A pectin lyase mutant delta dd pel z, its coding gene, preparation method and application

Abstract

The application discloses a pectin lyase mutant ADdPelZ, a coding gene thereof, a preparation method and application, wherein the pectin lyase mutant ADdPelZ is obtained by mutating an amino acid at the 168th position of a substrate binding pocket of a wild-type pectin lyase DdPelZ from a macromolecular branched valine into a positively charged arginine, and mutating an amino acid at the 319th position of a rigid region from a macromolecular branched valine into a small-molecular-weight alanine; the amino acid sequence and the nucleotide sequence of the mutant are respectively represented as SEQ ID NO. 1 and SEQ ID NO. 2. The mutant enzyme provided by the application has obvious improvements in enzyme activity and heat resistance under alkaline conditions, and solves the problems of low catalytic activity and insufficient heat resistance of the wild-type pectin lyase under alkaline conditions, thereby creating good conditions for application of the enzyme in cotton and hemp processing, pulping and papermaking, and feed processing.

Description

TECHNICAL FIELD

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

[0002] Pectinase is an important industrial enzyme, which has been widely used in papermaking industry, food processing, environmental protection and textile industry, etc. According to the optimum pH of enzymatic reaction, pectinase is divided into acid pectinase and alkaline pectinase. Most acid pectinases are applied in food and feed processing, and alkaline pectinases are applied in cotton and hemp processing in the textile industry. Alkaline pectinase is mainly involved in biological degumming of hemp and biological refining of cotton and hemp yarn in textile processing. Compared with traditional chemical methods, the application of biological enzyme method in hemp degumming has the advantages of green environmental protection, which 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, high-efficiency expression of the existing alkaline pectin(lyase) enzyme gene and molecular modification of the existing enzyme by molecular biology methods to improve the enzyme activity are effective methods to obtain excellent industrial pectinase.

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

[0004] In the prior art, the amino acid Y at positions 7 and / or 111 of endoglucanase NfEG12A is mutated to W in Chinese patent application No. 201810013726.7, so that the catalytic efficiency of the mutant thereof on beta-1, 3-1, 4-glucan and xyloglucan is increased by 0.5-0.8 times, respectively, and the mutant has good heat resistance and pH tolerance. In addition to improving the enzyme catalytic efficiency, enhancing the thermal stability can also be achieved by point mutation. In Chinese patent application No. 201910835766.4, the base at position 68 of the amino acid sequence of glucose oxidase GOD-M5 is mutated from Asp to Lys, the base at position 274 is mutated from Thr to Phe, the base at position 278 is mutated from Tyr to Thr, the base at position 94 is mutated from Ser to Ala, the base at position 31 is mutated from Thr to Val, and the base at position 88 is mutated from Gln to Arg, so that the thermal stability is greatly improved. In Chinese patent application No. 202011580654.8, the starch sucrolase derived from microorganism Calidithermus timidus DSM 17022 is used as a parent to construct a four-point mutant enzyme L382P / S414N / P618I / H631K, and the half-life of the mutant enzyme at 65℃ is increased from 23.58h to 43.72h, and the half-life at 70℃ is increased from 71min to 110min. In Chinese patent application No. 202210213233.4, a wild-type agarase is point-mutated by rational design to obtain an agarase mutant E122W, so that the enzyme activity and thermal stability of the agarase mutant are obviously improved, and the agarase mutant can efficiently catalyze the degradation of agar to prepare agar oligosaccharide. Therefore, according to the known or predicted structural information and catalytic mechanism of pectin lyase, the relationship between the structure and function is analyzed, the key amino acid sites affecting the catalytic activity and stability of pectin lyase are speculated, and point mutation is an effective way to obtain excellent pectin lyase.

[0005] Up to now, there is no report on mutation and modification of pectin lyase. SUMMARY

[0006] Therefore, the present application provides a pectin lyase mutant ΔDdPelZ, a coding gene thereof, a preparation method and an application.

[0007] To achieve the above-mentioned object, the technical scheme of the present application is as follows:

[0008] The present application provides a pectin lyase mutant ΔDdPelZ, which mutates the amino acid at position 168 of the substrate binding pocket of wild-type pectin lyase DdPelZ from valine with large molecular weight and branches to arginine with positive charge (and similar structure), and mutates the amino acid at position 319 of the rigid region from valine with large molecular weight and branches to alanine with small molecular weight.

