CN111848759A

CN111848759A - Cellulosomal dockerin mutant 36741 with improved activity and application thereof

Info

Publication number: CN111848759A
Application number: CN202010730985.9A
Authority: CN
Inventors: 汪俊卿; 王子睿; 杨翠平; 范翰; 王瑞明; 薛乐; 李楠; 姜彦君; 王大涛; 田文卓
Original assignee: Qilu University of Technology
Current assignee: Qilu University of Technology
Priority date: 2020-07-27
Filing date: 2020-07-27
Publication date: 2020-10-30
Anticipated expiration: 2040-07-27
Also published as: CN111848759B

Abstract

The invention relates to a fibrosome docking protein mutant 36741 with improved activity and application, wherein a fibrosome docking protein mutant 36741 is a fibrosome docking protein DocA, the nucleotide sequence is shown as SEQ ID No.3, and the mutant is subjected to site-directed mutagenesis to obtain a docking protein mutant 36741 with improved activity, and the nucleotide sequence is shown as SEQ ID No. 1; the docking protein can be combined and assembled with the mucin more efficiently, and the method has wide application prospect in the construction process of an extracellular multienzyme system of microorganisms.

Description

Cellulosomal dockerin mutant 36741 with improved activity and application thereof

Technical Field

The invention belongs to the field of genetic engineering and enzyme engineering, and particularly relates to a fibrosome dockerin mutant 36741 with improved activity and application thereof.

Background

The cellulosome is a cellulase system existing in anaerobic organisms, is a multienzyme complex structure formed by multiple cellulases and hemicellulases by virtue of an anchoring-adhesion mechanism, is attached to the bacterial cell wall through cell adhesion protein, and has a molecular weight of 2.0x10⁶～2.5x10⁶Da, the natural cellulose material can be efficiently and thoroughly degraded. The fibroid consists essentially of two parts: dockerin (Doc) containing enzymes or other accessory proteins and fibronectin (cohesin, Coh) containing structural proteins. A fibrosome is a cell surface organelle containing a variety of enzymes and associated cofactors which are linked to the enzymes by specific folding subunits. The enzyme molecule is integrated in the cellulosome [ Lytle B L, Volkman B F, Westler W M, et al. Secondary genomic DNA and calcium-induced folding of the Clostridium thermocellum Domain subunit purified by NMR spectroscopy. Arch Biochem Biophys,2000,379(2): 237-244]Thereby forming the multifunctional module with a supermolecular structure. The fibre-linked domain of the scaffold protein facilitates the binding of the enzyme to the substrate [ Yague E, Beguin P, Aubert J P cellulase-encoding gene CelH of Clostridiumthermocellum.Gene,1990,89:61～67]. The fibrosomes can also form a poly-fibrosome through mutual anchoring effect, and a layer of glycoprotein coat is wrapped to form a protruding structure on the surface of the cell.

Cellulosome tissues various degrading enzymes form multienzyme complexes, thereby efficiently degrading cellulosic materials. The mode of action of this supramolecular structure is key to understanding cellulose degradation and utilization of cellulose resources. Interactions between proteins in the fibrosomes, morphogenesis of the organization structure of the fibrosomes, etc. Researches find that the cellulosome can greatly improve the degradation rate of cellulose (which is several times higher than that of the cellulose degraded by pure cellulase), so that the utilization rate of the cellulose is obviously improved. However, the difference of the cellulosome produced by different cellulose-degrading bacteria is large, mainly due to the difference of the self-assembly modes, the difference of the structures [ luohui, enemy Tianlei, Lei, etc.. the research progress of cellulose anaerobic degradation [ J ]. Chinese biogas, 2008, 26 (2): 3-9].

Cellulosomes are usually present on the cell surface and are degraded by sufficient contact of the cellulose binding units with the cellulose surface. Cellulose is a renewable resource which is found at present in the largest quantity and has high utilization value, but only a small part of cellulose is developed and utilized by human beings, and most of cellulose becomes waste [ wu shui picture, penhui, shore blue ] research progress of crystalline cellulose degrading enzyme [ J ] Anhui agricultural science, 2007, 35 (9): 2532-2534]. The cellulose is extremely difficult to dissolve in water and degrade, and due to the existence of cellulose bodies, the cellulose can be efficiently degraded, so that the cellulose is converted into chemical products and fuels, and the environment and the ecological green sustainable development are realized. Although various cellosome elements are found at present, research and development of a more efficient extracellular self-assembly system and a manufacturing method thereof are still crucial to further application of the cellosome and the elements thereof.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a mutant 36741 of a cellulosome dockerin with improved activity and application thereof.

