CN118256479A - Pectase mutant and encoding gene and application thereof - Google Patents
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Abstract
The invention discloses a pectase mutant, a coding gene and application thereof, wherein the amino acid sequence of the pectase mutant is shown as SEQ ID NO. 3. The pectase mutant of the invention replaces serine with leucine at position 262 of pectase MarPel, which improves the optimal reaction temperature by 15 ℃ and half-life by 2.31 times under 60 ℃ compared with wild pectase MarPel, improves the melting temperature by 3 ℃ compared with wild pectase, greatly improves the thermal stability compared with wild pectase, meets the high temperature requirements of paper pulp industry and the like, and provides an effective method for industrial mass production of pectase.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a pectase mutant MarPel L S with high thermal stability, and a coding gene and application thereof.
Background
Pectin (pecin) is a generic name for a class of galacturonic acid-rich plant cell wall polysaccharides, whose basic backbone is a homopolymer of alpha-1, 4 glycosidic linkages. In the plant cell wall, pectin is mainly covalently bound to cellulose, hemicellulose, lignin, etc., forming protopectin. Its presence helps to maintain plant specific external structural features but increases the difficulty of deep processing of the plant.
Pectic enzymes are a generic term for a class of enzymes commonly found in microorganisms and plants that are capable of degrading pectin. Pectic enzymes are classified into two main classes, esterase and depolymerase, according to their mode of action, and depolymerase is classified into hydrolase and lyase. Wherein the hydrolase cleaves the alpha-1, 4-glycosidic bond in the polygalacturonic acid chain constituting the pectin by introducing water molecules, and the lyase cleaves the backbone alpha-1, 4-glycosidic bond of the pectin polymer by trans-elimination. Most of the pectinases reported so far use both methods to degrade pectin.
Although there are several documents at home and abroad reporting pectase, its wide application is still limited by enzymatic properties and production cost. Most of the existing pectic enzymes, whether lyase or hydrolase, fail to achieve the enzyme activity or thermal stability required for industrial degradation of pectin. Meanwhile, the production levels of the pectase enzyme-producing strains obtained by screening in China are different, and the production levels are quite distant from meeting the industrial application requirements.
Therefore, screening pectinase with high activity and high thermal stability and constructing high-yield engineering strains are regarded as important key technology for promoting the development of the pectinase in industrial application.
Disclosure of Invention
Based on the above, the invention aims to provide a pectase mutant, and a coding gene and application thereof, wherein the pectase mutant has high activity and high thermal stability.
The technical scheme for realizing the aim of the invention comprises the following steps.
In a first aspect of the invention, a pectase mutant is provided, and the amino acid sequence of the pectase mutant is shown as SEQ ID NO. 3.
In a second aspect of the invention, a coding gene of the pectase mutant is provided, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 4.
In a third aspect of the invention, there is provided a recombinant expression vector having inserted therein a gene encoding a pectase mutant, the nucleotide sequence of which is shown in SEQ ID NO. 4.
In a fourth aspect of the present invention, there is provided a method for preparing the recombinant expression vector described above, comprising the steps of:
(1) The pectase gene MarPel with the nucleotide sequence shown as SEQ ID NO. 2 is connected to pET28a (+) to obtain a recombinant expression vector pET28a (+) -MarPel;
(2) Using pETDuet-SUMO as a template, using SEQ ID NO. 7 and SEQ ID NO. 8 as primers, and amplifying to obtain a solvoprotein SUMO gene with a nucleotide sequence shown as SEQ ID NO. 6;
(3) Amplifying to obtain linearized pET28a (+) -MarPel by taking pET28a (+) -MarPel as a template and SEQ ID NO 9 and SEQ ID NO 10 as primers;
(4) Connecting the linearized pET28a (+) -MarPel and the proprotein SUMO gene fragment by utilizing GibsonAssembly seamless cloning technology to construct a recombinant expression vector pET28a (+) -SUMO-MarPel;
(5) And amplifying to obtain a pectase mutant expression vector pET28a (+) -SUMO-MarPel L S by using pET28a (+) -SUMO-MarPel as a template and SEQ ID NO. 11 and SEQ ID NO. 12 as primers.
In a fifth aspect of the present invention, there is provided an engineering bacterium expressing the recombinant expression vector described above.
The sixth aspect of the present invention provides a method for constructing the engineering bacteria, comprising the steps of: and (3) converting the pectase mutant expression vector pET28a (+) -SUMO-MarPelL262S into an escherichia coli expression host ESCHERICHIA COLIBL (DE 3) to obtain the recombinant vector.