[0009] Specifically, in the above technical solution, by point mutation of the amino acid at position 168 of the substrate binding pocket and the amino acid at position 319 of the rigid region (alpha-helix), specifically, on the basis of the pelZ gene sequence (GenBank accession number: KC900169) published in the GenBank database, the amino acids encoded thereby 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 which can bind to pectin molecules, thereby improving the catalytic efficiency; and the amino acid at position 319 is mutated from valine to alpha-helix preferred amino acid-alanine, thereby enhancing the stability of the region structure.

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

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

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

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

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

[0015] The application also provides an engineered bacterium containing the gene ΔpelZ or the vector.

[0016] The application also provides an application of the gene ΔpelZ and the enzyme encoded thereby in cotton and hemp processing, pulp and paper making, and feed processing.

[0017] The application also provides a method for producing the pectin lyase mutant ΔDdPelZ, comprising:

[0018] The nucleotide sequence shown as SEQ ID NO. 2 is used as an expression vector in the form of a plasmid capable of expressing the enzyme, and an expression host in the form of a strain capable of expressing the enzyme, so as to realize high-efficiency expression of the mutant shown as SEQ ID NO. 1.

[0019] In detail, in the above technical solution, the nucleotide sequence shown as SEQ ID NO. 2 is used as an expression vector in the form of pEASY-E1 or a plasmid capable of expressing the enzyme, and an expression host in the form of Escherichia coli BL21 (DE3) or a strain capable of expressing the enzyme, so as to realize high-efficiency expression of the mutant gene ΔpelZ.

[0020] Specifically, in the above technical solution, the pectin lyase gene pelZ is from a hemp degumming efficient strain Dickeya dadantii DCE-01 (preservation number: CGMCC 5522, patent number: ZL201110410078.7); the promoter of the used pEASY-E1 expression unit is a commonly used T7 promoter, under the action of the T7 promoter, the mutant enzyme can directly complete intracellular soluble expression in the host cell E.coli BL21 (DE3).

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

[0022] The mutant enzyme provided by the present application has obvious improvement in enzyme activity and heat resistance under alkaline conditions, and solves the problems of low catalytic activity and insufficient heat resistance of the wild-type pectin lyase under alkaline conditions, thereby creating good conditions for the application of the enzyme in cotton and hemp processing, pulp making, and feed processing.

[0023] By comparing the degradation abilities of wild enzyme DdPelZ and mutant enzyme ΔDdPelZ to polygalacturonic acid sodium salt and ramie pectin under alkaline conditions, it is found that under the conditions of pH 8.5 and temperature 45℃, the specific enzyme activity of the mutant enzyme ΔDdPelZ is 2405 U / mg and 4092 U / mg, respectively, which is 7.3 times and 8.5 times of that of the wild enzyme DdPelZ; under the condition of 55℃, the half-life of the mutant enzyme ΔDdPelZ is 4h, which is 2.4 times of that of the wild enzyme DdPelZ, i.e., the mutant enzyme has the characteristics of high heat resistance and high enzyme activity under alkaline conditions, which indicates that it has important application prospects in industrial production under high-temperature alkaline conditions. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The figure is a construction flowchart of the pectin lyase mutant engineering strain in the embodiment of the present application;

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

[0026] Figure 3 The figure is a schematic diagram of site-directed mutagenesis in the embodiment of the present application;

[0027] Figure 4 The figure is a SDS-PAGE map of the induced expression of the wild-type and mutant enzymes in the embodiment of the present application;

[0028] Figure 5 The figure is a comparison chart of the enzyme activity of the wild-type and mutant enzymes in the embodiment of the present application;

[0029] Figure 6 Figure for comparison of half-life of wild-type and mutant enzymes in embodiments of the present application. DETAILED DESCRIPTION

[0030] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to embodiments.

[0031] It should be understood that the specific embodiments described herein merely serve to explain the present application and do not limit the present application.

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

[0033] The terms "comprising", "including", or any other variation thereof, used in the present document are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements does not necessarily limit those elements to only those elements recited, but can include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

[0034] In addition, the technical features involved in each of the embodiments of the present application described below can be combined with each other as long as there is no conflict.

