A pectin methylesterase gene PagPME2 and its in vitro expression and purification method and application

Abstract

The present invention belongs to the field of genetic engineering technology, and specifically relates to a pectin methylesterase gene PagPME2 and an in vitro expression purification method and application thereof. The present invention clones the pectin methylesterase gene PagPME2 from 84K poplar for the first time, and uses molecular biology and biochemical techniques to prove that the PagPME2 gene does have the catalytic properties of pectin methylesterase, and is a new gene with pectin methylesterase function. The pectin methylesterase gene PagPME2 provided by the present invention encodes a pectin methylesterase enzyme activity of up to 24.69U, which can catalyze the demethylation reaction of galacturonic acid, the main component of pectin, and reduce the degree of methylation of pectin polysaccharides, thereby changing the physical and chemical properties of pectin molecules, and providing application value for subsequent in-depth exploration of the physicochemical properties of pectin methylesterase and its important role in food, medicine, textiles, biomass conversion, plant growth and development, and other fields.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and specifically relates to a pectin methylesterase gene PagPME2 and an in vitro expression and purification method and application thereof. Background Art

[0002] Pectin methylesterase (PMEs) belongs to PCWDE enzymes, classified as sugar esterase family CE-8, and is widely present in the cell walls of plants, bacteria and fungi. PMEs can catalyze the demethylation reaction of galacturonic acid, the main component of pectin, and reduce the degree of methylation of pectin polysaccharides. The cross-linking effect between demethylated pectin molecules will be weakened, thereby changing the physical and chemical properties of pectin molecules.

[0003] Pectin methylesterase can be divided into two categories, type I pectin methylesterase and type II pectin methylesterase, depending on whether it contains the PMEI domain. Among them, type I pectin methylesterase only contains the PME domain, and type II pectin methylesterase contains the PME domain and the PMEI domain. Studies have shown that pectin methylesterase plays an important role in regulating plant growth and development, fruit softening, and adverse stress. Zheng et al. (Zheng L, Chen Y, Ding L et al The transcription factor MYB156 controls the polar stiffening of guard cell walls inpoplar. Plant Cell 2023, 45: 198216.) found that pectin methylesterase PME6 can regulate the degree of pectin methylesterification in the end region of the poplar guard cell wall, thereby regulating the movement of stomata. In the model plant Arabidopsis thaliana, Tian et al. (Tian GW, Chen MH, Zaltsman A et al. Pollen specific pectin methylesterase involved in pollen tube growth. Developmental Biology, 2006 294 (1): 8391.) identified a total of 66 pectin methylesterase genes, including 22 type I pectin methylesterase genes and 44 type II pectin methylesterase genes. In addition, studies have shown that under stress conditions, the expression levels of a large number of PME genes in Arabidopsis thaliana will change significantly; factors such as temperature and insect pests can induce the expression of PME genes. However, most of the studies on pectin methylesterase sequences and their functions have been conducted in herbaceous plants such as Arabidopsis thaliana, and relatively few reports on pectin methylesterase sequences and their functions in woody plants. Summary of the invention

[0004] The purpose of the present invention is to provide a pectin methylesterase gene PagPME2 and an in vitro expression and purification method and application thereof, explore the pectin methylesterase sequence and function in woody plants, realize the in vitro expression and purification of pectin methylesterase in woody plants, and detect the enzyme activity, change the physicochemical properties of pectin molecules, and provide application value for subsequent in-depth exploration of the physicochemical properties of pectin methylesterase and its important role in food, medicine, textile, biomass conversion, plant growth and development and other fields.

[0005] The present invention provides a pectin methylesterase gene PagPME2, the amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0006] Preferably, the nucleotide sequence of the CDS region of the pectin methylesterase gene PagPME2 is as shown in SEQ ID NO.1.

[0007] The present invention also provides primers for amplifying the pectin methylesterase gene PagPME2 described in the above technical solution, wherein the primers include an upstream primer with a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.4.

[0008] The present invention also provides a recombinant vector, which comprises a basic vector and a pectin methylesterase gene PagPME2 inserted into the basic vector; the amino acid sequence encoded by the pectin methylesterase gene PagPME2 is shown in SEQ ID NO.2.

[0009] Preferably, the basic vector comprises a pmal-c5x vector.

