A pectin methylesterase mutant △DdPemA and its encoding gene, preparation method and application

Abstract

The present invention discloses a pectin methylesterase mutant ΔDdPemA, its encoding gene, preparation method, and application. The pectin methylesterase mutant ΔDdPemA is obtained by mutating the amino acid at position 200 in the center of the substrate binding pocket of the wild-type pectin methylesterase DdPemA from a high-molecular-weight, branched valine to a low-molecular-weight alanine, and by mutating the amino acid at position 339 at the substrate inlet and outlet of the substrate binding pocket from a high-molecular-weight, branched valine to a basic lysine. The amino acid sequence and nucleotide sequence of the wild-type pectin methylesterase DdPemA are SEQ ID NO. 1 and SEQ ID NO. 2, respectively. The mutant enzyme provided by the present invention has significantly improved enzyme activity under weakly acidic conditions, solving the problem of insufficient catalytic activity of the wild-type pectin methylesterase under weakly acidic conditions and creating favorable conditions for the application of the enzyme in the fields of food, textiles, and feed processing.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology, and more specifically relates to a pectin methylesterase mutant ΔDdPemA and its encoding gene, preparation method and application. Background Art

[0002] Low-ester pectin is an important functional raw material in the food and pharmaceutical industries. It is primarily obtained by deesterifying high-ester pectin. Traditional chemical deesterification methods for producing low-ester pectin suffer from low efficiency and poor quality. Enzymatic deesterification, however, offers advantages such as high efficiency, specificity, and environmental friendliness, making it a popular method.

[0003] In the prior art, Chinese invention patent application number 201610333837.7 uses enzyme hydrolysis combined with alkali-catalyzed deesterification reaction to prepare low-ester pectin with an esterification degree of <10%, which can reduce the negative impact of the existing alkaline deesterification that causes the molecular weight of pectin to decrease; Chinese invention patent application number 201710042929.4 uses pectin methylesterase hydrolysis, followed by crystallization-pressing-washing-vacuum drying-crushing process to produce apple low-ester pectin, and the apple low-ester pectin has good quality and color.

[0004] Pectin methylesterase is an important pectinase that hydrolyzes the methyl ester groups in pectin, releasing methanol and reducing the degree of methyl esterification. It is a key enzyme in the preparation of low-ester pectin from high-ester pectin. However, existing pectin methylesterases suffer from low enzymatic activity.

[0005] Therefore, it is necessary to obtain pectin methylesterase with high enzyme activity through modern molecular biology technology. Summary of the Invention

[0006] In view of this, the present invention provides a pectin methylesterase mutant ΔDdPemA and its encoding gene, preparation method and application.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] The present invention provides a pectin methylesterase mutant ΔDdPemA, in which the amino acid at position 200 in the center of the substrate binding pocket of the wild-type pectin methylesterase DdPemA is mutated from the large molecular weight and branched valine to the small molecular weight alanine, and the amino acid at position 339 at the substrate inlet and outlet of the substrate binding pocket is mutated from the large molecular weight and branched valine to the basic lysine; the amino acid sequence of the wild-type pectin methylesterase DdPemA is shown in SEQ ID NO.1.

[0009] Specifically, in the above technical solution, point mutations are performed on the amino acid at position 200 in the center of the substrate binding pocket and the amino acid at position 339 at the substrate inlet and outlet of the substrate binding pocket. Specifically, based on the pemA gene sequence published in the GenBank database (GenBank accession number: KC422449), the amino acids encoded therein are substituted with valine at position 200 and valine at position 339. The amino acid at position 200 is mutated from valine to alanine, which has similar chemical properties but a smaller molecular weight, to increase the space of the substrate binding pocket; the amino acid at position 339 is mutated from valine to lysine, which is basic, thereby promoting the binding of the catalytic center to the pectin substrate.

[0010] The present invention also provides a gene ΔpemA encoding the pectin methylesterase mutant.

[0011] Furthermore, in the above technical solution, the nucleotide sequence of the wild-type pectin methylesterase DdPemA is shown as SEQ ID NO.2.

[0012] The present invention also provides a vector containing the gene ΔpemA.

[0013] The present invention also provides a host cell containing the gene ΔpemA or the vector.

[0014] The present invention also provides an engineered bacterium containing the gene ΔpemA or the vector.

[0015] In another aspect, the present invention provides the application of the gene ΔpemA and the enzyme encoded thereby in food, textile and feed processing.