[0035] Materials and reagents:

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

[0037] The Ultra HiFidelity PCR Kit, the centrifugal column type bacterial genome extraction kit, the fast site-directed mutagenesis kit, the DNA Marker III, the 2xTaq PCR Mix reagent, the ordinary agarose gel NDA recovery kit, the plasmid small extraction kit, IPTG, and ampicillin sodium salt (Amp) were purchased from Tiangen Biotech Co., Ltd.

[0038] The polygalacturonate sodium salt, tryptone, yeast extract, and agar powder were purchased from Sigma Company.

[0039] The remaining chemical reagents were all analytical grade commercial products purchased from the National Pharmaceutical Group;

[0040] The primer synthesis and nucleic acid sequencing were completed by GenScript Biotech Co., Ltd.

[0041] As shown in Figure 1 The figure shows a construction flowchart of the pectin lyase mutant engineering strain in embodiments of the present application.

[0042] Example One: Construction of recombinant plasmid of 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 the genomic DNA was extracted according to the kit instructions.

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

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

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

[0047] The genomic DNA was used as a template to PCR amplify the pectin lyase original gene pelZ.

[0048] The PCR reaction system was as follows: 10x 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; supplemented with sterile ddH2O to a total volume of 50 μL, mixed and then placed in a PCR instrument for PCR reaction.

[0049] The parameters were set as follows:

[0050] (1) 94°C pre-denaturation for 4 min; (2) 94°C denaturation for 30 s; (3) 55°C annealing for 30 s; (4) 72°C extension for 1 min; repeat steps (2)-(4) for 30 cycles; (5) 72°C incubation for 10 min. Then stored at 4°C.

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

[0052] The PCR product of the target gene was subjected to A-tailing and gel purification. According to the instructions of the kit, the recombinant plasmid was obtained. The heat shock method was used to transfer it into E. coli BL21(DE3), and it was plated on LB medium containing 120 μg / mL Amp and incubated overnight. The positive transformants were picked and incubated overnight, and the plasmid was extracted and subjected to PCR verification. The positive plasmid with correct band size and insertion direction was submitted to GENEWIZ for sequence determination. The recombinant plasmid with correct target gene sequence was pEASY-E1-pelZ. Figure 2

[0053] Example Two: Site-directed mutation

[0054] The principle of site-directed mutation: the construction of point mutation plasmid uses Dpn I method. Figure 3

[0055] According to the amino acid site to be mutated, the PCR point mutation primers were designed 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] Among them, the underlined part represents the codon corresponding to the 168th arginine and the 319th alanine of the mutant gene coding.

[0061] The rapid site-directed mutation kit was used to introduce mutation sites by whole plasmid PCR with pEASY-E1-pelZ recombinant plasmid as the template.

[0062] ​​PCR reaction system: forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, 5×FastAlteration Buffer 5 μL, plasmid DNA 1 μL, Fast Alteration DNA Polymerase 0.5 μL, and ddH2O to 25 μL.

[0063] Parameter settings are as follows:

[0064] (1) 95℃ pre-denaturation 2 min; (2) 94℃ denaturation 20 s; (3) 60℃ rehydration 10 s; (4) 68℃ extension 2.5 min; repeat steps (2)-(4) for 18 cycles; (5) 68℃ incubation for 5 min, and the product is stored at 4℃.

[0065] Add 0.5 μL of restriction endonuclease Dpn I to the 25 μL of mutated PCR product, mix thoroughly, and then digest at 37℃ for 1 h; transfer 5 μL of the Dpn I digestion product into DH5α, evenly spread the transformed bacteria on an LB selection plate (Amp 120 μg / mL), and incubate at 37℃ overnight to obtain the transformants of the relevant mutant strains, extract the plasmid, and verify the correct mutant strains by sequencing. The successfully constructed recombinant plasmid is 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 and mutant enzymes and SDS-PAGE analysis

[0067] Inoculate single colonies of the original genetically engineered bacteria pEASY-E1-pelZ / BL21 and the mutant genetically engineered bacteria pEASY-E1-ΔpelZ / BL21 in LB liquid medium containing 120 mg / mL Amp, and incubate at 37℃ and 200 r / min until the OD 600 is 0.6. Add 1 mmol / L IPTG, and induce expression at 28℃ and 120 r / min for 12-15 h.

[0068] Transfer 1 mL of the induced mature fermentation bacteria into 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 centrifuge, and wash twice. Suspend the bacterial pellet with 40 μL of sterilized ddH2O, add 10 μL of 5×protein loading buffer, boil for 5 min, naturally cool, and store at -20℃ for standby use (Note: soak in a boiling water bath for 3 min before loading).