[0010] The present invention also provides a recombinant bacterium, wherein the basic bacterium comprises a basic bacterium and a pectin methylesterase gene PagPME2 or a recombinant vector introduced into the basic bacterium;

[0011] The amino acid sequence encoded by the pectin methylesterase gene PagPME2 is shown in SEQ ID NO.2;

[0012] The recombinant vector is the recombinant vector described in the above technical solution.

[0013] Preferably, the recombinant bacteria includes Escherichia coli.

[0014] The present invention also provides a method for expressing and purifying a pectin methylesterase gene PagPME2, comprising the following steps:

[0015] Inducing the expression of the recombinant bacteria described in the above technical solution using IPTG to obtain an induced expression product;

[0016] The expression product is purified to obtain purified pectin methylesterase PagPME2.

[0017] Preferably, the induction concentration of IPTG is 0.5 mM to 1.5 mM; the temperature for inducing expression is 19 to 37° C., and the rotation speed is 110 to 180 rpm.

[0018] The present invention also provides the use of the pectin methylesterase gene PagPME2 or the recombinant vector or recombinant bacteria described in the above technical solution or the pectin methylesterase PagPME2 obtained by the expression and purification method in changing the physicochemical properties of pectin molecules.

[0019] Beneficial effects:

[0020] The present invention cloned the pectin methylesterase gene PagPME2 from 84K poplar for the first time, and used molecular biology and biochemical techniques to prove that the PagPME2 gene does have the catalytic properties of pectin methylesterase, and is a new gene with pectin methylesterase function. The pectin methylesterase gene PagPME2 provided by the present invention encodes a pectin methylesterase with an enzyme activity of up to 24.69U, which can catalyze the demethylation reaction of galacturonic acid, the main component of pectin, and reduce the degree of methylation of pectin polysaccharides, thereby changing the physical and chemical properties of pectin molecules, and providing application value for subsequent in-depth exploration of the physicochemical properties of pectin methylesterase and its important role in food, medicine, textiles, biomass conversion, plant growth and development and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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.

[0022] Figure 1 This is the agarose gel electrophoresis result after amplification of 84K poplar pectin methylesterase gene PagPME2;

[0023] Figure 2 The sequencing results of the recombinant strain containing the prokaryotic expression vector of pmal-PagPME2;

[0024] Figure 3 To explore the induction conditions for prokaryotic expression of PagPME2 protein;

[0025] Figure 4 This is the result of prokaryotic expression induction of PagPME2 protein;

[0026] Figure 5 The SDS-PAGE electrophoresis results of the in vitro purified PagPME2 protein;

[0027] Figure 6 is the volume of NaOH solution consumed at different time points;

[0028] Figure 7 This is the relationship between the cumulative amount of NaOH consumed and time. DETAILED DESCRIPTION

[0029] The present invention provides a pectin methylesterase gene PagPME2, the amino acid sequence encoded by the gene is shown in SEQ ID NO. 2. In the present invention, the nucleotide sequence of the CDS region of the pectin methylesterase gene PagPME2 is shown in SEQ ID NO.1.

[0030] The sequence information of SEQ ID NO.1 to SEQ ID NO.2 of the present invention is as follows:

[0031]

[0032] SEQ ID NO.2: MAIRIMLTFLLVSFLLSSTILGYNHDEVNSWCSKTPNPQP CEYFLSHDPKNIPIQHESDFLKISIELALERAMHGRDNTYSLGSKCRNGLEKAAWEDCLELYQEIVLWLNKTTGSECTKYDAQTWLSTALTNLETCRTGFAEFGMTDYILPLMSNNVS ELISNTLAINKAPYSEPSFNGGFPSWVRPGDRKLLQSSSPASQANIVVAQDGSGNVKTIKEAIDAASKRSGSGRYVIYVKAGTYNENVEVGQKVKNVMVVGDGIGKTIVTGSKSVGGGS TTFKSATFAVVGDNFIARDMTFRNTAGANNHQAVALRSGSDLSVFYKCSFEGYQDTLYVHSQRQFYRECDIYGTVDFIFGNAAVVFQNCNIYARNPPNKTNTVTAQGRTDPNQNTGISI HNCKVTAASDLKSVQSSVKTYLGRPWQKYSRTVFMKTNLDSLIDSAGWMPWNGNFALDTLYYGEYGNTGPGSSTANRVNWKGYHVITSASVASQFTVASFISGNNWLPATNVPFTAGL.