The technical scheme of the invention

A fibrosome dockerin mutant 36741 is a fibrosome dockerin DocA, the nucleotide sequence is shown in SEQ ID NO.3, and site-directed mutagenesis is performed to obtain a dockerin mutant 36741 with improved activity, and the nucleotide sequence is shown in SEQ ID NO. 1.

A fibrosome docking protein mutant 36741 is a fibrosome docking protein DocA, the amino acid sequence is shown in SEQ ID NO.4, and the mutant carries out site-directed mutagenesis to obtain a docking protein mutant 36741 with improved activity, and the amino acid sequence is shown in SEQ ID NO. 2.

The DocA is derived from Clostridium thermocellum (Clostridia thermocellum) (GenBank:2CCL _ B), the nucleotide sequence of the DocA is shown as SEQ ID NO.3, and the amino acid sequence of the DocA is shown as SEQ ID NO. 4.

D in the above amino acid sequence₄₀VDKNGSINAAD₅₁Mutation to D₄₀TSNNGTINAAD₅₁。

The preparation method of the fibrosome dockerin mutant 36741 comprises the following steps:

carrying out site-directed mutagenesis by taking a nucleotide sequence in the docking protein as shown in SEQ ID No.3 to obtain a docking protein mutant 36741, designing a site-directed mutagenesis primer on the basis of the nucleotide sequence as shown in SEQ ID No.1, carrying out PCR by taking a pET28a (+) vector carrying a cellosome docking protein gene as a template to construct a recombinant mutant plasmid, transforming the mutant plasmid into escherichia coli BL21(DE3), selecting a positive clone for fermentation, collecting thalli after the fermentation is finished, crushing the thalli, and purifying to obtain the cellosome docking protein mutant.

According to the invention, in the preferable preparation method, the dockerin is mutated into a dockerin mutant 36741 by the dockerin with an amino acid sequence shown as SEQ ID No.4, and the amino acid sequence is shown as SEQ ID No. 2.

Preferably, in the above preparation method, after the fermentation is completed, the bacterial cells are collected by centrifugation, disrupted by sonication, and purified by affinity chromatography to obtain the mutant of the fibrosome dockerin.

According to a preferred embodiment of the present invention, the nucleotide sequence of the PCR amplification primer of dockerin mutant 36741 is as follows:

36741-F：TGCCGAT accagcaat AATGGC acc ATTAATGCC SEQ ID NO.7；

36741-R：CATTAAT ggt GCCATT attgctggt ATCGGCACGG SEQ ID NO.8。

the lower case letters in the primer nucleotide sequence are mutation sites.

Further preferably, in the preparation method, the PCR reaction system:

1 μ L of plasmid vector template, 2 μ L of forward mutation primer F, 2 μ L of reverse mutation primer R, 10 μ L of 5 XFastmutation buffer, 1 μ L of Fastmutation DNA Polymerase, ddH₂O34μL。

Further preferably, in the preparation method, the PCR reaction conditions are as follows:

pre-denaturation at 95 ℃ for 2 min; 94 ℃ for 20sec, 55 ℃ for 10sec, 68 ℃ for 3min, 18 cycles; extension was supplemented at 68 ℃ for 5 min.

According to the invention, preferably, after the PCR reaction is completed, the original template in the PCR amplification product needs to be digested by Dnp I enzyme, and after the digestion reaction system is mixed uniformly, the original template is digested for 1 hour at 37 ℃ to obtain the vector to be transformed.

Further preferably, the digestion system:

PCR amplification product 40. mu.L, Dnp I enzyme 1. mu.L.

Further preferably, the transformation of the mutant vector comprises transforming the above-mentioned vector to be transformed into E.coli BL21(DE3) cells, and plating the transformant with a medium containing kanamycin (50. mu.g.mL)^-1) After overnight culture at 37 ℃, single colony is picked out, sequencing and verification are carried out, and positive mutant is screened to obtain recombinant escherichia coli 36741-BL 21.

