CN109456930A - One plant for rapidly purifying the engineering bacteria and purification process of the preparation pure enzyme of enterococcus faecium glutamate decarboxylase native enzyme - Google Patents
One plant for rapidly purifying the engineering bacteria and purification process of the preparation pure enzyme of enterococcus faecium glutamate decarboxylase native enzyme Download PDFInfo
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- CN109456930A CN109456930A CN201811557855.9A CN201811557855A CN109456930A CN 109456930 A CN109456930 A CN 109456930A CN 201811557855 A CN201811557855 A CN 201811557855A CN 109456930 A CN109456930 A CN 109456930A
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 201
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 200
- 108091022930 Glutamate decarboxylase Proteins 0.000 title claims abstract description 131
- 241000194031 Enterococcus faecium Species 0.000 title claims abstract description 69
- 241000894006 Bacteria Species 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- 102000008214 Glutamate decarboxylase Human genes 0.000 title claims abstract description 11
- 238000000746 purification Methods 0.000 title abstract description 16
- 229920002101 Chitin Polymers 0.000 claims abstract description 75
- 239000000872 buffer Substances 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 34
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- 238000003776 cleavage reaction Methods 0.000 claims abstract description 28
- 230000007017 scission Effects 0.000 claims abstract description 27
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- 238000010521 absorption reaction Methods 0.000 claims abstract description 16
- 238000010008 shearing Methods 0.000 claims abstract description 7
- 102100035902 Glutamate decarboxylase 1 Human genes 0.000 claims description 114
- 241000588724 Escherichia coli Species 0.000 claims description 37
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 20
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- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 claims description 16
- 230000017730 intein-mediated protein splicing Effects 0.000 claims description 15
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- PMUNIMVZCACZBB-UHFFFAOYSA-N 2-hydroxyethylazanium;chloride Chemical compound Cl.NCCO PMUNIMVZCACZBB-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01015—Glutamate decarboxylase (4.1.1.15)
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Abstract
The invention discloses one plant for rapidly purifying the engineering bacteria and purification process of the preparation pure enzyme of enterococcus faecium glutamate decarboxylase native enzyme.The present invention provides the engineering bacterias for rapidly purifying enterococcus faecium GAD native enzymeEscherichia coli(DH5 α-LNSF2), the engineering bacteria were preserved in Guangdong Province's Culture Collection, deposit number on September 18th, 2018 are as follows: GDMCC No.60446.The pure enzyme of enterococcus faecium GAD native enzyme can be quickly produced using the engineering bacteria, method is that the shearing of self cleavage buffer is added, can be obtained the pure enzyme of enterococcus faecium GAD native enzyme using the affine absorption CBM-DnaB-GAD crude enzyme liquid of chitin.This method only needs step purifying to can be obtained the pure enzyme of enterococcus faecium GAD native enzyme, has many advantages, such as that quick, efficient, at low cost, yield is high, is with a wide range of applications in terms of the industrialized production of the pure enzyme of enterococcus faecium GAD native enzyme.
Description
Technical field
The invention belongs to technical field of food biotechnology, more particularly, to one plant for rapidly purifying preparation enterococcus faecium
The engineering bacteria and purification process of the pure enzyme of glutamate decarboxylase native enzyme.
