.

[0015] Beneficial effect: The present invention provides a β-glucosidase mutant, the amino acid sequence of the β-glucosidase mutant is shown in SEQ ID NO. 1. Compared with the wild-type β-glucosidase, the β-glucosidase mutant of the present invention has mutation sites V268A, E290V, and I450T. The results of the embodiment show that the enzyme activity of the β-glucosidase mutant of the present invention is 544U / L, which is 267.57% higher than the β-glucosidase activity of the wild-type strain of 148U / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 is the pNP standard curve;

[0018] Figure 2 For different Mn 2+ Agarose gel imaging results of CepPCR products at different concentrations; Lane M is DL 5000 DNA marker; Lanes 1 to 5: P1 fragment, Mn 2+ 0.10mM, 0.30mM, 0.50mM, 0.70mM, 0.90mM, respectively; Lanes 6-10: P2 fragment, Mn 2+ 0.10mM, 0.30mM, 0.50mM, 0.70mM, 0.90mM, respectively; Lanes 11-15: P3 fragment, Mn 2+ 0.10mM, 0.30mM, 0.50mM, 0.70mM, 0.90mM;

[0019] Figure 3It is the agarose gel electrophoresis diagram of Megawhop PCR products; Lane M is DL 15000 DNA marker; P1 is amplified by primers of fragment 1; P2 is amplified by primers of fragment 2; P3 is amplified by primers of fragment 3;

[0020] Figure 4 The agarose gel electrophoresis diagram of the colony PCR product; lane M is the DL 5000 DNA marker; + is the positive control; 1 to 15 are target gene mutant strains;

[0021] Figure 5 It is a single clone colony image;

[0022] Figure 6 The results of rescreening the mutant enzyme activity;

[0023] Figure 7 This is a comparison of β-glucosidase amino acid sequences. DETAILED DESCRIPTION

[0024] The present invention provides a β-glucosidase mutant, and the amino acid sequence of the β-glucosidase mutant is shown in SEQ ID NO.1: MKEVNEILSKLTLEEKVKLVVGVGMPGFFGNPP.

[0025]

[0026] The present invention also provides a recombinant expression vector, which includes the nucleic acid molecule described in the above scheme. As an embodiment, the expression vector includes pET28a. In a specific embodiment of the present invention, the nucleic acid molecule is inserted between NcoⅠ and XhoⅠ of the expression vector; the 5′ end of the nucleic acid molecule is also connected to the NcoⅠ restriction site recognition sequence CCATGG, and the linker TT, and the 3′ end of the nucleic acid molecule is also connected to the XhoⅠ restriction site recognition sequence CTCGAG. In the present invention, the linker TT can provide a more stable platform for the enzyme to bind to the site, improve the enzyme cutting stability, and increase the stability of the enzyme cutting site.

[0027] The present invention also provides a recombinant bacterium, the recombinant bacterium comprises the recombinant expression vector described in the above scheme. As an embodiment, the basic bacteria of the recombinant bacterium comprises competent E. coli. In a specific embodiment of the present invention, the competent E. coli is competent E. coli BL21 (DE3).

[0028] The present invention also provides a method for preparing the β-glucosidase mutant described in the above scheme, comprising the following steps: culturing the recombinant bacteria described in the above scheme, inducing the recombinant bacteria to express, and obtaining the β-glucosidase mutant.

[0029] The present invention also provides the use of the nucleic acid molecule or the recombinant expression vector or the recombinant bacteria in the above scheme in preparing the β-glucosidase mutant in the above scheme.

[0030] The present invention also provides the use of the β-glucosidase mutant or the nucleic acid molecule or the recombinant expression vector or the recombinant bacteria described in the above scheme in the production of ginsenoside Rg3. Compared with the wild type, the β-glucosidase mutant of the present invention has mutation sites V268A, E290V, and I450T, and the enzyme activity of the β-glucosidase mutant is 544U / L, which is 267.57% higher than the enzyme activity of the wild type strain 148U / L.

