Pectate lyase mutant DdPelZ as well as coding gene, preparation method and application thereof

Abstract

The invention discloses a pectin lyase mutant DdPelZ as well as a coding gene, a preparation method and application thereof. The pectin lyase mutant DdPelZ is obtained by mutating amino acid at the 168th site of a wild type pectin lyase DdPelZ substrate binding pocket from valine with large molecular weight and branched chain into arginine with positive charges; the amino acid at the 319th site of the rigid region is mutated into alanine with small molecular weight from valine with large molecular weight and branched chain; the amino acid sequence and the nucleotide sequence of the gene are SEQ ID NO.1 and SEQ ID NO.2 respectively. The enzyme activity and the heat resistance of the mutant enzyme provided by the invention are obviously improved under the alkaline condition, the problems of low catalytic activity and insufficient heat resistance of wild type pectin lyase under the alkaline condition are solved, and good conditions are created for application of the enzyme in cotton and linen processing, pulping and papermaking and feed processing.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and more specifically, relates to a pectin lyase mutant ΔDdPelZ and a coding gene thereof, a preparation method and an application thereof. Background Art

[0002] Pectinase is an important type of industrial enzyme, which has been widely used in the papermaking industry, food processing, environmental protection and textile industry. According to the optimal pH of the enzymatic reaction, pectinase is divided into acidic pectinase and alkaline pectinase. Most acidic pectinases are used in food and feed processing, while alkaline pectinases are used in cotton and linen processing in the textile industry. Alkaline pectinase is mainly involved in hemp biodegumming and cotton and linen yarn biorefining in textile processing. Compared with the traditional chemical method, the application of bioenzyme method in hemp degumming has the advantage of green environmental protection and represents the development trend; however, there are not enough alkaline pectin (lyase) enzymes that can be directly applied to industrial production on the market. The reason is that the enzyme activity is not high and the heat resistance is poor. Therefore, it is an effective method to obtain excellent industrial pectinase by efficiently expressing the existing alkaline pectin (lyase) enzyme genes and using molecular biological methods to molecularly modify the existing enzymes to improve the enzyme activity.

[0003] The main methods for molecular modification of industrial enzymes include directed evolution based on irrational design and site-directed mutagenesis based on rational design. Directed evolution introduces amino acid mutation sites (regions) through random mutations, and needs to be combined with high-throughput screening to obtain enzymes with improved thermal stability; site-directed mutagenesis refers to the use of PCR and other methods to change specific sites of the target DNA, including the addition, deletion and point mutation of bases, which has the advantages of high mutation rate and good repeatability, and has been widely used to improve enzyme performance.

[0004] In the prior art, the Chinese invention patent with application number 201810013726.7 mutates the amino acid Y at position 7 and / or position 111 of endoglucanase NfEG12A to W, so that the mutant has a catalytic efficiency of 0.5-0.8 times for β-1,3-1,4-glucan and xyloglucan, respectively, and has good heat resistance and pH tolerance. In addition to improving enzyme catalytic efficiency, enhancing thermal stability can also be achieved through point mutations. The Chinese invention patent with application number 201910835766.4 mutates the 68th base of the amino acid sequence of glucose oxidase GOD-M5 from Asp to Lys, the 274th base from Thr to Phe, the 278th base from Tyr to Thr, the 94th base from Ser to Ala, the 31st base from Thr to Val, and the 88th base from Gln to Arg, thereby greatly improving its thermal stability. The Chinese invention patent with application number 202011580654.8 uses the starch sucrase from the microorganism Calidithermus timidus DSM 17022 as the parent to construct the four-point mutant enzyme L382P / S414N / P618I / H631K. The half-life of the mutant enzyme at 65°C is increased from 23.58h to 43.72h, and the half-life at 70°C is increased from 71min to 110min. The Chinese invention patent with application number 202210213233.4 obtains an agarase mutant E122W by rationally designing a point mutation of wild agarase, which significantly improves the enzyme activity and thermal stability of the agarase mutant, and can efficiently catalyze the degradation of agar to prepare agar oligosaccharides. Therefore, based on the known or predicted structural information and catalytic mechanism of pectin lyase, the relationship between structure and function was analyzed, and the key amino acid sites that affect the catalytic activity and stability of pectin lyase were inferred. Site-directed mutagenesis is an effective way to obtain excellent pectin lyase.