The seventh aspect of the invention provides application of the pectase mutant, the coding gene, the recombinant expression vector or the engineering bacteria in pectin degradation or papermaking.
The invention has the following beneficial effects:
in the invention, pectase MarPel gene from ocean Marinimicrobium sp.EAC58 is used as a template, a pET28a (+) -SUMO-MarPel L262S recombinant expression vector with 6 histidine tags and a fusion gene of a pectase MarPel of a soluble protein is obtained through PCR amplification, and then the recombinant expression vector is transformed into E.coli BL21 (DE 3) through heat shock to obtain engineering bacteria, wherein the engineering bacteria expression product is cell soluble protein, and the protein is obtained through cell ultrasonic disruption, nickel column affinity chromatography, separation, purification and digestion, namely pectase mutant MarPel L262S (one leucine on pectase MarPel is replaced by serine). The optimal reaction temperature of the pectase mutant can reach 60 ℃, the optimal reaction temperature is improved by 15 ℃ compared with the optimal reaction temperature of the wild type MarPel, the half life under the condition of 60 ℃ is 15.75min, the half life under the condition of 60 ℃ is improved by 2.31 times compared with the half life of the wild type pectase under the temperature of 4.76min, and the melting temperature is improved by 3 ℃ compared with the wild type, so that the thermal stability is greatly improved compared with the wild type, the requirements of the paper pulp industry and other high temperature are met, and an effective method is provided for industrial mass production of pectase.
Drawings
FIG. 1 is an SDS-PAGE protein electrophoresis of wild-type pectinase MarPel and pectinase mutant MarPel L262S in example 2 of the present invention.
FIG. 2 shows the relative enzyme activities of pectase mutants MarPel L and 262S at different pH values in example 3 of the present invention.
FIG. 3 shows the relative enzyme activities of the wild-type pectinase MarPel and the pectinase mutant MarPel L262S at different temperatures in example 3 of the present invention.
FIG. 4 shows the relative enzyme activities of the wild-type pectinase MarPel and the pectinase mutant MarPel L262S at various times in example 4 of the present invention.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
In some embodiments of the invention, a pectase mutant is disclosed, the amino acid sequence of which is mutated at the site of occurrence of L262S in the amino acid composition of pectase MarPel, and the amino acid sequence of which is shown as SEQ ID NO. 3.
In other embodiments of the present invention, a gene encoding a pectinase mutant is disclosed, the nucleotide sequence of which is shown in SEQ ID NO. 4.
In other embodiments of the present invention, a recombinant expression vector is disclosed in which the coding gene of the pectase mutant is inserted, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 4.
In other embodiments of the present invention, an engineering bacterium expressing the recombinant expression vector is disclosed.
In other embodiments of the present invention, a method for constructing the engineering bacteria is disclosed, and reference ：Improving the specific activity and thermo-stability ofalkaline pectate lyase from Bacillus subtilis 168forbioscouring.Wang,X.,et al.,Biochemical Engineering Journal,2018.129:p.74-83, specifically includes the following steps:
(1) Synthesizing codon-optimized marine origin by using whole gene synthesis technology
Marinimicrobium sp.EAC58 pectase gene (amino acid sequence shown in SEQ ID NO:1, nucleotide sequence shown in SEQ ID NO: 2) is connected to pET28a (+) to obtain recombinant expression vector pET28a (+) -MarPel;
(2) The pectase gene MarPel with the nucleotide sequence shown as SEQ ID NO. 2 is connected to pET28a (+) to obtain a recombinant expression vector pET28a (+) -MarPel;
(3) Using pETDuet-SUMO as a template, using SEQ ID NO. 7 and SEQ ID NO. 8 as primers, and amplifying to obtain a solvoprotein SUMO gene with a nucleotide sequence shown as SEQ ID NO. 6;
(4) Amplifying to obtain linearized pET28a (+) -MarPel by taking pET28a (+) -MarPel as a template and SEQ ID NO 9 and SEQ ID NO 10 as primers;
(5) Connecting the linearized pET28a (+) -MarPel and the proprotein SUMO gene fragment by utilizing GibsonAssembly seamless cloning technology to construct a recombinant expression vector pET28a (+) -SUMO-MarPel;
(5) And amplifying to obtain a pectase mutant expression vector pET28a (+) -SUMO-MarPel L S by using pET28a (+) -SUMO-MarPel as a template and SEQ ID NO. 11 and SEQ ID NO. 12 as primers.