[0031] To further illustrate the present invention, a β-glucosidase mutant, nucleic acid molecule and application thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1

[0033]

[0034] (2) Use a pipette to inoculate the strain E. coli BL21 (DE3) -pET28a-GH3 (200 μL) into 20 mL of LB medium (containing 50 μg / mL kanamycin) at a 1% inoculation rate, and culture in a constant temperature shaker at 37°C and 180 rpm for 12 h to obtain an activated E. coli BL21 (DE3) -pET28a-GH3 bacterial solution.

[0035] (3) The activated E. coli BL21 (DE3)-pET28a-GH3 bacterial solution was transferred to a 96-well plate for induction culture of the strain. 1 mL of LB medium (containing 50 μg / mL kanamycin) was added to each well, and the activated bacterial solution (10 μL) was added at a 1% inoculum. The plate was sealed and cultured at 37°C and 700 rpm. When the OD 600 When the pH was 0.7, the bacterial solution was transferred to a 96-deep-well plate, and isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0.25 mM, and induced at 30°C for 6 h.

[0036] (4) Determination of β-glucosidase activity:

[0037] A. Standard curve determination: Weigh 0.1391g p-nitrophenol (p-NP) and dissolve it in distilled water, dilute to 100mL, and obtain 10mM p-NP. Take 9 10mL volumetric flasks, and pipette 0.1, 0.2, 0.6, 1.0, 1.5, 2.5, 3.5, 4.5, 5.5mL of p-NP solution into flasks 1 to 9, dilute to volume with distilled water and mix well. The p-NP concentrations corresponding to flasks 1 to 9 are 0.1, 0.2, 0.6, 1.0, 1.5, 2.5, 3.5, 4.5, 5.5mM. Take 150 μL pNP from each bottle, add 1 mL 1 M Na2CO3 and mix well, corresponding to 0.0150, 0.0300, 0.0900, 0.150, 0.225, 0.375, 0.525, 0.675, 0.825 μmoL pNP, respectively, measure the absorbance at 400 nm, and obtain the standard curve, as shown in Figure 1 As shown, the resulting equation is y = 0.2404x-0.02206, R 2 Reaching 0.9997, the linearity is good.

[0038] B. Take 50 μL of the bacterial solution from step (3) and mix with 100 μL of 5 mM p-nitrophenyl-β-D-pyranoglucoside (pNPG), and keep at 50°C for 30 min. After the incubation, add 1 mL of 1 M Na2CO3 to terminate the reaction and develop the color. The absorbance is measured at 400 nm to detect the activity of β-glucosidase. The definition of β-glucosidase activity is: the amount of enzyme required to hydrolyze the substrate to release 1 μmoL p-nitrophenol (p-NP) per minute under the conditions of 50°C and pH = 5.0 is one unit (U).

[0039] The absorbance detection results are shown in Table 1. Through calculation, it can be seen that the relative deviation is 8.15%, which is less than the set value of 12%, indicating that this method is suitable for high-throughput screening of β-glucosidase.

[0040] Table 1 Absorbance values ​​of original strains screened in 96-well plate

[0041] / 1 2 3 4 5 6 7 8 9 10 11 12 A 1.05 1.08 1.13 1.04 1.00 0.97 1.18 1.12 1.09 0.98 1.09 1.05 B 0.99 1.00 0.95 0.92 1.13 0.99 0.95 1.19 0.90 1.07 1.05 1.02 C 1.07 1.08 1.05 1.02 0.98 1.14 1.01 0.96 0.97 1.19 0.98 0.98 D 1.19 0.84 0.96 0.85 1.02 1.02 0.98 0.98 1.12 0.96 1.02 0.93 E 0.94 1.13 1.03 0.84 1.03 1.04 1.00 1.06 1.16 1.02 1.15 1.03 F 1.07 1.06 1.12 1.03 1.08 1.14 1.06 1.09 1.06 1.09 0.91 0.91 G 1.12 0.97 1.12 0.99 0.95 0.96 0.91 1.08 1.00 0.96 0.96 0.90 H 1.05 1.09 1.15 1.15 1.10 1.23 1.19 1.07 0.97 1.01 1.17 1.04

[0042] Example 2 Determination of CepPCR Amplification Conditions

[0043] (1) The plasmid was extracted from the strain E. coli BL21 (DE3)-pET28a-GH3 in Example 1 using a plasmid extraction kit (Plasmid Mini Kit I, OmegaBio-Tek D6943-02). The specific operation was carried out according to the instruction manual to obtain plasmid pET28a-GH3.

