A beta-glucosidase mutant, nucleic acid molecule and application thereof

Abstract

The present invention belongs to the field of genetic engineering technology, and in particular to a beta-glucosidase mutant, a nucleic acid molecule and an application thereof. The present invention provides a beta-glucosidase mutant, the amino acid sequence of the beta-glucosidase mutant being as shown in SEQ ID NO.1. The beta-glucosidase mutant of the present invention has mutation sites V268A, E290V and I450T compared with the wild-type beta-glucosidase, and the results of the examples show that the enzyme activity of the beta-glucosidase mutant of the present invention is 544U / L, which is 267.57% higher than the beta-glucosidase activity of 148U / L of the wild-type strain.

Description

Technical Field

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

[0002] Ginsenosides are among the most important secondary metabolites of ginseng. Currently, 180 ginsenosides have been isolated from ginseng roots. Among them, ginsenoside Rg3 exhibits diverse pharmacological activities, including anti-inflammatory, anti-aging, anti-cancer, and immune-modulating activities. Based on the spatial arrangement of the hydroxyl groups on the chiral carbon atoms of the Rg3 aglycone, there are optical isomers, 20(S)-Rg3 and 20(R)-Rg3. 20(S)-Rg3 is more water-soluble and has higher bioavailability than 20(R)-Rg3. Major ginsenosides such as Rb1, Rb2, and Rd can be converted into a mixture of ginsenosides 20(S)-Rg3 and 20(R)-Rg3 by acid treatment or heating. However, isolating each isomer from the racemic mixture is complex and time-consuming. 20(S)-Rg3 can also be chemically synthesized via betulintriol, but the synthesis is complex and yields low overall yields.

[0003] Currently, microbial enzymes can be used as catalysts to catalyze reactions on specific substrates to produce ginsenoside Rg3. For example, β-glucosidase can catalyze the conversion of ginsenoside Rb1 to ginsenoside Rg3. However, increasing the enzymatic activity of β-glucosidase remains an urgent challenge 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 β-glucosidase mutant, the amino acid sequence of the β-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 includes 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, which 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, the nucleic acid molecule, the recombinant expression vector or the recombinant bacteria described in the above scheme in the production of ginsenoside Rg3.

[0015] Beneficial effects: The present invention provides a β-glucosidase mutant, the amino acid sequence of which 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 examples show that the enzyme activity of the β-glucosidase mutant of the present invention is 544 U / L, which is 267.57% higher than the β-glucosidase activity of 148 U / L of the wild-type strain. 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 for use 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; Lanes 6-10: P2 fragment, Mn 2+ 0.10mM, 0.30mM, 0.50mM, 0.70mM, 0.90mM; Lanes 11-15: P3 fragment, Mn 2+ 0.10mM, 0.30mM, 0.50mM, 0.70mM, 0.90mM;

[0019] Figure 3The figure is an 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 figure is an 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 The figure is a monoclonal colony;

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

[0023] Figure 7 This is a diagram of the amino acid sequence alignment of β-glucosidase. DETAILED DESCRIPTION

[0024] The present invention provides a β-glucosidase mutant, 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 comprising the nucleic acid molecule described in the above embodiment. In one embodiment, the expression vector comprises pET28a. In a specific embodiment of the present invention, the nucleic acid molecule is inserted between Nco I and Xho I of the expression vector; the 5′ end of the nucleic acid molecule is further linked to the Nco I restriction site recognition sequence CCATGG and a linker TT, and the 3′ end of the nucleic acid molecule is further linked to the Xho I 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 stability of the enzyme cleavage, and increase the stability of the restriction site.