[0005] So far, there has been no report on mutational modification of pectin lyase. Summary of the invention

[0006] In view of this, the present invention provides a pectin lyase mutant ΔDdPelZ 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 lyase mutant ΔDdPelZ, which mutates the amino acid at position 168 of the substrate binding pocket of the wild-type pectin lyase DdPelZ from the large molecular weight and branched valine to the positively charged (and structurally similar) arginine, and mutates the amino acid at position 319 of the rigid region from the large molecular weight and branched valine to the small molecular weight alanine.

[0009] Specifically, in the above technical solution, the amino acid at position 168 of the substrate binding pocket and the amino acid at position 319 of the rigid region (α-helix) are point mutated, specifically based on the pelZ gene sequence published in the GenBank database (GenBank accession number: KC900169), the amino acids encoded therein are substituted, and the amino acid substitution points are valine at position 168 and valine at position 319. The amino acid at position 168 is mutated from valine to arginine that can bind to pectin molecules, thereby improving the catalytic efficiency; the amino acid at position 319 is mutated from valine to alanine, an α-helix preferred amino acid, thereby enhancing the stability of the structure of this region.

[0010] Furthermore, in the above technical solution, the amino acid sequence of the pectin lyase mutant ΔDdPelZ is shown in SEQ ID NO.1.

[0011] The present invention also provides a gene ΔpelZ encoding the pectin lyase mutant.

[0012] Furthermore, in the above technical solution, the nucleotide sequence of the gene ΔpelZ is shown in SEQ ID NO.2.

[0013] The present invention also provides a vector containing the gene ΔpelZ.

[0014] The present invention also provides a host cell containing the gene ΔpelZ or the vector.

[0015] The present invention also provides an engineering bacterium containing the gene ΔpelZ or the vector.

[0016] In another aspect, the present invention also provides the application of the gene ΔpelZ and the enzyme encoded thereby in cotton and linen processing, pulping and papermaking, and feed processing.

[0017] In another aspect, the present invention also provides a method for producing the pectin lyase mutant ΔDdPelZ, comprising:

[0018] The nucleotide sequence shown in SEQ ID NO.2 is used as an expression vector with a plasmid capable of expressing the enzyme and a strain capable of expressing the enzyme as an expression host to achieve efficient expression of the mutant shown in SEQ ID NO.1.

[0019] In detail, in the above technical scheme, the nucleotide sequence shown in SEQ ID NO.2 is used as an expression vector with pEASY-E1 or a plasmid capable of expressing the enzyme, 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 ΔpelZ.

[0020] Specifically, in the above technical solution, the pectin lyase gene pelZ comes from a hemp degumming efficient strain 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 T 7 Promoter, in T 7 Under the action of the promoter, the mutant enzyme can be directly expressed in the host cell E.coli BL21 (DE3) to achieve intracellular soluble expression.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] The mutant enzyme provided by the present invention has significantly improved enzyme activity and heat resistance under alkaline conditions, which solves the problems of low catalytic activity and insufficient heat resistance of wild-type pectin lyase under alkaline conditions, and creates good conditions for the application of the enzyme in cotton and linen processing, pulping and papermaking, and feed processing.