[0044] The β-glucosidase gene sequence (SEQ ID NO.3) was divided into three fragments, named P1, P2 and P3, wherein 1 to 720 bp of the sequence shown in SEQ ID NO.3 was the P1 fragment, with a length of 720 bp; 721 to 1440 bp was the P2 fragment, with a length of 720 bp; 1441 to 2180 bp was the P3 fragment, with a length of 740 bp. Primer pairs were designed at both ends of the three gene fragments of P1, P2 and P3, respectively, and CepPCR amplification was performed to obtain mutant gene fragments. The primer sequences for CepPCR amplification are shown in Table 2.

[0045] Table 2 CepPCR amplification primer sequences

[0046] name Sequence (5′-3′) Serial Number 1-F1 CCATGGTTATGAAGGAAGTGAATGAG SEQ ID NO.4 1-R1 AACCCTCAAAGCCCCAATCT SEQ ID NO.5 1-F2 TTGTTATGAGCGATTGGTATGCC SEQ ID NO.6 1-R2 GCTCAAACTCATCGTCGCTC SEQ ID NO.7 1-F3 TGATTCAGAAAGTTAGCAGCCAG SEQ ID NO.8

[0047] The CepPCR system is: Premix Taq DNA polymerase 25 μL, primer F 1 μL (1 μM), primer R 1 μL (1 μM), pET28a-GH3 30 ng, MnCl2 0.1-0.9 mM, and ultrapure water to 50 μL. The CepPCR program is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 68°C for 2 min, 30 cycles; final extension at 68°C for 10 min; storage at 4°C.

[0048] (2) The CepPCR product was analyzed by agarose gel electrophoresis as follows:

[0049] A. Weigh 0.25g agarose and add 25mL 1×TAE solution, heat in a microwave oven for 1min to ensure that the agarose is fully dissolved and a transparent and clear solution is obtained; after cooling to 50℃~60℃ at room temperature, add 2.5μL 10000×Gel Red nucleic acid stain, shake well, pour into the gelatin plate, insert a tooth comb, and remove any bubbles in time. Let it stand at room temperature until the gelatin solution is completely solidified (about 20~30min).

[0050] B. After the gel solidifies, gently pull out the tooth comb and place the colloid in an electrophoresis tank filled with 1×TAE solution, with the loading hole facing the negative electrode.

[0051] C. Mix 5 μL of sample with 1 μL of 6× Loading Buffer by pipetting, add 5 μL of the mixed sample to the loading well, adjust the voltage to 120V, and the time to 40 minutes. Figure 2 shown.

[0052] according to Figure 2 It can be seen that the CepPCR products with different manganese ion concentrations were imaged after agarose gel electrophoresis. Lanes 1 to 5 are the electrophoresis results of CepPCR products with template P1 added, lanes 6 to 10 are the electrophoresis results of epPCR products with template P2 added, and lanes 11 to 15 are the electrophoresis results of CepPCR products with template P3 added. Each group from left to right is the CepPCR system with Mn added. 2+ The concentrations are 0.10, 0.30, 0.50, 0.70, and 0.90 mM. Lane M is DL 5000 DNA marker. Figure 2 It can be seen that fragment P1 adds Mn 2+ The concentration range should be 0.10-0.50 mM. The band is brighter when the concentration is between 0.10 and 0.30 mM. 2+ The concentration range should be 0.10-0.70 mM. The band is brighter when the concentration is between 0.10 and 0.50 mM. 2+The concentration range should be 0.10-0.70 mM, and the bands are brighter when the concentration is between 0.10-0.30 mM. In summary, 0.3 mM was selected as the concentration of the final CepPCR system.

[0053] (3) Purification and recovery of CepPCR products

[0054] The CepPCR product was recovered using TaKaRa MiniBEST DNA Fragment Purification Kit Ver. 4.0. For specific experimental procedures, please refer to the kit instructions.