[0027] The present invention also provides a recombinant bacterium comprising the recombinant expression vector described in the above embodiment. In one embodiment, the base bacteria of the recombinant bacterium include competent Escherichia coli. In a specific embodiment of the present invention, the competent Escherichia coli is 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, 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, the nucleic acid molecule, 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 mutations at V268A, E290V, and I450T. The enzyme activity of the β-glucosidase mutant is 544 U / L, which is 267.57% higher than the enzyme activity of the wild-type strain of 148 U / 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 with reference to 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 volume. The plate was sealed and cultured at 37°C and 700 rpm. When the OD 600 When the pH value was 0.7, the bacterial solution was transferred to a 96-deep-well plate, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added to 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 0.1391 g of p-nitrophenol (p-NP) and dissolve it in distilled water to 100 mL to obtain 10 mM p-NP. Transfer 0.1, 0.2, 0.6, 1.0, 1.5, 2.5, 3.5, 4.5, and 5.5 mL of p-NP solution to nine 10 mL volumetric flasks, respectively. Dose distilled water to the volume and mix thoroughly. The corresponding p-NP concentrations in flasks 1 to 9 are 0.1, 0.2, 0.6, 1.0, 1.5, 2.5, 3.5, 4.5, and 5.5 mM. Take 150 μL of pNP from each bottle, add 1 mL of 1 M Na2CO3 and mix well, corresponding to 0.0150, 0.0300, 0.0900, 0.150, 0.225, 0.375, 0.525, 0.675, and 0.825 μmoL pNP, respectively. Measure the absorbance at 400 nm to obtain a standard curve, as shown in the following example: Figure 1 As shown, the resulting equation is y = 0.2404x - 0.02206, R 2 It reaches 0.9997, with good linearity.

[0038] B. Take 50 μL of the bacterial solution from step (3) and mix it with 100 μL of 5 mM p-nitrophenyl-β-D-pyranoglucoside (pNPG), and incubate at 50°C for 30 min. After 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 at 50°C and pH = 5.0 is one unit (U).

[0039] The absorbance detection results are shown in Table 1. Calculation shows 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 of the E. coli BL21 (DE3)-pET28a-GH3 strain in Example 1 was extracted using a plasmid extraction kit (Plasmid Mini Kit I, OmegaBio-Tek D6943-02). The specific operation was carried out according to the instructions to obtain plasmid pET28a-GH3.

[0044] The β-glucosidase gene sequence (SEQ ID NO. 3) was divided into three segments, designated P1, P2, and P3. The 1-720 bp segment of SEQ ID NO. 3 is the 720 bp P1 segment; the 721-1440 bp segment is the 720 bp P2 segment; and the 1441-2180 bp segment is the 740 bp P3 segment. Primer pairs were designed at each end of the P1, P2, and P3 gene segments, respectively, and CepPCR amplification was performed to obtain mutant gene segments. 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 TGATTCAGAAAGTTAGCAGCCAGT SEQ ID NO.8

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

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

[0049] A. Weigh 0.25 g agarose and add 25 mL of 1× TAE solution. Microwave for 1 minute to ensure the agarose is fully dissolved and a clear solution is obtained. After cooling to 50°C–60°C at room temperature, add 2.5 μL of 10000× Gel Red nucleic acid stain. Shake well and pour onto a gelatin plate. Use a tooth comb to remove any bubbles. Let stand at room temperature until the gelatin solution is completely solidified (approximately 20–30 minutes).

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

[0051] C. Mix 5 μL of sample with 1 μL of 6× Loading Buffer by pipetting, then 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 CepPCR 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 the fragment P1 adds Mn 2+ The concentration range should be 0.10~0.50mM, and the band is brighter when the concentration is between 0.10~0.30mM; 2+ The concentration range should be 0.10~0.70mM, and the band is brighter when the concentration is between 0.10~0.50mM; 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 final concentration of the 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 with a concentration of 100 μL was used as a large primer for Megawhop PCR to amplify the entire plasmid and obtain the mutated plasmid. The Megawhop PCR system is as follows: 25 μL PrimeSTARMax DNA polymerase, 40 ng pET28a-GH3, 500 ng CepPCR product (primer), and 50 μL ddH2O. 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 to 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 is a restriction nuclease that specifically excises methylated DNA strands and is commonly used to excise template DNA after PCR. Since the original template plasmid, derived from conventional E. coli, is dam-methylated, it is sensitive to DpnI and cleaved (DpnI recognizes a methylated GATC sequence, which occurs more than once in nearly all plasmids). However, the in vitro synthesized plasmid carrying the mutant sequence is not methylated and thus remains uncut. Therefore, subsequent transformations are successful, resulting in cloned mutant plasmids.