[0023] The invention compares the degradation abilities of wild enzyme DdPelZ and mutant enzyme ΔDdPelZ on polygalacturonic acid sodium salt and ramie pectin under alkaline conditions, and the results show that under the conditions of pH 8.5 and temperature 45 DEG C, the specific enzyme activities of the mutant enzyme ΔDdPelZ are 2405 U / mg and 4092 U / mg, respectively, which are 7.3 times and 8.5 times of the wild enzyme DdPelZ, respectively; when kept warm at 55 DEG C, the half-life of the mutant enzyme ΔDdPelZ is 4 hours, which is 2.4 times of the wild enzyme DdPelZ, that is, the mutant enzyme has the characteristics of high heat resistance and high enzyme activity under alkaline conditions, indicating that it has important application prospects in industrial production requiring high temperature and alkaline conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The flowchart of the construction of the pectin lyase mutant engineering strain in the embodiment of the present invention;

[0025] Figure 2 This is a construction map of the recombinant plasmid pEASY-E1-pelZ in the embodiment of the present invention;

[0026] Figure 3 Schematic diagram of the principle of site-directed mutagenesis in an embodiment of the present invention;

[0027] Figure 4 The SDS-PAGE patterns of the wild-type and mutant enzymes induced in the embodiments of the present invention are shown in FIG.

[0028] Figure 5 A comparison diagram of the enzyme activities of the wild-type and mutant enzymes in the embodiments of the present invention;

[0029] Figure 6 Graph comparing the half-lives of the wild-type and mutant enzymes in the examples of the present invention. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0031] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0032] In the examples, unless otherwise specified, all the means used are conventional means in the art.

[0033] As used herein, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

[0034] 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.

[0035] Materials and Reagents:

[0036] The vector pEASY-E1 and prokaryotic expression competent E. coli BL21 (DE3) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0037] Ultra HiFidelity PCR Kit, centrifugal bacterial genome extraction kit, rapid site-directed mutagenesis kit, DNA Marker III, 2× Taq PCR Mix reagent, ordinary agarose gel DNA recovery kit, plasmid mini-extraction kit, IPTG, and ampicillin sodium salt (Amp) were all purchased from Tiangen Biotechnology Co., Ltd.

[0038] Polygalacturonic acid sodium salt, tryptone, yeast extract, and agar powder were purchased from Sigma;

[0039] The remaining chemical reagents were all commercial products of analytical grade purchased from Sinopharm Group;

[0040] Primer synthesis and nucleic acid sequencing were performed by Qingke Biotechnology Co., Ltd.

[0041] like Figure 1 Shown is a flow chart for constructing a pectin lyase mutant engineering strain in an embodiment of the present invention.

[0042] Example 1: Construction of the recombinant plasmid of the original pelZ gene

[0043] 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 precipitate was collected; then genomic DNA was extracted according to the kit instructions.

[0044] Based on the cloned pel gene sequence (GenBank accession number: KC900169) and the expression profile of pEASY-E1, the following primers were designed using the bioinformatics software Primer Premier 5:

[0045] F: 5'-TCGAGCTCCATGAAACATACCCTCTGTTTGCTT-3' (SEQ ID NO.3)

[0046] R: 5'-TTATGCGGCCGCTTATTCCAGCTCTTTGGCCATT-3' (SEQ ID NO. 4).

[0047] The original gene pelZ of pectate lyase was amplified by PCR using genomic DNA as template.

[0048] PCR reaction system: 10×PCR Buffer, 5.0 μL; MgSO 4 (25mmol / L), 2.0μL; dNTPs (10mmol / L), 5.0μL; recombinant plasmid DNA template, 1.0μL; forward primer F (10μmol / L), 1.0μL; reverse primer R (10μmol / L), 1.0μL; KOD plus DNA polymerase, 1.0μL; sterilized ddH 2 O to a total volume of 50 μL, mix well and place on a PCR instrument for PCR reaction.

[0049] The parameter settings are:

[0050] (1) Pre-denaturation at 94°C for 4 min; (2) Denaturation at 94°C for 30 s; (3) Annealing at 55°C for 30 s; (4) Extension at 72°C for 1 min; Repeat steps (2)-(4) for 30 cycles; (5) Incubate at 72°C for 10 min. Then store at 4°C.