Background technique
Glutamate decarboxylase (glutamate decarboxylase, GAD, EC4.1.1.15) is exclusively catalyzed L- paddy ammonia
Decarboxylation occurs for acid (L-glutamic acid, L-Glu) α-carboxyl, in γ-aminobutyric acid (γ-aminobutyric
Acid, GABA) have in terms of biosynthesis and the fractionation of chiral material DL- glutamic acid (DL-glutamic acid, DL-Glu)
Important application (Brazilian Journal ofMicrobiology, 2012,43 (4): 1230-1241；AminoAcids,
2016,48(11):2519-2531；Chemistry and bioengineering, 2013,30 (11): 55-56.).Since lactic acid bacteria has preferably
Safety, convenient for applying in fermented food, lactic acid bacteria GAD receives very big concern, it has been found that Lactobacillus brevis
(Lactobacillus brevis) (LWT-Food Sci Technol, 2016,67:22-26), lactobacillus paracasei
(Lactobacillusparacasei) (Food Microbiol 2005,22:497-504), Lactobacillus rhamnosus
(Lactobacillus rhamnosus)(Braz J Microbiol 2013；44:183-187), Lactobacillus saki
(Lactobacillus sakei)(J Microbiol Biotechnol2015；25:696-703), saliva chain coccus thermophilous Asia
Kind (Streptococcus salivarius ssp.thermophilus) (Food Science, 2011,32 (1): 162-167), penta
Sugar-tablet coccus (Pediococcus pentosaceus) (hubei agricultural science, 2010,49 (6): 1450-1453), enterococcus faecium
(Enterococcus faecium) (Food Science, 2018,39 (4): 90-98) and lactobacillus plantarum
(Lactobacillusplantarum) microorganisms such as (Microbiol Biotechnol Lett, 2015,43:300-305) are equal
With GAD.
In biocatalysis field, zymologic property is the important evidence that technological parameter determines.Shadow of the thick enzyme due to heteroproteins
It rings, the zymologic property of measurement usually will appear relatively large deviation, and technological parameter is caused deviation, catalytic efficiency decline occur.While by
There are other enzymes or downstream metabolic enzyme in thick enzyme, it is also easy to produce Reverse transcriptase, product component complicates or the converted product of enzyme
Further metabolism, purpose product accumulation are reduced.In addition, some analyses are also required with enzyme with pure enzyme preparation.Therefore, the purifying of enzyme exists
Biocatalysis field has consequence, and the purifying process of enzyme is an important factor for restricting enzyme preparation production cost.
Microorganism GAD is endocellular enzyme, protein ingredient complex, isolates and purifies very difficult, is industrialized using wild strain
It is extremely low to prepare the pure enzyme yield of GAD, it is with high costs.For example, the purifying of saliva chain coccus thermophilous subspecies GAD is needed through ammonium persulfate point
Grade precipitating, isoelectric precipitation, DEAE-SephadexA-50 ion-exchange chromatography, HiPrep16/10Phenyl FF hydrophobic chromatography
With the 5 steps purifying such as Sephadex G-100 gel chromatography could obtain the pure GAD of electrophoresis (Amino Acids, 2008,34 (3):
473-478)；Lactococcus lactis GAD need ammonium sulfate precipitation, 3 DEAE-Sepharose CL-6B chromatography,
The means such as Sephacryl S-200 gel filtration could obtain the pure GAD of electrophoresis (Wuxi Light Industry Univ.'s journal, 2004,23 (3):
79-84).It can be seen that the natural enzyme purification difficulty of wild strain GAD is big, condition require high, the rate of recovery is low, purification not
Height, purifying cost are very high, and existing GAD purification process is not suitable for industrial-scale production.
Synthase expression vectors are melted using technique for gene engineering clone's purpose enzyme gene and affinity tag building, are transformed into large intestine
It is expressed in the expression systems such as bacillus, yeast, then utilizes affiliation carrier purified fusion enzyme, be a kind of skill of efficiently purifying enzyme
Art.For example, by by the gad gene of Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) with
PMD19-T simple vector constructs expression plasmid, and expression is to Ni in Escherichia coli (E.coli) DH5 ɑ2+-Chelating
Sepharose fast flow affinity fusion GAD only can efficiently purify recombination by step column chromatography
GAD (food and biotechnology journal, 2012,31 (3): 302-306).But the band affinity tag constructed by this method
Recombinase, still with affinity tag after affiliation carrier elutes, affine peptide fragment cannot be obtained with GAD peptide chain separation
Purifying enzyme is with the fusion enzyme for having affine peptide fragment, is not native enzyme, and Ni2+-Chelating sepharose fast
Flow carrier is expensive, safety is poor, simultaneously because the influence of affine peptide fragment, merging enzyme and native enzyme may deposit on zymologic property
In different, therefore, the new technology of native enzyme can be obtained by ad hoc approach shear removal affinity tag by needing research and development.