(6) And transforming pET28a (+) -SUMO-MarPelL S into an escherichia coli expression host ESCHERICHIA COLI BL (DE 3).
In other embodiments of the invention, the pectinase mutant, the encoding gene of the pectinase mutant, the recombinant expression vector or the application of engineering bacteria in pectin degradation are disclosed.
In other embodiments of the invention, the pectase mutants, the coding genes of the pectase mutants, recombinant expression vectors or engineering bacteria are disclosed for use in papermaking.
The sequences involved in the present invention are as follows:
SEQ ID NO:1：
MTVNLAAGSNVIRADATVSSGGPNFDYIEVSGGDGSSSSSSSSSSSSSSSSSSSV
PSDPGSPLSCQEANTVRGFASLGGGTTGGAGGDVVTVSSGTELEAALDNKGS
NPLTIYVDGTITPGNSSVSKFDIKDMNDVSIIGVGNNALFDGIGIKIWRANNVI
VRNVTMRYVRIGDKDHITIDGPSSNIWIDHNTFYNSLDVGKDYYDELVSGKN
DIDNITVSYNILRDSWKTSLWGSSDGDDSHRRVTFWGNHWENANSRLPLFRF
GEGHVINNYYDGILSTGINSRMGATIRIEGNVFENSQNPIVSFYSDEIGYWDVQ
DNIFSNITWEEGSGDDIIAGPNVQSTVSYTPPYSYSALPASDAKGHVLANAGA
GVISNCVAP
SEQ ID NO:2：
ATGACCGTTAACCTGGCGGCGGGTAGCAACGTTATCCGTGCGGATGCGACC
GTGTCCTCCGGCGGTCCGAACTTCGATTACATCGAAGTTTCTGGTGGTGAT
GGCAGCAGCTCCTCTTCCTCCAGCAGCTCCAGCAGCAGCTCTTCTTCCAGC
AGCTCTAGCGTTCCGAGCGATCCGGGTAGCCCGCTGAGCTGCCAGGAAGC
TAACACCGTGCGTGGCTTCGCATCTCTGGGTGGCGGCACCACCGGTGGTG
CGGGTGGCGACGTTGTGACCGTGAGCAGCGGCACCGAACTGGAAGCGGC
GCTGGACAACAAAGGCTCCAACCCGCTGACCATTTACGTTGATGGTACTAT
TACTCCGGGTAACTCTTCTGTTAGCAAATTCGACATCAAAGATATGAACGA
CGTGTCTATCATCGGTGTTGGCAACAACGCACTGTTCGATGGTATCGGCAT
CAAAATTTGGCGTGCGAACAACGTTATTGTTCGTAACGTTACCATGCGCTA
CGTTCGTATCGGTGATAAAGACCACATTACCATCGATGGTCCGAGCAGCAA
CATCTGGATCGATCATAACACCTTCTACAACTCTCTGGATGTTGGTAAAGAT
TACTATGATGAACTGGTTTCTGGCAAAAACGATATTGATAACATCACCGTTT
CTTACAACATCCTGCGTGACTCCTGGAAAACCAGCCTGTGGGGCAGCTCT
GATGGCGATGACTCTCACCGTCGTGTTACCTTTTGGGGCAACCACTGGGAA
AACGCGAACAGCCGTCTGCCGCTGTTCCGTTTCGGCGAAGGCCACGTGAT
CAACAACTATTACGATGGTATCCTGAGCACCGGTATCAACAGCCGCATGGG
CGCTACCATCCGTATCGAAGGCAACGTTTTCGAAAACAGCCAGAACCCGA
TCGTTAGCTTCTACTCTGATGAAATCGGCTACTGGGATGTGCAGGACAACA
TCTTCTCTAACATCACCTGGGAAGAAGGCTCTGGTGATGACATCATCGCTG
GCCCGAACGTTCAGAGCACCGTTAGCTACACCCCGCCGTATTCTTACTCTG
CTCTGCCGGCGTCTGATGCGAAAGGCCACGTTCTGGCGAACGCGGGTGCG
GGTGTTATCAGCAACTGCGTTGCGCCGTAA
SEQ ID NO:3：
MTVNLAAGSNVIRADATVSSGGPNFDYIEVSGGDGSSSSSSSSSSSSSSSSSSSV
PSDPGSPLSCQEANTVRGFASLGGGTTGGAGGDVVTVSSGTELEAALDNKGS
NPLTIYVDGTITPGNSSVSKFDIKDMNDVSIIGVGNNALFDGIGIKIWRANNVI
VRNVTMRYVRIGDKDHITIDGPSSNIWIDHNTFYNSLDVGKDYYDELVSGKN
DIDNITVSYNILRDSWKTSLWGSSDGDDSHRRVTFWGNHWENANSRLPSFRF
GEGHVINNYYDGILSTGINSRMGATIRIEGNVFENSQNPIVSFYSDEIGYWDVQ
DNIFSNITWEEGSGDDIIAGPNVQSTVSYTPPYSYSALPASDAKGHVLANAGA
GVISNCVAP
SEQ ID NO:4：
ATGACCGTTAACCTGGCGGCGGGTAGCAACGTTATCCGTGCGGATGCGACC
GTGTCCTCCGGCGGTCCGAACTTCGATTACATCGAAGTTTCTGGTGGTGAT
GGCAGCAGCTCCTCTTCCTCCAGCAGCTCCAGCAGCAGCTCTTCTTCCAGC
AGCTCTAGCGTTCCGAGCGATCCGGGTAGCCCGCTGAGCTGCCAGGAAGC