[0051] The obtained PCR product was detected by 1.0% agarose gel electrophoresis, and the gel was cut, and the target fragment was recovered using a DNA gel recovery kit.

[0052] 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 transferred into E.coli BL21 (DE3) by heat shock method, and plated on LB medium plates containing 120μg / mL Amp and cultured overnight. The positive transformants were selected and cultured overnight, and the plasmids were extracted and verified by PCR. The positive plasmids with the correct target band size and insertion direction were submitted to Qingke Biotechnology Company for sequence determination. The recombinant plasmid with the correct target gene sequence is pEASY-E1-pelZ ( Figure 2 ).

[0053] Example 2: Site-directed mutagenesis

[0054] Principle of site-directed mutagenesis: The construction of point mutation plasmids was carried out using the Dpn I method ( Figure 3 ).

[0055] The PCR point mutation primers are designed according to the amino acid site to be mutated as follows:

[0056] F V168A :5'-AAACCTGTCG CGT TCCGGGC-3' (SEQ ID NO. 5)

[0057] R V168A :5'-GCCCGGA ACG CGACAGGTTT-3' (SEQ ID NO. 6)

[0058] F v319A :5'-AAGGCC GCG GCGCAAACCTC-3' (SEQ ID NO.7)

[0059] R V319A :5'-GAGGTTTGCGC CGC GGCCTT-3' (SEQ ID NO. 8)

[0060] The underlined parts represent the codons corresponding to the 168th arginine and the 319th alanine encoded by the mutant gene.

[0061] The mutation site was introduced by whole plasmid PCR using a rapid site-directed mutagenesis kit and pEASY-E1-pelZ recombinant plasmid as a template.

[0062] The PCR reaction system was as follows: 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 5 μL 5× Fast Alteration Buffer, 1 μL plasmid DNA, 0.5 μL Fast Alteration DNA Polymerase, and ddH2 Add 0.1% HO to make up to 25 μL.

[0063] The parameter settings are:

[0064] (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 and store the product at 4°C.

[0065] Add 0.5 μL of restriction endonuclease Dpn I to 25 μL of the mutated PCR product, mix thoroughly, and digest at 37°C for 1 h; take 5 μL of Dpn I digestion product and transfer it into DH5α, evenly spread the transformation bacterial solution on the LB screening plate (Amp 120 μg / mL), and culture at 37°C overnight to obtain transformants of the relevant mutant strains, extract the plasmid, and sequence to verify the correct mutant strain. The successfully constructed recombinant plasmid was transferred into E. coli BL21 (DE3) to obtain the genetically engineered mutant strain pEASY-E1-ΔpelZ / BL21 to be expressed.

[0066] Example 3: Induced expression of wild-type enzyme and mutant enzyme and SDS-PAGE analysis

[0067] Single colonies of the original genetically engineered bacteria pEASY-E1-pelZ / BL21 and the mutant genetically engineered bacteria pEASY-E1-ΔpelZ / BL21 were inoculated into LB liquid medium containing 120 mg / mL Amp and cultured at 37°C and 200 r / min until OD 600 To 0.6, add 1mmol / L IPTG, 28℃, 120r / min to induce expression for 12-15h.

[0068] Take 1 mL of induced mature fermentation liquid in a 1.5 mL centrifuge tube, centrifuge at 10000 r / min for 5 min, discard the supernatant, add 500 μL of physiological saline, vortex and shake, centrifuge, wash twice. Use 40 μL of sterilized ddH 2 O resuspended bacterial precipitate, added 10 μL of 5× protein loading buffer, boiled for 5 min, cooled naturally, and stored at -20°C for later use (Note: use boiling water bath for 3 min before loading).