More preferably, the mutant is expressed and purified by inoculating the strain containing kanamycin (50. mu.g.mL) with the strain whose accuracy was verified as described above^-1) The cultured cells were cultured overnight at 37 ℃ in the liquid LB medium, and then transferred to the liquid LB medium and cultured to OD at 37 ℃₆₀₀When the concentration is approximately equal to 1, 1 mu m.mL is added^-1The IPTG (isopropyl-beta-D-thiogalactoside) is induced for 8 hours at 26 ℃ and 200rpm, and collected after inductionThe bacterial cells of (4) were then resuspended in 2 x PBS buffer, disrupted by ultrasonication, centrifuged at 10000rpm for 10min, and the protein in the supernatant after centrifugation was purified using a nickel ion affinity column and desalted by dialysis using PBS-EP + buffer to obtain purified mutant of fibronectin 36741.

Use of the aforementioned mutant of cellulosome dockerin 36741 for interacting with fibronectin to construct a protein complex.

Preferably, according to the invention, the concentration of calcium ions required in the above-mentioned applications is 10^-4M～10^-2M。

The invention has the beneficial technical effects

DocA is a fibrosome docking protein currently reported to have high docking activity and has been widely used. The invention provides a dockerin mutant 36741 with calcium ion concentration of 5 × 10^-4Docking activity with mucin CohA at M was 4.12 times that of DocA and was found at a calcium ion concentration of 10^-4M～10^-2M shows higher activity in the environment, and the better activity can improve the assembly efficiency and the finishing strength of the cellulosome, thereby having wider application prospect.

Drawings

FIG. 1 is a line graph showing the binding capacity of the mutant fibronectin and fibronectin at different calcium ion concentrations in example 4

Detailed Description

The invention is further illustrated with reference to specific examples, without limiting the scope of protection.

Sources of materials

The vectors DocA-pET28a and CohA-pET28a were extracted from Escherichia coli DH 5. alpha. or stored in the Escherichia coli DH 5. alpha. in the laboratory, which is an important laboratory in microbial engineering, Shandong, university of Qilu Industrial science, and those skilled in the art can construct the vectors according to the prior art or purchase them from the laboratory.

The contents of the examples, which are not specified in specific conditions, were carried out under conventional conditions; the reagents or instruments used are not indicated by the manufacturer, and are all common commercial products.

Example 1

Mutant primer design and mutant vector construction

Primer design of dockerin mutants was performed using dockerin DocA (vector DocA-pET28a, nucleotide sequence shown in SEQ ID NO. 9) ligated to pET28a (+) vector, respectively, as a template (Table one). Wherein the DocA is derived from Clostridium thermocellum (Clostridia thermocellum) (GenBank:2CCL _ B), and the optimization of nucleotide sequence is carried out according to the codon preference of Escherichia coli, the DocA nucleotide sequence is shown as SEQ ID NO.3, and the DocA amino acid sequence is shown as SEQ ID NO. 4.

TABLE-mutant primer Table

Note: lower case letters are mutation sites.

And accurately adding the plasmid DocA-pET28a, the designed mutation primer, the PCR enzyme and the buffer according to the addition amount of each component in the second table to prepare a PCR reaction solution, and carrying out PCR amplification according to the PCR reaction conditions in the third table.

PCR reaction system for epidiaschisis

PCR reaction conditions for the epitrimutation

After the PCR reaction is completed, the original plasmid template methylated by Escherichia coli DH5 alpha in the PCR product needs to be digested by Dnp I enzyme, the digestion reaction system is shown as table four, and after the system is prepared and fully mixed, the mixture is digested for 1h at 37 ℃ to obtain the vector to be transformed.

TABLE IV digestion System

Taking escherichia coli BL21(DE3) competence at-80 ℃ and quickly dissolving the enzyme in ice at 4 ℃, sucking a proper amount of enzyme digestion solution by a micropipette and injecting the enzyme digestion solution into the molten escherichia coli BL21(DE3) competence, uniformly mixing the tube walls of the flickers, accurately heating the mixture for 90sec at 42 ℃, carrying out ice bath for 2min, adding 400 mu L of liquid LB culture medium (1% peptone, 1% yeast extract powder, 0.5% NaCl and the balance of water) into an ultraclean workbench after the ice bath, recovering the mixture in a shaking table at 37 ℃ for about 1h, centrifuging the mixture for 5min at 4000rpm after the recovery is finished, removing 400 mu L of supernatant, and directly coating the rest 200 mu L of the mixture to a solution containing kanamycin (50 mu g. mL)^-1) After overnight culture on the solid medium (1% peptone, 1% yeast extract, 0.5% NaCl, 2% agar, balance water), a single colony was picked up and cultured, and sent to sequencing company for sequencing and verifying the amino acid sequence D in DocA₄₀VDKNGSINAAD₅₁Mutation to D₄₀TSNNGTINAAD₅₁(ii) a Screening out positive clones to obtain recombinant Escherichia coli 36741-BL 21.