TAACACCGTGCGTGGCTTCGCATCTCTGGGTGGCGGCACCACCGGTGGTG
CGGGTGGCGACGTTGTGACCGTGAGCAGCGGCACCGAACTGGAAGCGGC
GCTGGACAACAAAGGCTCCAACCCGCTGACCATTTACGTTGATGGTACTAT
TACTCCGGGTAACTCTTCTGTTAGCAAATTCGACATCAAAGATATGAACGA
CGTGTCTATCATCGGTGTTGGCAACAACGCACTGTTCGATGGTATCGGCAT
CAAAATTTGGCGTGCGAACAACGTTATTGTTCGTAACGTTACCATGCGCTA
CGTTCGTATCGGTGATAAAGACCACATTACCATCGATGGTCCGAGCAGCAA
CATCTGGATCGATCATAACACCTTCTACAACTCTCTGGATGTTGGTAAAGAT
TACTATGATGAACTGGTTTCTGGCAAAAACGATATTGATAACATCACCGTTT
CTTACAACATCCTGCGTGACTCCTGGAAAACCAGCCTGTGGGGCAGCTCT
GATGGCGATGACTCTCACCGTCGTGTTACCTTTTGGGGCAACCACTGGGAA
AACGCGAACAGCCGTCTGCCGAGCTTCCGTTTCGGCGAAGGCCACGTGAT
CAACAACTATTACGATGGTATCCTGAGCACCGGTATCAACAGCCGCATGGG
CGCTACCATCCGTATCGAAGGCAACGTTTTCGAAAACAGCCAGAACCCGA
TCGTTAGCTTCTACTCTGATGAAATCGGCTACTGGGATGTGCAGGACAACA
TCTTCTCTAACATCACCTGGGAAGAAGGCTCTGGTGATGACATCATCGCTG
GCCCGAACGTTCAGAGCACCGTTAGCTACACCCCGCCGTATTCTTACTCTG
CTCTGCCGGCGTCTGATGCGAAAGGCCACGTTCTGGCGAACGCGGGTGCG
GGTGTTATCAGCAACTGCGTTGCGCCGTAA
SEQ ID NO:5：
MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFA
KRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGA
SEQ ID NO:6：
ATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAG
AAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAG
AGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAA
GCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTAC
GACGGTATTAGGATTCAAGCTGATCAGACCCCTGAAGATTTGGACATGGAG
GATAACGATATTATTGAGGCTCACAGAGAACAGATTGGTGGAGCT
Pectic substances used in the following examples were purchased from Sigma-Aldrich (CAS number: 9000-69-5), and were conventional, unless otherwise specified; the consumables, reagents and instruments used, if not specified, can be obtained from commercial sources; the instrument used for measuring the protein content is a Nano Drop micro-spectrophotometer. The property research of the mutant pectase, which is tested by the invention, is that the mutant pectase protein liquid is subjected to salt replacement and dissolution promoting label removal if no special description exists; the method of measuring the activity is ultraviolet absorption measurement at 235nm unless otherwise specified.
The invention is described in detail below with reference to the drawings and the specific embodiments.
EXAMPLE 1 construction of recombinant expression vector for mutant pectase MarPel L262S Gene
The method comprises the following steps:
1. The complete gene synthesis technology is adopted by the Shanghai biological company, the pectase MarPel gene from Marinimicrobiumsp.EAC58 is synthesized on an expression vector pET28a (+) after codon optimization (the amino acid sequence is shown as SEQ ID NO:1, the nucleotide sequence is shown as SEQ ID NO: 2), and the pET28a (+) -MarPel recombinant expression vector is constructed.