[0033] The present invention also provides primers for amplifying the pectin methylesterase gene PagPME2 described in the above technical solution, wherein the primers include an upstream primer with a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.4.

[0034] The present invention is based on 84K poplar genome information, uses PrimerPremier 5 to design amplification primers, clones the pectin methylesterase gene PagPME2 from 84K poplar for the first time, and uses molecular biology and biochemical techniques to prove that the PagPME2 gene does have the catalytic properties of pectin methylesterase and is a new gene with pectin methylesterase function.

[0035] The present invention also provides a recombinant vector, comprising a basic vector and a pectin methylesterase gene PagPME2 inserted into the basic vector; the amino acid sequence encoded by the pectin methylesterase gene PagPME2 is shown in SEQ ID NO. 2. In the present invention, the basic vector preferably comprises a pmal-c5x vector. In the present invention, the pectin methylesterase gene PagPME2 is preferably inserted between the BamHI and HindIII sites of the pmal-c5x vector.

[0036] The present invention also provides a recombinant bacterium, the recombinant bacterium comprises a basic bacterium and a pectin methylesterase gene PagPME2 or a recombinant vector introduced into the basic bacterium; the amino acid sequence encoded by the pectin methylesterase gene PagPME2 is shown in SEQ ID NO.2; the recombinant vector is the recombinant vector described in the above technical solution. In the present invention, the basic bacterium preferably comprises Escherichia coli, and more preferably Escherichia coli BL21.

[0037] The present invention also provides a method for expressing and purifying a pectin methylesterase gene PagPME2, comprising the following steps:

[0038] Inducing the expression of the recombinant bacteria described in the above technical solution using IPTG to obtain an induced expression product;

[0039] The expression product is purified to obtain purified pectin methylesterase PagPME2.

[0040] The present invention uses IPTG to induce expression of the recombinant bacteria described in the above technical solution to obtain an induced expression product. In the present invention, the induction concentration of the IPTG is preferably 0.5mM to 1.5mM, and more preferably 1.0mM; the temperature of the induced expression is preferably 19 to 37°C, and more preferably 25 to 37°C, and more preferably 30 to 37°C; the time of the induced expression is preferably 10 to 14h, and more preferably 12h; the rotation speed of the induced expression is 110 to 180rpm, and more preferably 120 to 160rpm, and more preferably 150rpm.

[0041] After obtaining the induced expression product, the present invention preferably centrifuges the expression product to collect the precipitate. In the present invention, the centrifugal speed is preferably 4500 rpm and the time is preferably 10 min.

[0042] After obtaining the precipitate, the present invention preferably uses a lysis solution to resuspend the precipitate, and after the crushing treatment, the solid-liquid separation is performed, and the supernatant is collected. In the present invention, the lysis solution preferably includes Tris-HCl (pH 7.4) 25mM, NaCl 250mM, EDTA (pH 7.4) 1mM and DTT 15mM; the crushing treatment is preferably ultrasonic crushing; the ultrasonic power of the ultrasonic crushing is preferably 130W; the ultrasonic crushing time is preferably 15min, each working time is 10s, and the rest time is 10s. The method of solid-liquid separation in the present invention is preferably centrifugation; the speed of the centrifugation is preferably 10000rpm, and the time is preferably 10min.

[0043] After obtaining the supernatant, the present invention preferably purifies the supernatant to obtain purified pectin methylesterase PagPME2. In the present invention, the purification method preferably includes: after the supernatant and the filler are mixed and incubated, column chromatography, impurity removal and elution are carried out under the action of gravity; the filler is preferably DextrinBeads6FF; the time of the mixed incubation is preferably 2h, and the temperature is preferably 4°C; the cleaning solution used for impurity removal includes Tris-HCI (pH 7.4) 25mM, NaCl 250mM and EDTA (pH 7.4) 1mM; the eluent used for elution preferably includes Tris-HCI (pH 7.4) 25mM, maltose 10mM and EDTA (pH7.4) 1mM.

[0044] The expression and purification method provided by the invention can realize the expression and purification of the pectin methylesterase gene PagPME2 in vitro, and has the advantages of being easy and rapid to operate and being able to maintain the catalytic activity of the pectin methylesterase.