Most of cellulases include three structural domains: cellulose binding domain (cellulose-binding
Domain, CBD), flexible connecting area and catalyst structure domain (J Biol Chem, 1992,267:6743-6749；J
Bacteriol,1993,175:5762-5768；Bioresour Technol,2011,102:2910-2915).Cellulose is opposite
Cheaply, chemical inertness has lower non-specific affinity to most protein, commercially can be with a variety of different shapes
Formula exists.It therefore, can be with cellulose using the cellulose binding module of CBD (cellulose-binding module, CBM)
CBM can develop as affinity tag by special and irreversible combination characteristic, and construction of fusion protein is affine with cellulose generation
Absorption prepares immobilised enzymes (Int J Mol Sci, 2012,13:358-368；Biotechnol Prog,2009,25:68-74；
J Ind Microbiol Biotechnol,2008,35:1455-1463).The technology is concentrated mainly at present utilizes building band
The fusion GAD (i.e. CBM-GAD) of CBM affinity tag and in terms of realizing the immobilization of GAD.Although also achieving fusion after immobilization
The separation of CBM-GAD and other albumen, but how to separate GAD with CBM and cellulose, still lack possible technique.Only protecting
GAD and CBM are efficiently separated under the premise of staying GAD active, the purifying purpose of GAD native enzyme could be finally realized, to obtain
The pure enzyme of GAD native enzyme.
There are greatest differences in structure by the GAD of separate sources, even if same microorganism belonging to genus, the subunit of the GAD of different strains
Composition and molecular weight etc. all have greatest differences.For example, Escherichia coli GAD contains 6 identical subunits,
Molecular weight is 53kDa (Biochemistry, 1970,9 (2): 226-232)；Streptococcus pneumonia (Streptococcus
Pneumonia) GAD (59% is similar, and 28% is consistent) similar to mammal GAD65 molecular weight subunit (54kDa) and sequence
(FEMS Microbiol Lett,1995,133(1-2):113-138)；Neuraspora crassa (Neurospora crassa) GAD
The measurement molecular weight of subunit is about 33kDa, kinetic property (The Journal OfBiological similar to E.coli GAD
Chemistry,1991,266(8):5135-5139)；Bacillus perfringens (Clostridiumperfringens) GAD molecule
Amount is 290kDa (Biochem J, 1970,118:135-141)；Lactobacillus brevis (Lactobacillus brevis) GAD has two
A subunit, molecular weight subunit are 60kDa (Biosci Biotech Biochem, 1997,61 (7): 1168-1171)；Lactic acid cream
Only one subunit of the GAD of coccus lactic acid subspecies (Lactococcus lactis subsp.lactis), molecular weight subunit is about
For 54kDa (Microbiology, 1999,145:1375-1380), although however Lactococcus lactis (Lactococcus
Lactis) the GAD of SYFS1.009 equally also only one subunit, but molecular weight subunit is 65kDa (Xu Jianjun, doctorate
Paper, Southern Yangtze University, 2 months 2004), illustrate even if there is also differents between the GAD of the different subspecies of same species of microorganism.
Therefore, it has been reported that be not necessarily suitable for enterococcus faecium using CBM as the method for affinity tag immobilized microorganism enzyme
GAD needs the certain enzyme for specified microorganisms to conduct a research, inquires into suitable method.