2. The pETDuet-SUMO recombinant plasmid (preserved in the laboratory of the applicant and prepared by a method known in the art) is used as a template, primers (an upstream primer SEQ ID NO:7 and a downstream primer SEQ ID NO: 8) are designed, the SUMO gene is obtained through PCR amplification, the amplified product is subjected to agarose gel electrophoresis, and 297bp (the nucleotide sequence is shown as SEQ ID NO:6 and the amino acid sequence is shown as SEQ ID NO: 5) is recovered and purified.
Upstream primer (SEQ ID NO: 7):
5’CAAATGGGTCGCGGATCCATGTCGGACTCAGAAGTCAATC 3’
Downstream primer (SEQ ID NO: 8):
5’CCAGGTTAACGGTCATAGCTCCACCAATCTGTTCTC 3’
The PCR amplification reaction system is shown in Table 1 (primer concentration: 10 mM), and the PCR amplification reaction procedure is shown in Table 2.
TABLE 1
| Composition of the components | Volume of |
| SUMO Gene | 2μL |
| Upstream primer | 2μL |
| Downstream primer | 2μL |
| 2×PrimeStarMax | 25μL |
| ddH2O | 19μL |
TABLE 2
| Program | Temperature (. Degree. C.) | Time (min) |
| 1. Pre-denaturation | 98 | 5 |
| 2. Denaturation (denaturation) | 98 | 0.5 |
| 3. Annealing | 55 | 0.5 |
| 4. Extension (procedure 2-4, 30 cycles) | 72 | 2000bp/min |
| Final extension | 72 | 5 |
| Preservation of | 4 | Until the mixture is taken out |
3. PCR amplification (reaction system and procedure are the same) is carried out by taking pET28a (+) -MarPel as a template, designing primers (an upstream primer SEQ ID NO:9 and a downstream primer SEQ ID NO: 10), linearizing pET28a (+) -MarPel, carrying out agarose gel electrophoresis on an amplified product, and recovering and purifying 6437bp.
Upstream primer (SEQ ID NO: 9): 5'ATGACCGTTAACCTGGCGG 3'
Downstream primer (SEQ ID NO: 10): 5'GGATCCGCGACCCATTTGC 3'
4. The purified and recovered linear pET28a (+) -MarPel and the purified and recovered SUMO DNA fragment are connected by GibsonAssembly seamless cloning technology (the seamless cloning system is shown in Table 3) to construct a recombinant expression vector pET28a (+) -SUMO-MarPel. Sequencing proves that the 5' end of the recombinant expression vector contains 6 histidine tag genes and a pectase fusion gene SUMO-MarPel of a soluble protein SUMO gene, namely MarPel (the nucleotide sequence is shown as SEQ ID NO: 2) and a soluble protein SUMO gene (the nucleotide sequence is shown as SEQ ID NO: 6) are inserted between BamHI and XholI sites of the vector pET28a (+).
TABLE 3 Table 3
| Component (A) | Volume of |
| SUMOFragment + LinerpET a (+) -MarPel (molar ratio 3:1) | 0.5～5μL |
| 2×SeamlessMasterMix | 5μL |
| ddH2O | Is added to 10 mu L |
5. PCR amplification was performed using pET28a (+) -SUMO-MarPel as a template and designing primers (upstream primer SEQ ID NO:11 and downstream primer SEQ ID NO: 12) (the reaction system is shown in Table 4, and the reaction procedure is shown in Table 2).
Upstream primer (SEQ ID NO: 11): 5'CTGCCGTCTTTCCGTTTCGGCGAAGG 3'
Downstream primer (SEQ ID NO: 12): 5'CGGAAAGACGGCAGACGGCTGTTCG 3'
TABLE 4 Table 4
| Composition of the components | Volume of |
| pET28a(+)-SUMO-MarPel | 2μL |
| Upstream primer | 2μL |
| Downstream primer | 2μL |
| 2×PrimeStarMax | 25μL |
| ddH2O | 19μL |
And (3) carrying out agarose gel electrophoresis on the amplified product, and carrying out template digestion and purification on the amplified product after confirming that the amplification is successful. Sequencing the purified product by Shanghai biological company, and constructing a pET28a (+) -SUMO-MarPel L262S mutant pectase gene recombinant expression vector, wherein the nucleotide sequence of pectase mutant MarPel L262S is shown as SEQ ID NO. 4, the amino acid sequence is shown as SEQ ID NO. 3, and compared with wild pectase MarPel, the 262 th lysine mutation is serine.