[0069] Analyze the prepared sample by discontinuous SDS-PAGE (5% concentrated gel and 12% separation gel) (such as Figure 4The results showed that both the original genetically engineered strain pEASY-E1-pelZ / BL21 and the mutant genetically engineered strain pEASY-E1-ΔpelZ / BL21 could successfully express specific protein bands.

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

[0071] The induced mature fermentation broth was centrifuged at 3000 r / min at 4°C for 10 min, and the supernatant was collected as the crude enzyme solution.

[0072] In order to compare the biological catalytic ability of wild-type enzyme and mutant enzyme under alkaline conditions, enzyme activity determination and protein content determination were carried out under the same conditions.

[0073] Under alkaline conditions, the degradation abilities of wild-type enzyme DdPelZ and mutant enzyme ΔDdPelZ to polygalacturonic acid sodium salt and ramie pectin were compared (as shown in FIG. 2). Figure 5

[0074] Enzyme activity determination method: 5 mg / mL polygalacturonic acid sodium or ramie pectin solution was prepared using 0.05 mol / L glycine-sodium hydroxide buffer (pH 8.5). 1 mL of the preheated substrate was taken to 50°C, 10 μL of appropriately diluted enzyme solution was added, and the reaction was carried out at 50°C for 10 min. Then, 2 mL of DNS was immediately added. Color development was carried out in a boiling water bath for 5 min, and rapid cooling was carried out in an ice water bath. The same enzyme solution that was inactivated by boiling was used as a negative control, and the OD 520 .

[0075] Pectin lyase activity was defined as: the amount of enzyme required to release 1 μmol of reducing sugar per minute of unsaturated galacturonic acid from the substrate was 1 enzyme activity unit, which was expressed as U.

[0076] Protein content determination method: Coomassie brilliant blue G-250 method; 100 μL of enzyme solution diluted to an appropriate multiple with 0.15 mol / L sterilized NaCl solution was added to 5 mL of Coomassie brilliant blue G-250 reagent, mixed well, color developed for 5 min, and the OD 595 .

[0077] The results showed that under the conditions of pH 8.5 and 45°C, the specific enzyme activity of the mutant ΔDdPelZ was 2405 U / mg and 4092 U / mg, which was 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 that of the wild-type enzyme DdPelZ.

[0078] ​Example 6: Comparison of heat tolerance of wild-type and mutant enzymes

[0079] The crude enzyme solution was incubated at 55°C, and samples were taken at regular intervals to determine the residual enzyme activity (as described in Example 1) and the time at which the enzyme activity was retained at 50% (i.e. the half-life T 1 / 2 ). Figure 6

[0080] The results show that the half-life of ΔDdPelZ is 4h at 55°C, which is 2.4 times that of the wild-type DdPelZ. Thus, the temperature tolerance of the mutant enzyme ΔDdPelZ is also greatly improved compared to the wild-type enzyme DdPelZ.

[0081] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent.

[0082] It should be noted that for those skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present application patent should be subject to the appended claims.​

Claims

1. A pectin lyase mutant Δ Dd PelZ characterized in that, Wild-type pectolyase Dd The amino acid at position 168 of the PelZ substrate binding pocket was mutated from a large, branched valine to a positively charged arginine, and the amino acid at position 319 of the rigid region was mutated from a large, branched valine to a small, unbranched alanine; The amino acid sequence is shown as SEQ ID NO.

1.

2. A gene encoding the pectin lyase mutant of claim 1 Δ pel Z.

3. The gene Δ according to claim 2 pel Z characterized in that, The nucleotide sequence is shown as SEQ ID NO.

2.

4. A vector comprising the gene of claim 2 or 3 pel Z.

5. A host cell comprising the gene Δ pel Z or the vector of claim 4.

6. An engineered bacterium comprising the gene Δ pel Z or the vector of claim 4.

7. The gene of claim 2 or 3 pel Use of Z and the enzyme encoded by Z in cotton processing, pulp and paper making, and feed processing.

8. A method of producing the pectolyase mutant gene Δ of claim 1 pel Z, characterized in that, The nucleotide sequence shown as 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 is used as an expression host, so as to realize high-efficiency expression of the mutant shown as SEQ ID NO. 1.

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