By upper analysis as it can be seen that although theoretically special, the irreversible binding characteristic using CBM and cellulose, passes through
The approach of the fusion protein of building and expression with CBM label realizes the separation of CBM- fusion protein, but still has in practice
It is many can not anticipation property:
Can (1) the gadB gene of purpose bacterial strain be cloned into? there are diversity for microorganism gadB gene, if can use
Is technology of the same race expressed in different escherichia expression systems?
(2) whether the CBM-GAD fusion enzyme transfer vector plasmid constructed scientific feasible?
(3) can the CBM-GAD fusion enzyme transfer vector plasmid that constructed high efficient expression in escherichia expression system
Does CBM-GAD merge enzyme?
Can (4) for CBM with after GAD amalgamation and expression, CBM change the foldable structure, enzyme activity and zymologic property of GAD?
(5) after merging the affine absorption of enzyme with CBM-GAD using cellulose, how GAD native enzyme to be cut with CBM and cellulose
Is cutting to obtain GAD native enzyme? what method or measure should be taken in terms of CBM-GAD transfer vector plasmid building?
(6) after GAD native enzyme and CBM and cellulose are cut off, if can occur to fold and inactivate again?
It can be seen that there is still a need for solve above-mentioned key according to specific microorganism GAD situation to ask in the research and development of technology
Topic.
In patent application CN201710510283.8, inventor from pickles separation screening to high GAD vigor
Enterococcusfaecium LNSF2 wild strain, and patent culture presevation has been carried out to the bacterial strain, it is preserved in the micro- life in Guangdong Province
Object Culture Collection Center, deposit number are as follows: GDMCC NO.60203.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the natural enzyme purification difficulty of wild strain GAD greatly, condition requires height,
The GAD native enzyme rate of recovery is low, purification is not high and purifying cost is very high and existing GAD purification process is not suitable for industry
The problem of changing large-scale production provides one plant of engineering bacteria and purifying for rapidly purifying the preparation pure enzyme of enterococcus faecium GAD native enzyme
Method.
The present invention by with cellulose binding module gene cbm3, include peptide gene dnaB, Enterococcus
The GAD gene gadB of faecium LNSF2 and the pRPOCDN expression vector voluntarily constructed construct jointly can high efficient expression band fiber
The enterococcus faecium GAD of plain binding modules (CBM) and intein (DnaB) merges the engineering bacteria of enzyme (CBM-DnaB-GAD), fermentation training
Preparation CBM-DnaB-GAD is supported, the affine absorption of specificity is carried out by CBM and chitin (chitin, CHI), forms chitin-
The insoluble immobilised enzymes of CBM-DnaB-GAD is washed the foreign protein of removal adherency with buffer, can be obtained pure chitin-CBM-
DnaB-GAD immobilised enzymes recycles the characteristic of DnaB, is handled using self cleavage liquid, and GAD is cut out to come, will by filtering
GAD is separated with solid-state chitin-CBM-DnaB, finally realizes efficiently purifying, prepares the pure enzyme of enterococcus faecium GAD native enzyme.
An object of the present invention is to provide one plant of engineering that can be used for rapidly purifying preparation enterococcus faecium GAD native enzyme
Bacterium.
The second object of the present invention is to provide a kind of method for constructing above-mentioned engineering bacteria.
The third object of the present invention is to provide above-mentioned engineering bacteria and is rapidly purifying enterococcus faecium GAD native enzyme or preparing dung intestines
Application in the pure enzyme of coccus GAD native enzyme.