[0045] The present invention also provides the use of the pectin methylesterase gene PagPME2 or the recombinant vector or the recombinant bacteria or the pectin methylesterase PagPME2 obtained by the expression and purification method described in the above technical scheme in changing the physicochemical properties of pectin molecules. The pectin methylesterase gene PagPME2 provided by the present invention has an enzyme activity of up to 24.69U, which can catalyze the demethylation reaction of galacturonic acid, the main component of pectin, and reduce the degree of methylation of pectin polysaccharides, thereby changing the physical and chemical properties of pectin molecules, providing application value for subsequent in-depth exploration of the physicochemical properties of pectin methylesterase and its important role in food, medicine, textiles, biomass conversion, plant growth and development and other fields.

[0046] To further illustrate the present invention, a pectin methylesterase gene PagPME2 and its in vitro expression and purification method and application 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.

[0047] Example 1

[0048] Cloning of pectin methylesterase gene PagPME2 and construction of prokaryotic expression vector

[0049] 1.84K RNA Extraction and Reverse Transcription from Poplar Root Tissue

[0050] (1) The roots of 84K poplar tissue culture seedlings grown for 3 weeks were selected as the material for RNA extraction, and the root tissue RNA was extracted using the PLANTpure Universal Plant Total RNA Rapid Extraction Kit (purchased from Beijing Aidelai Biotechnology Co., Ltd.).

[0051] (2) Use The One-Step gDNA Removal and cDNA Synthesis SuperMixKit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) was used to reverse transcribe the above RNA into cDNA; wherein, the reverse transcription reaction system was: Total RNA 7μL, Anchored oligo(dT) 18 Primer 1.5μL, 2×TS Reaction Mix 10μL, TransScript RT / RI Enzyme Xix 1.5μL, gDNA Remover 1.5μL and RNase-free H2O 3μL; the reverse transcription reaction procedure is: mix the reaction system evenly, incubate at 42℃ for 30min, heat at 85℃ for 5s, obtain cDNA by reverse transcription, and store at -80℃.

[0052] 2.84K Poplar pectin methylesterase gene PagPME2 cloning.

[0053] (1) Based on the 84K poplar genome information, the upstream primer PagPM E2-F (5'-CGCGATATCGTCGACGGAATGGCTATTCGAATTATGCTG-3', SEQ ID NO. 3) and the downstream primer PagPME2-R (5'-CTTTCGTTTTATTTGATCAAAGGCCGG CGGTGAAT-3', SEQ ID NO. 4) were designed using Primer Premier 5;

[0054] (2) Using the cDNA obtained in step 1 as a template, PCR amplification was performed using the upstream primers and downstream primers mentioned above; wherein the PCR reaction system was: PrimeSTAR Max Primix (2×) 25 μL, 10 μM PagPME2-F 1 μL, 10 μM PagPME2-R 1 μL, 483 ng / μL cDNA 2 μL, and ddH2O 21 μL; the PCR reaction program was: 98°C 2 min; 98°C 10 s, 57°C 15 s, 72°C 10 s, 30 cycles; 72°C 5 min; 4°C∞;

[0055] (3) The PCR amplified products were subjected to agarose gel electrophoresis, and the target bands obtained by PCR amplification ( Figure 1 ) was recovered by agarose gel to obtain the pectin methylesterase gene PagPME2.

[0056] 3. PagPME2 gene was connected to the prokaryotic expression vector pmal-c5x

[0057] (1) The prokaryotic expression vector pmal-c5x was linearized using endonucleases BamHI and HindIII. The linearization system was: 30 μL of 476 ng / μL pmal-c5x vector, 1 μL of 20,000 units / ml BamHI, 1 μL of 20,000 units / ml HindIII, 5 μL of rCutSmart, and 13 μL of ddH2O. The linearization conditions were: 37°C for 15 to 20 min.

[0058] (2) The digested products were subjected to agarose gel electrophoresis, and the undigested pmal-c5x vector was used as a control to confirm that the vector was successfully linearized. The linearized vector was then recovered using the Adlay PCR Product Recovery Kit.