[0069] The prepared samples were analyzed by discontinuous SDS-PAGE (5% stacking gel and 12% separation gel) (e.g. Figure 4As 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-pelZ / BL21 and the mutant genetically engineered bacteria (mutant enzyme engineering strain) pEASY-E1-Δpe1Z / BL21 could successfully express specific protein bands.

[0070] Example 4: Comparison of catalytic ability of wild-type enzyme and mutant enzyme in vitro

[0071] 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.

[0072] 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.

[0073] Under alkaline conditions, the degradation abilities of wild enzyme DdPelZ and mutant enzyme ΔDdPelZ on polygalacturonic acid sodium salt and ramie pectin were compared (e.g. Figure 5 shown).

[0074] Enzyme activity determination method: Use 0.05mol / L glycine-sodium hydroxide buffer (pH 8.5) to prepare 5mg / mL sodium polygalacturonate or ramie pectin solution. Take 1mL substrate and preheat to 50℃, add 10μL of appropriately diluted enzyme solution, react at 50℃ for 10min, and immediately add 2mL DNS. Color development in boiling water bath for 5min, and quickly cool in ice water bath. Use the same enzyme solution inactivated by boiling to do the same reaction as negative control, and measure the OD of the sample. 520 .

[0075] The activity of pectin lyase is defined as: the amount of enzyme required to release 1 μmol of reducing sugar of unsaturated galacturonic acid per minute is 1 enzyme activity unit, expressed as U.

[0076] 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, color for 5 minutes, and measure the OD of each tube solution at 595 nm 595 .

[0077] The results showed that at pH 8.5 and 45℃, the specific enzyme activities of the mutant ΔDdPelZ were 2405U / mg and 4092U / mg, respectively, which were 7.3 times and 8.5 times that of the wild-type pectinase DdPelZ; therefore, the catalytic degradation ability of the mutant enzyme ΔDdPelZ was greatly improved compared with the wild enzyme DdPelZ.

[0078] Example 6: Comparison of heat resistance of wild enzyme and mutant enzyme

[0079] The crude enzyme solution was kept warm at 55°C, and samples were taken at regular intervals to determine the residual enzyme activity (e.g. Figure 6 The time for the enzyme activity to retain 50% (i.e., half-life T 1 / 2 ) to characterize the thermal stability of enzymes.

[0080] The results showed that when kept warm at 55°C, the half-life of ΔDdPelZ was 4h, which was 2.4 times that of the wild-type DdPelZ; therefore, the temperature tolerance of the mutant enzyme ΔDdPelZ was greatly improved compared with the wild-type enzyme DdPelZ.

[0081] The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the invention patent.

[0082] It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these modifications and improvements all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A pectin lyase mutant ΔDdPelZ, characterized in that: The amino acid at position 168 of the substrate binding pocket of the wild-type pectate lyase DdPelZ was mutated from a large molecular weight and branched valine to a positively charged arginine, and the amino acid at position 319 of the rigid region was mutated from a large molecular weight and branched valine to a small molecular weight alanine.

2. The pectin lyase mutant ΔDdPelZ according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

3. A gene ΔpelZ encoding the pectin lyase mutant according to claim 2.

4. The gene ΔpelZ according to claim 3, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

2.

5. A vector containing the gene ΔpelZ according to claim 3 or 4.

6. A host cell containing the gene ΔpelZ according to claim 3 or 4 or the vector according to claim 5.

7. An engineered bacterium containing the gene ΔpelZ according to claim 3 or 4 or the vector according to claim 5.

8. Use of the gene ΔpelZ and the enzyme encoded by it according to claim 3 or 4 in cotton and linen processing, pulp and paper making, and feed processing.

9. A method for producing the pectin lyase mutant gene ΔpelZ according to claim 2, characterized in that: The nucleotide sequence shown in SEQ ID NO.2 is used as an expression vector with a plasmid capable of expressing the enzyme and a strain capable of expressing the enzyme as an expression host to achieve efficient expression of the mutant shown in SEQ ID NO.1.

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