The fourth object of the present invention, which is to provide, utilizes the quick pure of the above-mentioned engineering bacteria preparation pure enzyme of enterococcus faecium GAD native enzyme
Change method.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The present invention is with stress induced promoter PrpoS, cellulose binding module CBM coded sequence cbm3, intein DnaB
GAD gene gadB and T7 the terminator sequence recombination to construct of coded sequence dnaB, Enterococcusfaecium LNSF2
PRPOCDN-efagadB recombinant plasmid, Transformed E .coli DH5 ɑ construct engineering bacteria Escherichia coli (DH5 α-
LNSF2), Escherichia coli (the DH5 α-of enzyme is merged by cultivating and collecting high efficient expression CBM-DnaB-GAD
LNSF2) thallus, ultrasonic disruption extracts CBM-DnaB- under ice-water bath in disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution
GAD merges enzyme crude enzyme liquid, then mixes the affine absorption of progress with the chitin being filtered dry and form chitin-CBM-DnaB-GAD, collects
And chitin-CBM-DnaB-GAD is washed sufficiently with disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution, self cleavage buffering is then added
Liquid carries out self cleavage reaction, and filter liquor is the pure enzyme of enterococcus faecium GAD native enzyme.
Promoter PrpoS (743bp) in the pRPOCDN plasmid constructed in the present invention contains RpoS transcriptional control system
5'- non-translational region, this region plays an important role in response of the bacterium to stress conditions；Promoter PrpoS independent of
Any inducer additionally added can effectively drive foreign protein in the expression of logarithmic growth phase；Using independent of luring
The promoter driving protein expression for leading agent advantageously reduces cost；CBM is a kind of effective Separation of Proteins label, energy and crust
It is known as the absorption of specificity, is used for the affine absorption of recombinant protein in the present invention；DnaB is a kind of intein, can be used for parent
With the excision of label, to obtain the pure enzyme of enterococcus faecium GAD native enzyme.
A kind of method the present invention provides building for rapidly purifying the engineering bacteria of enterococcus faecium GAD native enzyme, including
Following steps:
S11. GAD the gene gadB, construction recombination plasmid pMD- of Enterococcusfaecium LNSF2 are cloned
efagadB；
S12. building includes stress induced promoter PrpoS, cellulose binding module CBM coded sequence cbm3, includes
The pRPOCDN expression vector of Peptide D naB coded sequence dnaB, T7 terminator sequence；
S13. using pMD-efagadB as template amplification gadB gene, using the carrier that pRPOCDN is linearized as template amplification,
Construction recombination plasmid pRPOCDN-efagadB；
S14. by the competent cell of recombinant plasmid pRPOCDN-efagadB conversion Escherichia coli, building expression CBM-
The engineering bacteria Escherichia coli (DH5 α-LNSF2) of DnaB-GAD fusion enzyme.
Wherein, CBM is cellulose binding module, and DnaB is intein, and GAD is glutamate decarboxylase.
Further, in one preferably embodiment, the competent cell of the Escherichia coli is Escherichia
Coli DH5 ɑ competent cell, i.e., using Escherichia coli DH5 ɑ as the table of recombinant plasmid pRPOCDN-efagadB
Up to system.
The engineering bacteria Escherichia coli (DH5 α-LNSF2) was preserved in the micro- life in Guangdong Province on September 18th, 2018
Object Culture Collection Center, deposit number are as follows: GDMCC No.60446, preservation address are as follows: the compound of Xianlie Middle Road, Guangzhou City 100
5 building, No. 59 building.
The engineering bacteria Escherichia coli (DH5 α-LNSF2) are as follows: containing by stress induced promoter PrpoS,
The gene gadB of CBM coded sequence cbm3, intein DnaB coded sequence dnaB, Enterococcusfaecium LNSF2GAD
With pRPOCDN-efagadB recombinant plasmid made of T7 terminator sequence recombination to construct.
The total length of the pRPOCDN-efagadB recombinant plasmid is 5652bp, inserts cellulose before gadB gene
Binding modules gene cbm3, peptide gene dnaB is included, fusion overall length is 2409bp, melts synthase full-length 802aa, Theoretical molecular
Amount is about 90867D；GadB gene length is 1401bp, and amino acid number 466aa, theoretical molecular weight is about 53710D；
The engineering bacteria Escherichia coli (DH5 α-LNSF2) can high efficient expression CBM-DnaB-GAD fusion enzyme.