EXAMPLE 2 construction of engineering bacterium expressing pectase mutant MarPel L262S and expression of pectase mutant MarPel L262S
The E.coli BL21 (DE 3) competent cells used in this example were purchased from Tiangen Biochemical technologies (Beijing) Inc.
1. Competent cells stored in a-80℃refrigerator were thawed on ice for 5min.
2. 10 Mu L of pET28a (+) -SUMO-MarPel L262S mutant pectase gene recombinant expression vector constructed in the example 1 is added, and the mixture is gently mixed and then placed on ice for 30min. After 90s in a water bath at 42 ℃, the mixture was immediately placed in ice for 3min.
3. Mu.L of sterile LB medium was added to each tube and resuscitated at 220rpm for 1h at 37 ℃. And centrifuging at 4000rpm for 2min, only leaving 200 mu L of supernatant, coating the whole bacterial liquid on a plate after re-suspension, and placing the plate in a 37 ℃ incubator for overnight culture to obtain engineering bacteria ESCHERICHIA COLI L (DE 3)/pET 28a (+) -SUMO-MarPel L262S expressing pectase mutants MarPel L S.
4. Single bacterial colony is selected and placed into 5mL LB liquid medium containing 50 mug/mL kanamycin, cultured for 10-12h at 37 ℃ and 220rpm, 600 mu L bacterial liquid is taken and mixed with 600 mu L sterile 50% glycerol, and the mixture is prepared into glycerinum, and frozen in a refrigerator at-80 ℃ for standby.
5. Expression of pectase mutant MarPel L S
(1) Preparation of seed liquid
And (3) marking and activating the frozen glycerol engineering bacteria ESCHERICHIA COLI BL (DE 3)/pET 28a (+) -SUMO-MarPel L S in an LB solid plate culture medium, and then picking single bacterial colonies into 5mL LB liquid culture medium containing 50 mug/mL kanamycin, and culturing at 37 ℃ and 220rpm for 10-12 hours to obtain a first-stage seed culture solution. Inoculating the seed solution into a 250mL conical flask with the specification of 50mL seed culture medium of liquid loading amount according to 1% (v/v) inoculum size, and carrying out shaking culture at 37 ℃ and 220rpm until OD 600 = 0.6 to obtain a secondary seed culture solution;
(2) Fermentation culture
Inoculating the second-level seed liquid into 500mL fermentation medium (LB liquid medium) with the specification of 2L liquid loading according to the inoculation amount of 5% (v/v), adding 1mM IPTG with the final concentration for induction when the culture is carried out at the temperature of 37 ℃ and 220rpm until OD 600 = 0.6-0.8, and carrying out induction at the temperature of 18 ℃ for 22 hours, thereby obtaining the engineering bacteria fermentation liquid.
And (3) centrifuging the fermentation broth obtained in the steps for 20min at the temperature of 4 ℃ and the rpm of 8000 by using a refrigerated centrifuge to obtain thalli.
(3) Purification of pectase mutant MarPel L S
The cells were buffered with a buffer (50 mM Gly-NaOH+0.3M NaCl, pH 8.0) to give a wet weight of cells: buffer = 1:10 (W/V) complete resuspension. Placing the bacterial liquid on ice for ultrasonic crushing with the crushing power of 455W, working for 4s, stopping for 4s, and crushing for 20min. When the bacterial liquid is changed from a milky state to transparent, the bacterial liquid is centrifuged for 20min at 11000rpm and 4 ℃, and the supernatant protein liquid is collected and filtered. Treating the supernatant protein liquid by nickel column affinity chromatography, pre-eluting by BufferA (20 mM Tris-HCl+50mM imidazole, pH 8.0) to remove the impurity protein, and eluting by using BufferB (20 mM Tris-HCl+0.5M imidazole, pH 8.0) to obtain fusion protein liquid; desalting and fusing pectase solution obtained after salt exchange of the protein eluent by a salt exchange column, digesting the solution overnight by SUMO protease at 4 ℃, treating the solution again by a nickel column affinity chromatography, collecting the flow-through solution, namely pectase mutant MarPel L S (the amino acid sequence of which is shown as SEQ ID NO:3, the nucleotide sequence of which is shown as SEQ ID NO: 4), taking 40 mu L of pectase mutant MarPel L262S protein solution, and carrying out SDS-PAGE electrophoresis analysis, wherein the result is shown as figure 1.