[0059] (3) The PagPME2 fragment obtained in step 2 was connected to the linearized pmal-c5x vector in step (2) by homologous recombination. The homologous recombination reaction system was as follows: 3 μL of linearized pmal-c5x vector, 4 μL of PagPME2 fragment, 10 μL of 2×MultifSeamlessAssemblyMix and 3 μL of ddH2O; the homologous recombination reaction conditions were: 50°C for 15 min.

[0060] (4) The homologous recombination product was transformed into BL21 competent cells by heat shock transformation, and the recombinant strain was sequenced and verified. The sequencing result was positive ( Figure 2 ) was a positive strain, recorded as pmal-c5x-PagPME2 recombinant strain, and stored at -80°C for future use.

[0061] Example 2

[0062] Inducible expression of pectin methylesterase gene PagPME2

[0063] 1. Study on the effects of temperature and IPTG concentration on the induced expression of target genes

[0064] (1) Take 2 mL of the pmal-c5x-PagPME2 recombinant strain obtained in Example 1 and inoculate it into 100 mL of liquid LB medium containing 50 mg / L ampicillin for amplification culture at a temperature of 37° C. and a shaking speed of 180 rpm;

[0065] After 5 hours of amplification, the OD of the bacterial solution was tested every 30 minutes. 600 The value of bacterial solution OD 600 When the value reaches 0.6-0.8, 2 mL of bacterial solution before induction was taken and stored at -20°C as a negative control sample, and then the inducer IPTG was added for induction. The final concentration of the inducer was 0.5 mM, 1.0 mM and 1.5 mM;

[0066] After adding the inducer, the gene prokaryotic expression induction was continued at 37°C and a shaking speed of 180 rpm;

[0067] After 12 hours of induction, 2 mL of bacterial solution was taken as the experimental sample after induction. The negative control sample and the experimental sample obtained in this step were subjected to SDS-PAGE electrophoresis. The results were as follows: Figure 3 Shown in the middle left picture.

[0068] (2) 2 mL of the pmal-c5x-PagPME2 recombinant strain obtained in Example 1 was inoculated into 100 mL of liquid LB medium containing 50 mg / L ampicillin for amplification culture at a temperature of 37° C. and a shaking speed of 180 rpm;

[0069] After 5 hours of amplification, the OD of the bacterial solution was tested every 30 minutes. 600 The value of bacterial solution OD 600 When the value reaches 0.6-0.8, 2 mL of bacterial solution before induction is taken and stored at -20°C as a negative control sample, and then the inducer IPTG is added for induction. The final concentration of the inducer is 0.5 mM, 1.0 mM or 1.5 mM.

[0070] After adding the inducer, the induction temperature was adjusted to 28°C, and the gene prokaryotic expression was induced at a shaking speed of 180 rpm;

[0071] After 12 hours of induction, 2 mL of bacterial solution was taken as the experimental sample after induction. The negative control sample and the experimental sample obtained in this step were subjected to SDS-PAGE electrophoresis. The results were as follows: Figure 3 As shown in the middle right picture.

[0072] according to Figure 3 It can be seen that under the induction condition of 37°C, different concentrations of inducers can induce the PagPME2 target protein (the target protein band is located between 75kDa and 95kDa, marked by a red arrow), and the induction effect is best when the concentration of the inducer IPTG is 1.0mM. In contrast, when induced at 28°C, the inducer IPTG at various concentrations could not induce the PagPME2 target protein. Therefore, for the induced expression of the pectin methylesterase gene PagPME2 described in the present invention, the best induction conditions are: 37°C, induced by 1.0mM IPTG.

[0073] 2. Inducible expression of pectin methylesterase gene PagPME2

[0074] Take 2 mL of the pmal-c5x-PagPME2 recombinant strain obtained in Example 1 and inoculate it into 100 mL of liquid LB medium containing 50 mg / L ampicillin for amplification culture at a temperature of 37° C. and a shaking speed of 180 rpm;

[0075] After 5 hours of amplification, the OD of the bacterial solution was tested every 30 minutes. 600 The value of bacterial solution OD 600 When the value reaches 0.6-0.8, take 2 mL of bacterial solution before induction and store it at -20°C as a negative control sample, then add the inducer IPTG for induction, and the final concentration of the inducer is 1.0 mM;

[0076] After adding the inducer, the bacterial solution was transferred to 37°C and the shaking speed was 180 rpm to induce gene prokaryotic expression;

[0077] After 12 hours of induction, 2 mL of bacterial solution was taken as the experimental sample after induction. The negative control sample and the experimental sample obtained in this step were subjected to SDS-PAGE electrophoresis. The results were as follows: Figure 4 As shown, the remaining bacterial solution was used for subsequent protein purification in vitro.