The bacterial strain of the enterococcus faecium wild strain is Enterococcusfaecium LNSF2, and separation screening is from pickles,
Guangdong Province's Culture Collection, deposit number are preserved on June 19th, 2017 are as follows: GDMCC No.60203 is protected
Hide address are as follows: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100；Gene gadB source bacterial strain of the bacterial strain as GAD.
As a kind of preferable embodiment, the side for rapidly purifying the engineering bacteria of enterococcus faecium GAD native enzyme is constructed
Method, comprising the following steps:
The GAD gene gadB of S11.Enterococcusfaecium LNSF2 is cloned: using efa-1, efa-2 as primer, from
Amplification obtains gadB gene, purifying, with pMD19-T Simple in Enterococcusfaecium LNSF2 genome
Vector connection, construction recombination plasmid pMD-efagadB are converted, sequencing；
S12. coli expression carrier pRPOCDN is constructed: building pRPOCDN plasmid is carried out based on pUC57 plasmid,
Include stress induced promoter PrpoS, cellulose binding module CBM coded sequence cbm3, intein DnaB coded sequence
DnaB, T7 terminator sequence；
S13. recombinant plasmid pRPOCDN-efagadB construct: using pRPOCDN as template, design primer Infu-22 and
Infu-27, amplification prepare linearized vector pRPOCDN；Design primer Infu-13 and Infu-24, using pMD-efagadB as mould
Plate expands gadB gene, purifying；It carries out linearized vector pRPOCDN and the clone pure gadB gene obtained to be assembled into recombination matter
Grain pRPOCDN-efagadB；
S14. engineering bacteria Escherichia coli (DH5 α-LNSF2) is constructed: by recombinant plasmid pRPOCDN-efagadB
Transformed E .coli DH5 ɑ competent cell, the engineering bacteria Escherichia coli of building expression CBM-DnaB-GAD fusion enzyme
(DH5α-LNSF2)；
Wherein, CBM is cellulose binding module, and DnaB is intein, and GAD is glutamate decarboxylase.
Correspondingly, the engineering bacteria Escherichia coli (DH5 α-LNSF2) is rapidly purifying enterococcus faecium GAD days
Application in right enzyme or the preparation pure enzyme of enterococcus faecium GAD native enzyme, also within protection scope of the present invention.
The present invention also provides the method for using above-mentioned engineering bacteria to rapidly purify the preparation pure enzyme of enterococcus faecium GAD native enzyme,
The following steps are included:
S1. under conditions of ice-water bath, it is crushed Escherichia coli (DH5 α-LNSF2) thallus in buffer, mentions
CBM-DnaB-GAD is taken to merge enzyme crude enzyme liquid；
S2.CBM-DnaB-GAD fusion enzyme crude enzyme liquid is mixed and stirred for the chitin being filtered dry, and affine absorption occurs and is formed
Chitin-CBM-DnaB-GAD；
S3. chitin-CBM-DnaB-GAD is washed, the shearing of self cleavage buffer, filtering is added, obtained filter liquor is
The pure enzyme of enterococcus faecium GAD native enzyme.
The CBM is cellulose binding module, and DnaB is intein, and GAD is glutamate decarboxylase.
Preferably, self cleavage buffer described in step S3 is disodium hydrogen phosphate-sodium dihydrogen phosphate containing NaCl and EDTA
Buffer.
Preferably, the concentration of self cleavage buffer described in step S3 is 0.2~0.3mol/L.
It is highly preferred that the concentration of self cleavage buffer described in step S3 is 0.2mol/L.
Preferably, the pH of self cleavage buffer described in step S3 is 5.5~6.5.
It is highly preferred that the pH of self cleavage buffer described in step S3 is 6.5.
Preferably, final concentration of 0.4~0.6mol/L of the NaCl.
It is highly preferred that the final concentration of 0.5mol/L of the NaCl.
Preferably, final concentration of 0.5~3mmol/L of the EDTA.