[0016] In another aspect, the present invention provides a method for producing the pectin methylesterase mutant gene ΔpemA, comprising:

[0017] The nucleotide sequence is expressed in a plasmid capable of expressing the enzyme as an expression vector and a strain capable of expressing the enzyme as an expression host, thereby achieving efficient expression of the mutant wild-type pectin methylesterase DdPemA shown in SEQ ID NO.1.

[0018] In detail, in the above technical solution, the nucleotide sequence is expressed in pEASY-E1 or a plasmid capable of expressing the enzyme as an expression vector, and Escherichia coli BL21 (DE3) or a strain capable of expressing the enzyme is used as an expression host to achieve efficient expression of the mutant gene △pemA.

[0019] Specifically, in the above technical solution, the pectin methylesterase gene pemA comes from a highly efficient strain of hemp degumming bacteria, Dickeya dadantii DCE-01 (deposit number: CGMCC 5522, patent number: ZL201110410078.7); the promoter of the pEASY-E1 expression unit used is the commonly used T7 promoter. Under the action of the T7 promoter, the mutant enzyme can be directly expressed in the host cell E. coli BL21 (DE3) to achieve intracellular soluble expression.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The mutant enzyme provided by the present invention has significantly improved enzyme activity under weakly acidic conditions, solving the problem of insufficient catalytic activity of wild-type pectin methylesterase under weakly acidic conditions, and creating good conditions for the application of the enzyme in the fields of food, textile, feed processing, etc.

[0022] The present invention compares the degradation abilities of the wild enzyme DdPemA and the mutant enzyme △DdPemA on orange pectin (DE≥85%) and ramie pectin under weakly acidic conditions. The results show that at a pH of 6.0 and a temperature of 40°C, the specific enzyme activities of the mutant enzyme △DdPemA are 1898 and 2439 U / mg, respectively, which are 5.8 times and 6.2 times that of the wild enzyme DdPemA. That is, the mutant enzyme has the characteristic of high enzymatic activity under weakly acidic conditions, indicating that it has important application prospects in industrial production where the esterification degree of pectin needs to be reduced under weakly acidic conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Flowchart for the construction of pectin methylesterase mutant engineered strains according to the embodiment of the present invention;

[0024] Figure 2 This is a construction map of the recombinant plasmid pEASY-E1-pemA in the embodiment of the present invention;

[0025] Figure 3 Schematic diagram of the principle of site-directed mutagenesis in an embodiment of the present invention;

[0026] Figure 4 The SDS-PAGE patterns of the wild-type and mutant enzymes induced in the examples of the present invention are shown;

[0027] Figure 5 Graph comparing the enzyme activities of the wild-type and mutant enzymes in the examples of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to the embodiments.

[0029] It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0030] In the examples, unless otherwise specified, all means used are conventional means in the art.

[0031] As used herein, the terms "comprise," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a listed element is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0032] In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] Materials and reagents:

[0034] The vector pEASY-E1 and prokaryotic expression competent E. coli BL21 (DE3) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0035] The Ultra HiFidelity PCR Kit, spin column bacterial genome extraction kit, rapid site-directed mutagenesis kit, DNA Marker III, 2× TaqPCR Mix reagent, conventional agarose gel DNA recovery kit, plasmid miniprep kit, IPTG, and ampicillin sodium salt (Amp) were all purchased from Tiangen Biotechnology Co., Ltd.

[0036] Orange pectin (DE ≥ 85%), tryptone, yeast extract, and agar powder were purchased from Sigma;

[0037] The remaining chemical reagents were commercial products of analytical grade purchased from Sinopharm Group;

[0038] Primer synthesis and nucleic acid sequencing were performed by Qingke Biotechnology Co., Ltd.

[0039] like Figure 1 Shown is a flow chart for constructing a pectin methylesterase mutant engineered strain in an embodiment of the present invention.

[0040] Example 1: Construction of recombinant plasmid of original pemA gene

[0041] D. dadantii DCE-01 was cultured to the logarithmic growth phase, 1.5 mL of bacterial solution was centrifuged at 12000 rpm for 1 min, and the bacterial pellet was collected; then genomic DNA was extracted according to the kit instructions.

[0042] Based on the cloned pemA gene sequence (GenBank accession number: KC422449) and the expression profile of pEASY-E1, the following primers were designed using the bioinformatics software Primer Premier 5:

[0043] F: 5'-TCGAGCTCCATGTGTATGTTAAAAACGATCTCAGG-3' (SEQ ID NO.3)

[0044] R: 5'-TTATGCGGCCGCTCAGGGGAGTGTCGGCGT-3' (SEQ ID NO. 4).