As can be seen from the results of FIG. 1, the high-purity wild pectase and its mutant MarPel L262S can be obtained by nickel column affinity chromatography, which lays a foundation for subsequent research and application.
Example 3 enzyme Activity of pectase mutant MarPel L262S under different pH and temperature conditions
This example shows the enzyme activity of the pectase mutant MarPel L S262S obtained in example 2 at various temperatures and pH conditions.
1、pH
The enzyme solution of the pectase mutant is used as a solution to be tested, the activity detection of the mutant pectase is carried out at 60 ℃, the corresponding absorbance of MarPel L S in BR buffers (pH 7.0-12.0) with different pH values and 50mM KCl-NaOH buffer (pH 12.0-13.0) is measured, and all the experiments are arranged in three parallel. The highest enzyme activity was defined as 100% and the rest expressed as relative enzyme activity, and the results are shown in FIG. 2.
From the results of fig. 2, pectase mutant MarPel L S showed no detectable activity under acidic conditions and had greater activity under alkaline conditions. When pH <7 or pH >12, substantially no enzyme activity was detected; when the pH is more than or equal to 8 and less than or equal to 12, the enzyme activity is correspondingly improved along with the improvement of the pH, and when the pH is 12.0, the enzyme activity is maximum, which indicates that the optimal reaction pH of the pectase mutant MarPel L S is 12.0.
2. Temperature (temperature)
The enzyme solution of the wild pectase MarPel and the enzyme solution of the pectase mutant MarPel L262S are used as the solutions to be detected, and the pectase activity detection is carried out at different temperatures, wherein the difference is only that different reaction temperatures (40-75 ℃ and different enzyme activities are measured at intervals of 5 ℃) are used in the reaction conditions. The highest enzyme activity was recorded as 100%, and the relative enzyme activities at different temperatures were calculated. The results are shown in FIG. 3.
As can be seen from the results of FIG. 3, the relative enzyme activity of the pectase mutant MarPel L S at 55-60℃is 100%, the activity increases with increasing temperature in the range of 45-60℃and the enzyme activity is maximum at 55-60 ℃; the enzyme activity keeps more than 80% of the maximum enzyme activity at the temperature of 60-65 ℃; above 70 ℃, the enzyme activity drops rapidly. Therefore, the optimal temperature of the pectase mutant MarPel L S is 55-60 ℃, the enzyme activity is kept to be more than 80% at 60-65 ℃, compared with the optimal temperature of the wild pectase MarPel at 45 ℃, the optimal temperature of the pectase mutant MarPel L S is improved, the thermal stability is improved, and the pectase mutant MarPel L S is more beneficial to industrial application, for example, has better application prospect in pulp degumming.
In this example, the enzyme activity measurement method is specifically as follows:
(1) Preparing a substrate: 1g of pectin is weighed into a 50mL beaker, added with 0.05M Gly-NaOH (pH 10.5) dissolved in 30mL, fully stirred and dissolved, adjusted to pH 7.0 by 50mM NaOH solution, and prepared into a substrate of 2% (w/v) by water to a volume of 50 mL.
(2) Control group: 1.7mL of 50mM Gly-NaOH (pH 10.5) buffer solution is added into a 4mL Ep tube, 200 mu L of 2% pectin substrate is added, the metal bath is preheated for 5min at 60 ℃ for enzyme activity measurement at different temperatures, 2mL of 80mM HCl solution is added, 100 mu L of enzyme solution which is properly diluted is added for reaction for 10min, and the absorbance value at 235nm is measured in 2 min;
(3) Experimental group: 1.7mL of 0.05M Gly-NaOH (pH 10.5) buffer was added to 4mL of Ep tube, 200. Mu.L of 2% pectin substrate was added, the mixture was preheated in a metal bath at 60℃for 5min (at different temperatures for enzyme activity measurement), and 100. Mu.L of an appropriately diluted enzyme solution was added to react for 10min. The reaction was quenched by the addition of 2mL of 80mM HCl solution, and its absorbance at 235nm was measured over 2 min.
(4) One standard enzyme activity unit (1U) of pectinase is defined as: under the above conditions, the amount of enzyme required to produce 1. Mu. Mol of unsaturated polygalacturonic acid from pectin is allowed to react for 1 min. The pectinase activity (U/m L) is calculated as follows:
Wherein: 4600 (L.mol -1·cm-1) is the molar absorption coefficient of unsaturated polygalacturonic acid at 235nm
Example 4 stability comparison of pectase mutant MarPel L262S with wild-type pectase MarPel
This example compares the half-life and melting temperature of wild-type pectinase MarPel with that of pectinase mutant MarPel L S.