It is highly preferred that the final concentration of 1mmol/L of the EDTA.
Preferably, the temperature of shearing described in step S3 is 20~32 DEG C.
It is highly preferred that the temperature of shearing described in step S3 is 30 DEG C.
Preferably, the time of shearing described in step S3 is 6~15h.
It is highly preferred that the time 12h of shearing described in step S3.
Preferably, chitin described in step S2 is unmodified, modified through concentrated phosphoric acid or is modified through NaOH/ urea mixed solution
Chitin.
It is highly preferred that chitin described in step S2 is the chitin modified through concentrated phosphoric acid.
The method of modifying of the chitin modified through concentrated phosphoric acid are as follows: be slowly added to while stirring in chitin ice-cold dense
Phosphoric acid, ice bath, stirring to transparence, then ice water is added while stirring, it is centrifuged, chitin precipitating is collected, after washing using ice water
It is resuspended in distilled water again, then uses Na2CO3Solution adjusts pH to 6~7, filters and removes liquid, then is sufficiently washed with distilled water
The chitin modified through concentrated phosphoric acid can be obtained in chitin.
The method of modifying through NaOH/ urea mixed solution modified chitin are as follows: mix chitin with NaOH/ urea
Solution mixing, repeats freeze thawing to chitin into transparence, ice water is added while stirring, is centrifuged, and collects chitin precipitating, uses ice water
It is resuspended in distilled water after washing, then adjusts pH to 6~7 with HCl solution, filtered and remove liquid, then sufficiently washed with distilled water
Chitin is washed, the chitin modified through NaOH/ urea mixed solution can be obtained.
Preferably, buffer described in step S1 is disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution.
It is highly preferred that the concentration of buffer described in step S1 is 10~100mmol/L.
It is further preferred that the concentration of buffer described in step S1 is 20mmol/L.By disodium hydrogen phosphate-di(2-ethylhexyl)phosphate
The concentration of hydrogen sodium buffer is extracted to CBM-DnaB-GAD fusion enzyme extract and CBM-DnaB-GAD merges enzyme and chitin
The influence of affine adsorption capacity is studied, and discovery disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution concentration is 20mmol/L, ionic strength
Low, the fusion enzyme of extraction is more excellent using Tris-HCl buffer to the affine adsorption capacity ratio of chitin.
It is highly preferred that the pH of buffer described in step S1 is 8.
Preferably, the method being crushed described in step S1 are as follows: the ultrasonic disruption in ice-water bath.
Preferably, CBM-DnaB-GAD described in step S2 merges enzyme crude enzyme liquid volume (mL) and the crust quality being filtered dry
The ratio for measuring (g) is 4 ︰ 1.
It is extracted using disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution as CBM-DnaB-GAD fusion enzyme crude enzyme liquid and is cut certainly
Liquid buffer is cut, at low cost, highly-safe, the shortcomings that overcoming Tris-HCl buffer.
In addition, it is natural to rapidly purify preparation enterococcus faecium GAD using above-mentioned engineering bacteria as a kind of preferable embodiment
The method of the pure enzyme of enzyme, comprising the following steps:
The expression of S1.CBM-DnaB-GAD fusion enzyme: by Escherichia coli (DH5 α-LNSF2) access LB culture
It in base, is placed in shaken cultivation case and cultivates, high efficient expression CBM-DnaB-GAD merges enzyme；
S2. extract CBM-DnaB-GAD and merge enzyme crude enzyme liquid: by the fermentation liquid to be fermented with LB culture medium, thalline were collected by centrifugation,
It is dispersed with stirring washing thalline, thalline were collected by centrifugation again, is 1 ︰ 15 according to wet thallus weight (g) and buffer volume (mL) ratio
Disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution, ultrasonic disruption cell under conditions of ice-water bath is added, centrifugation takes supernatant,
As CBM-DnaB-GAD merges enzyme crude enzyme liquid；
S3. it forms chitin-CBM-DnaB-GAD: chitin is impregnated with water, be centrifuged or filter removal residual moisture, it will
CBM-DnaB-GAD merges enzyme crude enzyme liquid volume (mL) to be mixed with the ratio that the ratio for the chitin quality (g) being filtered dry is 4 ︰ 1,
It has a rest stirring, makes CBM-DnaB-GAD fusion enzyme and chitin that affine absorption sufficiently occur and form chitin-CBM-DnaB-GAD, from
Chitin-CBM-DnaB-GAD is collected in the heart or filtering；
S4. it obtains the pure enzyme of enterococcus faecium GAD native enzyme: first is washed sufficiently with disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution
Then shell element-CBM-DnaB-GAD is added self cleavage buffer and is sheared, filtered, obtained filter liquor is enterococcus faecium
The pure enzyme of GAD native enzyme.