Example 2

Inducible expression and purification of muteins

The correctly verified strain was inoculated to a strain containing kanamycin (50. mu.g.mL)^-1) The cultured cells were cultured overnight at 37 ℃ in 50mL of liquid LB medium, and then transferred to a new 50mL of liquid LB medium at 37 ℃ in an amount of 2% by volume, and cultured to OD₆₀₀When the concentration is approximately equal to 1, the mixture is added until the final concentration is 1 mu m.mL^-1The IPTG was placed in a shaker at 26 ℃ and 200rpm for 8 hours, and the induced cells were collected. The induced cells were resuspended in 10mL 2 XPBS buffer, disrupted by ultrasonication, centrifuged at 10000rpm for 10min, and the protein in the supernatant after centrifugation was purified by a nickel ion affinity column and desalted by dialysis using PBS-EP + buffer (from GE). Purified fibrosome dockerin 36741 was obtained.

Example 3

Construction and induced expression of expression vector of fibronectin

Transformation of the CohA-pET28a vector was carried out in the same manner as in example 1 using pET28a (+) vector to which the cellulosome fibronectin CohA was ligated, and similarly transformation of DocA-pET28a vector was prepared to obtain recombinant E.coli DocA-BL21 and CohA-BL21, and inducible expression and purification of genes were carried out in the same manner as in example 2. Purified fibrosome mucin CohA, fibrosome dockerin DocA was obtained. Wherein the cellulosome mucin CohA is derived from a Clostridium thermocellum (Clostridium thermocellum) scaf gene (GenBank: MH049738.1), the nucleotide sequence of the CohA is shown as SEQ ID NO.5, and the amino acid sequence of the CohA is shown as SEQ ID NO. 6.

Example 4

Analysis of binding Capacity of Fibrosomal docking protein and mucin at different calcium ion concentrations

Analyzing the binding capacity between the mutant of the cellosome docking protein and the fibronectin with different calcium ion concentrations by using a biomacromolecule interaction instrument, selecting a proper CM5 chip as an anchoring chip, calculating the approximately required concentration of the protein CohA according to a formula, performing gradient dilution on acetic acid-sodium acetate buffer solutions with different pH values according to the approximately protein concentration to serve as the protein to be anchored, determining the optimal anchoring concentration and pH value according to the anchoring condition, and then anchoring the CohA according to the mucoprotein CohA in Biacore micromolecule application operation manual. The anchored chip was loaded into a molecular interaction apparatus in 1 XPBS-HP + solution (available from GE), the dockerin and mutants were diluted to nearly the same anchoring concentration as the protein CohA, and bound to different concentrations of CaCl₂After standing at 4 ℃ for 30min, the binding was determined on the machine and judged according to the AbsResp value (i.e., the binding interaction intensity value), and the results are shown in fig. 1 (table v). The results indicate that dockerin mutant 36741 had calcium ion concentrations greater than or equal to 10, relative to unmutated dockerin DocA^-4The binding capacity to the mucin CohA is obviously enhanced when M is used, wherein the calcium ion concentration is 5X 10^-4The docking activity of the binding protein CohA at M was 4.12 times that of DocA.

Analysis of binding Capacity of Fivessilebody dockerin mutant and mucin at different calcium ion concentrations

SEQUENCE LISTING

<110> university of Qilu Industrial science

<120> cellulosome dockerin mutant 36741 with improved activity and application thereof