[0062] Successfully validated Megawhop PCR products were digested with Dpn I using the following digestion system: 25 μL of 10× CutSmart Buffer, PCR product <2 μg, 2 μL of Dpn I (20 units), and ultrapure water to a volume of 50 μL. The Dpn I digestion protocol was as follows: 37°C for 2.5 hours, 85°C for 30 minutes, and 4°C for ∞.

[0063] (3) Transformation into 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.). The specific operation is carried out according to the instructions. Each 100 μL of the transformed bacterial liquid is evenly spread on solid LB medium containing kanamycin and cultured at 37°C for 12 to 17 hours, not more than 20 hours. The growth of the colonies is observed, and the gene mutation library can be obtained.

[0065] (4) Verification of positive conversion rate

[0066] From the solid LB medium in step (3), more than ten single colonies were randomly selected and placed in 20 μL of ultrapure water. The cells were heated at 95°C for 5 minutes and centrifuged for 1 minute. The supernatant contained the plasmid released by the high-temperature cell disruption and could be used as a template for colony PCR. The pET28a-GH3 plasmid was used as a positive control. The colony PCR system was 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 was as follows: pre-denaturation at 98°C for 90 seconds; denaturation at 98°C for 45 seconds, annealing at 55°C for 45 seconds, and extension at 72°C for 4 minutes, for 30 cycles; final extension at 72°C for 10 minutes; and storage at 4°C.

[0067] The above PCR products were subjected to agarose gel electrophoresis, and the results were as follows: Figure 4 The length of the positive control pET28a-GH3 gene fragment is 2180 bp. In lanes 1 to 15 of the experimental group, there are 13 bands of the same length as the positive control lanes, indicating that the plasmid pET28a-GH3mutant was successfully constructed, with a positive rate of approximately 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, elute the solid culture medium plate with colonies grown in step (3) with LB liquid medium (containing kana), collect the eluate in a sterilized centrifuge tube, and extract the plasmid using a plasmid extraction kit (Plasmid MiniKit I, OmegaBio-Tek D6943-02). Specific operations are carried out according to the instructions.

[0070] B. All plasmids containing the mutant library in step A were transformed into E. coli BL21 (DE3) competent cells. The transformation method is described in the instructions. Then the bacterial solution was spread on solid LB medium containing kana resistance and incubated at 37°C for 16 hours. A large number of E. coli BL21 (DE3)-pET28a-GH3 mutant colonies were grown. The obtained single colonies are mutant monoclonal colonies with kana resistance. The results are as follows: Figure 5 As shown; store the solid culture medium in a 4°C refrigerator and wait for subsequent bacterial activation culture.

[0071] (6) Mutant culture and induced expression

[0072] A. Activation: Open a sterilized sealed 96-well plate on a sterile workbench and add 1 mL of liquid LB medium (containing Kana resistance) to each well. Use a sterilized toothpick to pick the bacteria from step (5) into the liquid medium and seal the wells with sterile sealing film. Incubate at 37°C and 700 rpm overnight for activation.

[0073] B. Induced expression: Add 1 mL of LB medium (containing Kana resistance) to each 96-deep-well plate, inoculate the activated bacterial solution into a new 96-deep-well plate in the same order at a 1% inoculum volume, and culture at 37°C and 700 rpm. When the OD 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 test: Mix 50 μL of bacterial solution with 100 μL of 5 mM pNPG and incubate at 50°C for 30 min. Add 1 mL of 1 M Na2CO3 to terminate the reaction and develop color. Measure the absorbance at 400 nm. 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 for rescreening. 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 better enzyme activity was sent to Guangzhou Aiji Biotechnology Co., Ltd. for sequencing to obtain the gene sequence of the mutant strain. The gene sequence was converted into an amino acid sequence using Snapgene software. The amino acid sequence of the mutant β-glucosidase 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, and 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 provides a detailed description of the present invention, 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 scope of protection 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 the 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 bacterium according to claim 6 or 7 in producing ginsenoside Rg3.

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