[0055] Example 3

[0056] (1) Megawhop PCR: Select the best Mn in the CepPCR system 2+ The CepPCR purified product of the concentration was used as a large primer for Megawhop PCR to amplify the whole plasmid to obtain the mutated plasmid. The Megawhop PCR system is: PrimeSTARMax DNA polymerase 25μL, pET28a-GH340ng, CepPCR product (primer) 500ng, ddH2O supplemented to 50μL. The program settings of the Megawhop PCR are shown in Table 3, where the annealing temperature of the P1 fragment is 62°C, the annealing temperature of the P2 fragment is 66°C, and the annealing temperature of the P3 fragment is 66°C. The test results are shown in Table 3. Figure 3 shown.

[0057] Table 3 Megawhop PCR program

[0058]

[0059] according to Figure 3 It can be seen that the DNA length obtained by the amplification product is about 7500-10000 bp. It is known that the size of pET28a-GH3 is about 7500 bp, which is consistent with the band in the figure. It is preliminarily judged that the amplification is successful.

[0060] (2) DpnⅠ digestion and purification

[0061] DpnⅠ enzyme is a restriction nuclease that can specifically excise methylated DNA chains and is often used to excise template DNA after PCR. Since the original template plasmid is derived from conventional E. coli and is modified by dam methylation, it is sensitive to DpnI and is chopped up (the DpnI recognition sequence is methylated GATC, which appears in almost all plasmids and more than once), and the plasmid with a mutant sequence synthesized in vitro is not methylated and is not cut, so it can be successfully transformed in the subsequent transformation, and the mutant plasmid clone can be obtained.

[0062] The successfully verified Megawhop PCR product was subjected to DpnⅠ digestion. The DpnⅠ digestion system was: 10×CutSmartBuffer 25μL, PCR product <2μg, DpnⅠ (20unit) 2μL, and ultrapure water to 50μL. The DpnⅠ digestion program was: 37℃ 2.5h; 85℃ 30min; 4℃∞.

[0063] (3) Transformation of E. coli TOP10 competent cells

[0064] Take 1 μL of the digested product from step (2) and transform it into E. coli Top10 heat shock competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), and perform the specific operation according to the instructions. Spread each 100 μL of the transformed bacterial solution evenly on a solid LB medium containing kanamycin, and culture at a constant temperature of 37°C for 12 to 17 hours, not more than 20 hours, and observe the growth of the colonies, thereby obtaining a gene mutation library.

[0065] (4) Positive conversion rate verification

[0066] From the solid LB medium in step (3), more than ten single colonies were randomly selected and placed in 20 μL ultrapure water, heated at 95°C for 5 min, and centrifuged for 1 min. The supernatant contained plasmids released by high temperature cell disruption, which can be used as a template for colony PCR, with pET28a-GH3 plasmid as a positive control. The colony PCR system is as follows: 5 μL of Premix Taq DNA polymerase, 1 μL of 1-F1 (1 μM), 1 μL of 1-R3 (1 μM), 1 μL of template, and 2 μL of ultrapure water. The colony PCR program is: pre-denaturation at 98°C for 90 s; denaturation at 98°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 4 min, 30 cycles; final extension at 72°C for 10 min; storage at 4°C.

[0067] The above PCR products were subjected to agarose gel electrophoresis. Figure 4 The length of the positive control pET28a-GH3 gene fragment is 2180bp. In lanes 1 to 15 of the experimental group, there are 13 bands of the same length as the positive control lane, indicating that the plasmid pET28a-GH3mutant was successfully constructed, with a positive rate of about 86.67%, which meets the requirements for library construction.

[0068] (5) Transformation into E. coli BL21 (DE3) competent cells

[0069] A. In a clean bench, the solid culture medium plate on which the colonies grew in step (3) was eluted with LB liquid culture medium (containing kana), the eluate was collected in a sterilized centrifuge tube, and the plasmid was extracted using a plasmid extraction kit (Plasmid MiniKit I, OmegaBio-Tek D6943-02). The specific operation was carried out according to the instructions.