1. Half-life period
The relative enzyme activities were measured at 60℃and at 10min intervals using the wild-type pectinase MarPel and pectinase mutant MarPel L S as the solutions to be measured, the half-lives of the wild-type pectinase MarPel and pectinase mutant MarPel L S at 60℃were calculated as the maximum 100% of the enzyme activities at the beginning (0 min) according to the method of example 4, and the results are shown in FIG. 4.
2. Melting temperature
Melting temperature (Tm) values for wild-type pectinase MarPel and pectinase mutant MarPel L S were determined using circular dichroism (ECD). The purified MarPel WT was salified with MarPel L262S in 50mM phosphate buffer (pH 8.0). The pectinase was diluted with 50mM phosphate buffer (pH 8.0) to a CD absorbance of about 1.5, and the measurement was started. Setting the initial measurement temperature at 20 ℃, ending the measurement temperature at 96 ℃, setting the step length at 2 ℃ and heating up at a speed of 1 ℃/min.
The half-life and melting temperature comparison results of the wild-type pectinase MarPel and the pectinase mutant MarPel L S are shown in table 5.
TABLE 5
| MarPel | 60℃t1/2(min) | Tm(℃) |
| WT | 4.76 | 46.7±0.2 |
| L262S | 15.75 | 49.7±1.0 |
From the results of fig. 4 and table 5, it can be seen that the half-life of the wild-type pectinase MarPel at 60 ℃ was 4.76min and the half-life of the pectinase mutant MarPel L262S was 15.75min; thus, compared to the wild type, the mutant pectinase has better kinetic stability at 60 ℃ and a significantly longer half-life. The results in Table 5 show that the pectinase mutant MarPel L S is more structurally stable than the wild-type pectinase MarPel, with a melting temperature of 49.7℃in 50mM phosphate buffer, increased by about 3 ℃. Thus, the thermal stability of pectinase mutant MarPel L S is significantly improved compared to the wild-type enzyme.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A pectase mutant is characterized in that the amino acid sequence of the pectase mutant is shown as SEQ ID NO. 3.
2. Use of the pectase mutant of claim 1 in pectin degradation or paper making.
3. The coding gene of the pectase mutant is characterized in that the nucleotide sequence of the coding gene is shown as SEQ ID NO. 4.
4. Use of a gene encoding the pectinase mutant of claim 2 in pectin degradation or paper making.
5. The recombinant expression vector inserted with the coding gene of the pectase mutant is characterized in that the nucleotide sequence of the coding gene is shown as SEQ ID NO. 4.
6. The method for preparing the recombinant expression vector according to claim 5, comprising the steps of:
(1) The pectase gene MarPel with the nucleotide sequence shown as SEQ ID NO. 2 is connected to pET28a (+) to obtain a recombinant expression vector pET28a (+) -MarPel;
(2) Using pETDuet-SUMO as a template, using SEQ ID NO. 7 and SEQ ID NO. 8 as primers, and amplifying to obtain a solvoprotein SUMO gene with a nucleotide sequence shown as SEQ ID NO. 6;
(3) Amplifying to obtain linearized pET28a (+) -MarPel by taking pET28a (+) -MarPel as a template and SEQ ID NO 9 and SEQ ID NO 10 as primers;
(4) Connecting the linearized pET28a (+) -MarPel and the proprotein SUMO gene fragment by utilizing GibsonAssembly seamless cloning technology to construct a recombinant expression vector pET28a (+) -SUMO-MarPel;
(5) And amplifying to obtain a pectase mutant expression vector pET28a (+) -SUMO-MarPel L S by using pET28a (+) -SUMO-MarPel as a template and SEQ ID NO. 11 and SEQ ID NO. 12 as primers.
7. Use of the recombinant expression vector of claim 5 in pectin degradation or paper making.
8. An engineered bacterium expressing the recombinant expression vector of claim 5.
9. The method for constructing engineering bacteria as claimed in claim 8, comprising the steps of: the pectase mutant expression vector pET28a (+) -SUMO-MarPel L S prepared in claim 6 is transformed into an escherichia coli expression host ESCHERICHIA COLI BL (DE 3) to obtain the pectase mutant.
10. The use of the engineering bacteria of claim 8 in pectin degradation or paper making.
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