[0078] Example 3

[0079] Purification of protein encoded by pectin methylesterase gene PagPME2 in vitro

[0080] 1. Take 200 mL of the bacterial solution after induction for 12 h in step 2 of Example 2, centrifuge at 4500 rpm for 10 min, collect the bacterial precipitate, and resuspend the bacterial precipitate with 20 mL of lysis buffer (Tris-HCl (pH 7.4) 25 mM, NaCl 250 mM, EDTA (pH 7.4) 1 mM and DTT 15 mM).

[0081] 2. The resuspended bacterial solution was ultrasonically disrupted, and the ultrasonic disruptor was set according to the following parameters: ultrasonic intensity was 20%, working time was 10s, rest time was 10s, and ultrasonic time was 15min.

[0082] 3. After ultrasonic disruption, the bacterial solution was centrifuged at 10,000 rpm for 10 min. The supernatant was mixed with DextrinBeads 6, a filler used in the chromatography column, and incubated at 4°C for 2 h.

[0083] 4. Load the bacterial liquid filler mixture in step 3 into the chromatography column. After all the bacterial liquid flows out under gravity, add 20 times the volume of the filler and the impurity protein cleaning solution (Tris-HCI (pH 7.4) 25mM, NaCl 250mM and EDTA (pH 7.4) 1mM) to the chromatography column to wash away other impurity proteins that are not bound to the chromatography column filler.

[0084] 5. After the impurity protein is cleaned, the outlet at the bottom of the chromatography column is closed, and the protein eluent (Tris-HCI (pH 7.4) 25mM, maltose 10mM and EDTA (pH 7.4) 1mM) is added, incubated for 10 minutes, and then the effluent (i.e., the target protein PagPME2 solution) is collected.

[0085] 6. After the eluted protein solution (effluent) was concentrated using a 50 kDa ultrafiltration tube, SDS-PAGE electrophoresis was performed to detect the size of the target protein. The results showed that the size of the protein band contained in the eluate was consistent with the expected size ( Figure 5 ).

[0086] 7. Take 20 mL of pure protein eluate and add it to the concentrated protein effluent, and then use a 50 kDa ultrafiltration tube to concentrate it to about 200 μL to remove a small amount of impurities in the protein effluent, and obtain a secondary concentrated protein solution (i.e., purified pectin methylesterase PagPME2 protein solution), which is stored at -20 ° C for subsequent in vitro enzyme activity detection experiments.

[0087] Example 4

[0088] Detection of pectin methylesterase gene PagPME2 in vitro

[0089] The methyl ester group (-COOCH3) of plant pectin polysaccharides undergoes demethylation under the catalysis of pectin methylesterase to generate carboxyl groups (-COOH), which ionize in the solution to generate hydrogen ions (H + ), thus causing the pH of the solution to decrease. By adding additional NaOH to the solution, the decrease in the pH of the solution caused by pectin methylesterase catalysis can be inhibited, so that the pH of the solution is maintained within a certain range. Therefore, the activity of pectin methylesterase can be characterized by calculating the consumption of NaOH. The experimental operation steps are as follows:

[0090] (1) Take 15 μL of the purified pectin methylesterase PagPME2 protein solution obtained in Example 3, dilute it in equal proportion with the protein eluent, and then detect the concentration of pectin methylesterase PagPME2 protein by UV method, which is 4.98 mg / mL.

[0091] (2) Reagent 1 (main component is galacturonan) in the pectin methylesterase detection kit (purchased from Suzhou Gres Biotechnology Co., Ltd., catalog number G0707D) was heated to 37°C.

[0092] (3) Prepare two new 10 mL centrifuge tubes, add 20 μL of the 4.98 mg / mL pectin methylesterase solution from step (1) above to one tube (referred to as the experimental group), and add 20 μL of the protein eluate without pectin methylesterase PagPME2 to the other tube (referred to as the control group).