[0070] B. All plasmids containing mutant sub-library in step A were transformed into E.coliBL21(DE3) competent cells. The transformation method is described in the instruction manual. Then the bacterial solution was spread on solid LB medium containing kana resistance and cultured at 37℃ for 16h until a large number of E.coliBL21(DE3)-pET28a-GH3 mutant colonies were grown. The obtained single colonies were mutant monoclonal colonies with kana resistance. The results were as follows: Figure 5 As shown; the solid culture medium was stored in a 4°C refrigerator awaiting subsequent bacterial activation culture.

[0071] (6) Mutant culture and induced expression

[0072] A. Activation: Open the sterilized sealed 96-well plate on a sterile workbench, add 1 mL of liquid LB medium (containing Kana resistance) to each well, pick the bacteria in step (5) into the liquid medium with a sterilized toothpick, and seal with a sterile sealing film. The culture temperature is 37°C, the rotation speed is 700 rpm, and the activation culture is carried out overnight.

[0073] B. Induced expression: Add 1 mL of liquid LB medium (containing Kana resistance) to each 96-well plate, inoculate the activated bacterial solution in the same order with a 1% inoculum into a new 96-well plate, and culture at 37°C and 700 rpm. 600 When the value was 0.7, IPTG was added (final concentration 0.25 mM, 10 μL IPTG was added to 1 mL of culture medium; 200 μL IPTG was added to 20 mL of culture medium), and the induction culture was continued at 30°C for 6 h.

[0074] C. Enzyme activity detection: Take 50 μL of bacterial solution and mix with 100 μL of 5 mM pNPG, and keep at 50℃ for 30 min. Add 1 mL of 1 M Na2CO3 to terminate the reaction and develop color, and measure the absorbance at 400 nm. The results of enzyme activity detection of mutant strains are as follows: Figure 6 shown.

[0075] according to Figure 6 It can be seen that after error-prone PCR mutation, 25 mutants with excellent enzyme activity were initially screened out for re-screening. CK is a wild-type bacterium of β-glucosidase, among which the enzyme activity of Q-17 strain is 544U / L, which is 267.57% higher than that of the wild-type strain of 148U / L.

[0076] (7) Mutant amino acid comparison

[0077] The mutant Q-17 strain with good enzyme activity was sent to Guangzhou Aiji Biotechnology Co., Ltd. for sequencing to obtain the gene sequence of the mutant strain, and the gene sequence was converted into an amino acid sequence using Snapgene software. The mutant β-glucosidase amino acid sequence was then compared with the wild type using the Clustal Omega website (https: / / www.ebi.ac.uk / jdispatcher / msa / clustalo). The results are as follows: Figure 7 The results showed that the amino acid sequence mutation sites of the β-glucosidase expressed by the Q-17 strain mutant were: V268A, E290V, I450T, and the amino acid sequence after mutation was shown in SEQ ID NO.1.

[0078] It can be seen that the enzyme activity of the β-glucosidase mutant of the present invention is 544 U / L, which is 267.57% higher than that of the wild-type strain 148 U / L. The β-glucosidase mutant of the present invention can be used to produce ginsenoside Rg3.

[0079] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A β-glucosidase mutant, characterized in that The amino acid sequence of the β-glucosidase mutant is shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding the β-glucosidase mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule according to claim 2 or 3.

5. The recombinant expression vector according to claim 4, characterized in that: The expression vector includes pET28a.

6. A recombinant bacterium, characterized in that: The recombinant bacteria comprises the recombinant expression vector according to claim 4 or 5.

7. The recombinant bacterium according to claim 6, characterized in that The basic bacteria of the recombinant bacteria include competent Escherichia coli.

8. The method for preparing a β-glucosidase mutant according to claim 1, characterized in that: The method comprises the following steps: culturing the recombinant bacteria according to claim 6 or 7, inducing the recombinant bacteria to express, and obtaining a β-glucosidase mutant.

9. Use of the nucleic acid molecule according to claim 2 or 3, the recombinant expression vector according to claim 4 or 5, or the recombinant bacterium according to claim 6 or 7 in preparing the β-glucosidase mutant according to claim 1.

10. Use of the β-glucosidase mutant according to claim 1, the nucleic acid molecule according to claim 2 or 3, the recombinant expression vector according to claim 4 or 5, or the recombinant bacteria according to claim 6 or 7 in producing ginsenoside Rg3.

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