[0045] The original gene pemA of pectin methylesterase was amplified by PCR using genomic DNA as template.

[0046] The PCR reaction system was as follows: 10×PCR Buffer, 5.0 μL; MgSO4 (25 mmol / L), 2.0 μL; dNTPs (10 mmol / L), 5.0 μL; recombinant plasmid DNA template, 1.0 μL; forward primer F (10 μmol / L), 1.0 μL; reverse primer R (10 μmol / L), 1.0 μL; KOD plus DNA polymerase, 1.0 μL; add sterile ddH2O to a total volume of 50 μL, mix well, and place on a PCR instrument for PCR reaction.

[0047] The parameter settings are:

[0048] (1) Pre-denaturation at 95°C for 4 minutes; (2) Denaturation at 94°C for 30 seconds; (3) Annealing at 55°C for 30 seconds; (4) Extension at 72°C for 1 minute; Repeat steps (2)-(4) for 30 cycles; (5) Incubate at 72°C for 10 minutes. Then store at 4°C.

[0049] The obtained PCR products were detected by 1.0% agarose gel electrophoresis, and the gel was cut and the target fragment was recovered using a DNA gel recovery kit.

[0050] The target gene PCR product was treated with A and purified by gel cutting. According to the instructions of the kit, the recombinant plasmid was obtained. The product was transformed into E. coli BL21 (DE3) by heat shock method, and plated on LB medium plates containing 110 μg / mL Amp and cultured overnight. After picking positive transformants and culturing overnight, the plasmid was extracted and verified by PCR. The positive plasmid with the correct target band size and insertion direction was submitted to Qingke Biotechnology Co., Ltd. for sequence determination. The recombinant plasmid with the correct target gene sequence is pEASY-E1-pemA ( Figure 2 ).

[0051] Example 2: Site-directed mutagenesis

[0052] Principle of site-directed mutagenesis: The construction of point mutation plasmids was carried out using the Dpn I method ( Figure 3 ).

[0053] The PCR point mutation primers are designed according to the amino acid site to be mutated as follows:

[0054] F V200A :5'-GCGGCACA GCT GACTTTATC-3' (SEQ ID NO. 5)

[0055] R V200A :5'-GATAAAGTC AGC TGTGCCGC-3' (SEQ ID NO. 6)

[0056] F V339K :5'-GAGCGGCG AAG AGCAAAGAC-3' (SEQ ID NO. 7)

[0057] R V339K :5'-GTCTTTGCT CTT CGCCGCTC-3' (SEQ ID NO. 8)

[0058] The underlined parts represent the codons corresponding to the 200th alanine and 339th lysine encoded by the mutant gene.

[0059] The mutation site was introduced by whole-plasmid PCR using a rapid site-directed mutagenesis kit and the pEASY-E1-pemA recombinant plasmid as a template.

[0060] The PCR reaction system was as follows: 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 5 μL of 5×FastAlterationBuffer, 1 μL of plasmid DNA, 0.5 μL of FastAlteration DNA Polymerase, and the volume was made up to 25 μL with ddH2O.

[0061] The parameter settings are:

[0062] (1) Pre-denaturation at 95°C for 2 min; (2) Denaturation at 94°C for 20 s; (3) Annealing at 60°C for 10 s; (4) Extension at 68°C for 2.5 min; Repeat steps (2)-(4) for 18 cycles; (5) Incubate at 68°C for 5 min. The product was stored at 4°C.

[0063] Add 0.5 μL of restriction endonuclease DpnI to 25 μL of the mutated PCR product, mix thoroughly, and digest at 37°C for 1 hour; take 5 μL of Dpn I digestion product and transform it into DH5α, evenly spread the transformed bacterial liquid on LB screening plate (containing Amp 110 μg / mL), and culture at 37°C overnight to obtain transformants of the relevant mutant strains. Extract the plasmid, sequence and verify the correct mutant strain, and transform the successfully constructed recombinant plasmid into E. coli BL21 (DE3) to obtain the genetically engineered mutant strain pEASY-E1-△pemA / BL21 to be expressed.

[0064] Example 3: Induced expression of wild-type enzyme and mutant enzyme and SDS-PAGE analysis

[0065] Single colonies of the original genetically engineered bacteria pEASY-E1-pemA / BL21 and the mutant genetically engineered bacteria pEASY-E1-△pemA / BL21 were inoculated into LB liquid medium containing 110 mg / mL Amp and cultured at 37°C and 200 rpm until OD 600 To 0.6, add 1mmol / LIPTG, 28℃, 120r / min to induce expression for 12-15h.