<160>9

<170>PatentIn version 3.5

<210>1

<211>261

<212>DNA

<213> Artificial sequence

<400>1

atggtgctgc tgggtgacgt gaatggtgac ggtaccatta atagcaccga tctgaccatg 60

ctgaaacgtt ctgttctgcg tgccattacc ctgaccgatg atgccaaagc ccgtgccgat 120

accagcaata atggcaccat taatgccgcc gatgttctgc tgctgtctcg ctatctgctg 180

cgtgttattg ataaaggagg aggcggctcg ggaggaggcg gctcgggagg aggcggctcg 240

catcatcatc atcatcatta a 261

<210>2

<211>86

<212>PRT

<213> Artificial sequence

<400>2

Met Val Leu Leu Gly Asp Val Asn Gly Asp Gly Thr Ile Asn Ser Thr

1 5 10 15

Asp Leu Thr Met Leu Lys Arg Ser Val Leu Arg Ala Ile Thr Leu Thr

20 25 30

Asp Asp Ala Lys Ala Arg Ala Asp Thr Ser Asn Asn Gly Thr Ile Asn

35 40 45

Ala Ala Asp Val Leu Leu Leu Ser Arg Tyr Leu Leu Arg Val Ile Asp

50 55 60

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

65 70 75 80

His His His His His His

85

<210>3

<211>261

<212>DNA

<213> Artificial sequence

<400>3

atggtgctgc tgggtgacgt gaatggtgac ggtaccatta atagcaccga tctgaccatg 60

ctgaaacgtt ctgttctgcg tgccattacc ctgaccgatg atgccaaagc ccgtgccgat 120

gtggataaaa atggcagcat taatgccgcc gatgttctgc tgctgtctcg ctatctgctg 180

cgtgttattg ataaaggagg aggcggctcg ggaggaggcg gctcgggagg aggcggctcg 240

catcatcatc atcatcatta a 261

<210>4

<211>86

<212>PRT

<213> Artificial sequence

<400>4

Met Val Leu Leu Gly Asp Val Asn Gly Asp Gly Thr Ile Asn Ser Thr

1 5 10 15

Asp Leu Thr Met Leu Lys Arg Ser Val Leu Arg Ala Ile Thr Leu Thr

20 25 30

Asp Asp Ala Lys Ala Arg Ala Asp Val Asp Lys Asn Gly Ser Ile Asn

35 40 45

Ala Ala Asp Val Leu Leu Leu Ser Arg Tyr Leu Leu Arg Val Ile Asp

50 55 60

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

65 70 75 80

His His His His His His

85

<210>5

<211>516

<212>DNA

<213> Artificial sequence

<400>5

atgagcgacg gtgtggtggt ggaaattggc aaagtgaccg gtagcgtggg taccaccgtg 60

gaaattccgg tgtactttcg cggtgttccg agcaaaggta tcgccaactg cgattttgtt 120

ttccgctacg atccgaacgt gctggaaatc atcggcatcg atccgggtga catcatcgtg 180

gacccgaatc cgaccaagag cttcgatacc gccatttacc cggaccgcaa aatcattgtg 240

ttcctgttcg cagaggatag cggtaccggt gcctatgcca tcaccaaaga tggcgtgttc 300

gcaaagattc gcgcaaccgt gaaaagtagc gcaccgggct acattacctt tgacgaggtg 360

ggtggctttg ccgataacga tctggtggaa cagaaggtga gcttcattga cggtggcgtt 420

aacgtgggta atgccacacc gaccaagggt ggaggaggcg gctcgggagg aggcggctcg 480

ggaggaggcg gctcgcatca tcatcatcat cattaa 516

<210>6

<211>171

<212>PRT

<213> Artificial sequence

<400>6

Met Ser Asp Gly Val Val Val Glu Ile Gly Lys Val Thr Gly Ser Val

1 510 15

Gly Thr Thr Val Glu Ile Pro Val Tyr Phe Arg Gly Val Pro Ser Lys

20 25 30

Gly Ile Ala Asn Cys Asp Phe Val Phe Arg Tyr Asp Pro Asn Val Leu

35 40 45

Glu Ile Ile Gly Ile Asp Pro Gly Asp Ile Ile Val Asp Pro Asn Pro

50 55 60

Thr Lys Ser Phe Asp Thr Ala Ile Tyr Pro Asp Arg Lys Ile Ile Val

65 70 75 80

Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile Thr Lys

85 90 95

Asp Gly Val Phe Ala Lys Ile Arg Ala Thr Val Lys Ser Ser Ala Pro

100 105 110

Gly Tyr Ile Thr Phe Asp Glu Val Gly Gly Phe Ala Asp Asn Asp Leu

115 120 125

Val Glu Gln Lys Val Ser Phe Ile Asp Gly Gly Val Asn Val Gly Asn

130 135 140

Ala Thr Pro Thr Lys Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Gly Gly Gly Gly Ser His His His His His His