[0093] (4) Add 25 μL of Reagent 2 and 4 mL of heated Reagent 1 to the centrifuge tube in step (3) above, mix by inverting the tube, and then adjust the pH of the mixed solution to 7.8 with Reagent 3.

[0094] (5) The mixed solution of step (4) was incubated at 37°C for 2.5 h. During this period, the pH drop of the solution was detected every 15 min, and the pH of the solution was adjusted to 7.8 with NaOH (concentration of 20 mM). The volume of NaOH solution consumed each time was recorded, and the cumulative amount of NaOH consumed versus time was plotted. The enzyme activity of pectin methylesterase was calculated according to the following formula (1 μmol of NaOH consumed per mg of PagPME2 protein per hour is one enzyme activity unit U). The results are as follows: Figures 6-7 shown.

[0095] Enzyme activity of pectin methylesterase PagPME2 = (V×M) / (T×m);

[0096] Wherein, V represents the cumulative amount of NaOH consumed in 1 hour (μmol), M represents the dilution factor of the pectin methylesterase protein solution (the dilution factor is 1), T represents the time of the catalytic reaction (1h), and m represents the mass of the PagPME2 protein added to the reaction system (mg).

[0097] according to Figures 6-7 It can be seen that within 1 hour before the catalytic reaction, the experimental group needs to continuously add NaOH solution to maintain the pH value of the pectin solution stable at around 7.8, indicating that under the condition of sufficient substrate, PagPME2 has a higher catalytic activity; in the control group, there was no pH drop, and the enzyme activity of 84K poplar pectin methylesterase PagPME2 was calculated to be 24.69U based on the above formula.

[0098] According to the above content, it can be seen that the pectin methylesterase gene PagPME2 provided by the present invention encodes pectin methylesterase with an enzyme activity of up to 24.69U, which can catalyze the demethylation reaction of galacturonic acid, the main component of pectin, reduce the degree of methylation of pectin polysaccharides, and thus change the physical and chemical properties of pectin molecules.

[0099] 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 pectin methylesterase gene PagPME2, the amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

2. The pectin methylesterase gene PagPME2 according to claim 1, characterized in that The nucleotide sequence of the CDS region of the pectin methylesterase gene PagPME2 is shown in SEQ ID NO.

1.

3. A recombinant vector, characterized in that: The recombinant vector comprises a basic vector and a pectin methylesterase gene PagPME2 inserted into the basic vector; the amino acid sequence encoded by the pectin methylesterase gene PagPME2 is shown in SEQ ID NO.

2.

4. The recombinant vector according to claim 3, characterized in that The basic vector includes the pma1-c5x vector.

5. A recombinant bacterium, characterized in that: The recombinant bacteria include basic bacteria and pectin methylesterase gene PagPME2 or a recombinant vector introduced into the basic bacteria; The amino acid sequence encoded by the pectin methylesterase gene PagPME2 is shown in SEQ ID NO.2; The recombinant vector is the recombinant vector according to claim 3 or 4.

6. The recombinant bacterium according to claim 5, characterized in that The basic bacteria include Escherichia coli.

7. A method for expressing and purifying the pectin methylesterase gene PagPME2, characterized in that: The steps include: Inducing the expression of the recombinant bacteria according to claim 5 or 6 using IPTG to obtain an induced expression product; The expression product is purified to obtain purified pectin methylesterase PagPME2.

8. The expression purification method according to claim 7, characterized in that: The induction concentration of IPTG is 0.5 mM-1.5 mM; the temperature of the induced expression is 19-37° C., and the rotation speed is 110-180 rpm.

9. Use of the pectin methylesterase gene PagPME2 according to claim 1 or 2, or the recombinant vector according to claim 3 or 4, or the recombinant bacteria according to claim 5 or 6, or the pectin methylesterase PagPME2 obtained by the expression and purification method according to claim 7 or 8 in changing the physicochemical properties of pectin molecules, wherein: The pectin methylesterase PagPME2 encoded by the pectin methylesterase gene PagPME2 or the pectin methylesterase PagPME2 according to claim 7 or 8 can catalyze the demethylation reaction of galacturonic acid, the main component of pectin, reduce the degree of methylation of pectin polysaccharides, and thus change the physical and chemical properties of pectin molecules.

Images