[0066] Place 1 mL of induced mature fermentation broth in a 1.5 mL centrifuge tube and centrifuge at 10,000 rpm for 5 minutes. Discard the supernatant and add 500 μL of saline, vortex, and centrifuge twice. Resuspend the pellet in 40 μL of sterile ddH2O, add 10 μL of 5× protein loading buffer, boil for 5 minutes, cool naturally, and store at -20°C until ready for use (Note: Boil in a boiling water bath for 3 minutes before loading).

[0067] The prepared samples were analyzed by discontinuous SDS-PAGE (5% stacking gel and 12% separation gel) (e.g. Figure 4 As shown in the figure, the pEASY-E1 / BL21 strain without the target gene was treated in the same way as a blank control. The results showed that both the original genetically engineered bacteria (wild enzyme engineering strain) pEASY-E1-pemA / BL21 and the mutant genetically engineered bacteria (mutant enzyme engineering strain) pEASY-E1-△pemA / BL21 could successfully express specific protein bands.

[0068] Example 4: Comparison of catalytic ability of wild-type enzyme and mutant enzyme in vitro

[0069] Take the induced mature fermentation liquid, centrifuge it at 3000r / min and 4℃ for 10min, and collect the supernatant, which is the crude enzyme liquid.

[0070] In order to compare the biocatalytic abilities of the wild-type enzyme and the mutant enzyme under alkaline conditions, enzyme activity assays and protein content determinations were performed under the same conditions.

[0071] Under weak acidic conditions, the degradation abilities of wild enzyme DdPemA and mutant enzyme △DdPemA on orange pectin (DE≥85%) and ramie pectin were compared (e.g. Figure 5 shown).

[0072] Pectin methylesterase activity assay: 5 mg / mL of orange pectin (DE ≥ 85%) or ramie pectin substrate is prepared in a pH 6.0 citric acid-Na2HPO4 buffer. 10 mL of 0.5% pectin substrate is equilibrated in a 40°C waterbath for 5 minutes. 2 mL of the diluted enzyme solution is added and the reaction is incubated at 40°C for 30 minutes. The reaction is terminated by boiling and the resulting carboxyl groups are titrated with 0.02 mol / L NaOH. A boiling-inactivated enzyme is treated in the same manner as a negative control. One unit of enzyme activity (U) is defined as the amount of enzyme required to release 1 μmol of carboxylic acid per minute from the substrate.

[0073] Protein content determination method: Coomassie Brilliant Blue G-250 method; add 100 μL of enzyme solution diluted to an appropriate multiple with 0.15 mol / L sterile NaCl solution to 5 mL of Coomassie Brilliant Blue G-250 reagent, mix well, develop color for 5 minutes, and measure the OD of each tube solution at 595 nm. 595 .

[0074] The results showed that at pH 6.0 and 40℃, the specific enzyme activities of the mutant △DdPemA were 1898 U / mg and 2439 U / mg, respectively, which were 5.8 times and 6.2 times that of the wild-type pectinase DdPemA; therefore, the catalytic degradation ability of the mutant enzyme △DdPemA was significantly improved compared with the wild-type enzyme DdPemA.

[0075] The above-mentioned embodiments only express several implementation methods of the present invention. The description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the invention patent.

[0076] It should be noted that those skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims.

Claims

1. A pectin methylesterase mutant ΔDdPemA, characterized in that: The amino acid at position 200 in the center of the substrate binding pocket of wild-type pectin methylesterase DdPemA was mutated from a large molecular weight, branched valine to a small molecular weight alanine, and the amino acid at position 339 at the substrate inlet and outlet of the substrate binding pocket was mutated from a large molecular weight, branched valine to a basic lysine. The amino acid sequence of the wild-type pectin methylesterase DdPemA is shown in SEQ ID NO.

1.

2. The gene ΔpemA encoding the pectin methylesterase mutant according to claim 1.

3. A vector containing the gene ΔpemA according to claim 2. A host cell containing the gene ΔpemA according to claim 2 or the vector according to claim 3.

5. An engineered bacterium containing the gene ΔpemA according to claim 2 or the vector according to claim 3.

6. A method for producing the pectin methylesterase mutant according to claim 1, characterized in that: Cultivate the engineered bacteria of claim 5 to achieve expression of the pectin methylesterase mutant ΔDdPemA.

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