165 170

<210>7

<211>34

<212>DNA

<213> Artificial sequence

<400>7

tgccgatacc agcaataatg gcaccattaa tgcc 34

<210>8

<211>35

<212>DNA

<213> Artificial sequence

<400>8

cattaatggt gccattattg ctggtatcgg cacgg 35

<210>9

<211>5528

<212>DNA

<213> Artificial sequence

<400>9

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatatacc atggtgctgc tgggtgacgt gaatggtgac 5100

ggtaccatta atagcaccga tctgaccatg ctgaaacgtt ctgttctgcg tgccattacc 5160

ctgaccgatg atgccaaagc ccgtgccgat gtggataaaa atggcagcat taatgccgcc 5220

gatgttctgc tgctgtctcg ctatctgctg cgtgttattg ataaaggagg aggcggctcg 5280

ggaggaggcg gctcgggagg aggcggctcg catcatcatc atcatcatta agaattcgag 5340

ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc 5400

tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc 5460

ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat 5520

atccggat 5528

Claims

1. A fibrosome dockerin mutant 36741 is characterized in that a fibrosome dockerin DocA with a nucleotide sequence shown in SEQ ID NO.3 is subjected to site-directed mutagenesis to obtain a dockerin mutant 36741 with improved activity, wherein the nucleotide sequence is shown in SEQ ID NO. 1.

2. A fibrosome docking protein mutant 36741 is a fibrosome docking protein DocA, the amino acid sequence is shown in SEQ ID NO.4, and the mutant carries out site-directed mutagenesis to obtain a docking protein mutant 36741 with improved activity, and the amino acid sequence is shown in SEQ ID NO. 2.

3. A method of making the dockerin mutant 36741 of any one of claims 1-2, comprising the steps of:

4. The method of claim 3, wherein the dockerin is mutated to dockerin mutant 36741 from dockerin having the amino acid sequence shown in SEQ ID No.4 and the amino acid sequence shown in SEQ ID No. 2.

5. The method according to claim 3, wherein the strain is collected by centrifugation after the fermentation is completed, and the strain is subjected to ultrasonication and affinity chromatography for purification to obtain the mutant of the fibrosome dockerin.

6. The method of claim 3, wherein the nucleotide sequence of the PCR amplification primer of dockerin mutant 36741 is as follows:

36741-F is shown in SEQ ID NO. 7;

36741-R is shown in SEQ ID NO. 8.

7. The method according to claim 6, wherein the PCR reaction system comprises:

8. The method of claim 7, wherein the PCR reaction conditions are:

pre-denaturation at 95 ℃ for 2 min; 94 ℃ for 20sec, 55 ℃ for 10sec, 68 ℃ for 3min, 18 cycles; supplementary extension at 68 ℃ for 5 min;

preferably, after the PCR reaction is finished, the original template in the PCR amplification product needs to be digested by Dnp I enzyme, and after a digestion reaction system is uniformly mixed, the original template is digested for 1 hour at the temperature of 37 ℃ to obtain a vector to be transformed;

preferably, the digestion system:

PCR amplification product 40. mu.L, Dnp I enzyme 1. mu.L;

preferably, the transformation of the mutant vector is carried out by transforming the above-mentioned vector to be transformed into E.coli BL21(DE3) cells, plating the transformant with a transformant containing kanamycin (50. mu.g.mL)^-1) After overnight culture at 37 ℃ on the LB solid culture medium, selecting a single colony, carrying out sequencing verification, and screening a positive mutant to obtain recombinant Escherichia coli 36741-BL 21;

preferably, the mutant is expressed and purified by inoculating the correctly verified strain containing kanamycin (50. mu.g.mL)^-1) The cultured cells were cultured overnight at 37 ℃ in the liquid LB medium, and then transferred to the liquid LB medium and cultured to OD at 37 ℃₆₀₀When the concentration is approximately equal to 1, 1 mu m.mL is added^-1IPTG (isopropyl thiogalactoside) at 26 DEG CInducing the strain for 8 hours under the condition of 200rpm, collecting the induced strain, then re-suspending the strain by using 2 XPBS buffer solution, crushing the strain by using ultrasonic waves, centrifuging the strain for 10 minutes at 10000rpm, purifying the protein in the supernatant after centrifugation by using a nickel ion affinity column, and dialyzing and desalting the protein by using PBS-EP + buffer solution to obtain the purified mutant of the cellulosome dockerin 36741.

9. Use of the dockerin mutant 36741 of any one of claims 1-2 to interact with mucin to construct a protein complex.

10. The use of claim 9, wherein the desired calcium ion concentration is 10^-4M～10^-2M。