Compared with prior art, the invention has the following advantages:
(1) the Escherichia coli (DH5 α-LNSF2) that the present invention constructs is to have the wild strain of high GAD vigor
Based on the excellent gadB gene of Enterococcusfaecium LNSF2, and the promoter PrpoS used is transcribed comprising RpoS
The 5 ' of regulator control system-non-translational region can effectively drive foreign protein right independent of any inducer additionally added
The expression in number growth period, CBM-DnaB-GAD merge expression of enzymes amount height, and GAD is energetic, the CBM-DnaB- of culture medium fermentation expression
The GAD vigor of GAD has reached (6041.82 ± 354.71) IU/L, can significantly reduce being produced into for CBM-DnaB-GAD fusion enzyme
This.
(2) chitin used in the present invention does not need to be modified can also merge enzyme generation specific bond, liquid with CBM-DnaB-GAD
Liquid flowability is good, overcomes the defect of liquid fluidity difference when cellulose needs to be modified and apply, simplifies purifying process；Crust
There are larger differences with the chemical structure of cellulose for element, however present invention discover that CBM can be specifically bound with chitin, it is close
Cellulose is significantly stronger than with power.
(3) design is scientific and reasonable for engineering bacteria of the invention building, has organically combined CBM to the strong pathoklisis of chitin
The self cleavage function of matter and intein DnaB only can need to specifically isolate CBM- using one step affinity chromatography of chitin
DnaB-GAD forms insoluble chitin-CBM-DnaB-GAD, then carries out self cleavage under the effect of self cleavage liquid and cut off GAD,
GAD native enzyme dissolves in self cleavage liquid, is simply separated by solid-liquid separation with insoluble chitin-CBM-DnaB, can be obtained dung
The pure enzyme of enterococcus GAD native enzyme.
(4) it is pure to have reached electrophoresis for the enterococcus faecium GAD native enzyme purity that is prepared of the present invention, and relative molecular weight and reason
Almost the same by molecular weight, the key technology for solving the CBM label that cannot cut off CBM- fusion enzyme and obtaining GAD native enzyme is asked
Topic；In addition, purifying process is simple, purification efficiency is high, and cost substantially reduces, and can be applied to industrialized production enterococcus faecium GAD days
The pure enzyme of right enzyme solves the problems, such as that the pure enzyme production hardly possible of GAD, cost remain high.
Detailed description of the invention
Fig. 1 is the design route schematic diagram of the natural enzyme purification of enterococcus faecium GAD；Wherein, 1: chitin, 2: cellulose combines
Module, 3: intein, 4:GAD, 5: self cleavage.
Fig. 2 is the electrophoretogram of the GAD gene gadB of PCR amplification Enterococcusfaecium LNSF2；Wherein, swimming lane M
For DNA molecular amount standard Marker, swimming lane 1 is the GAD gene for the Enterococcus faecium LNSF2 that PCR amplification obtains
gadB。
Fig. 3 is the structural schematic diagram of recombinant plasmid pRPOCDN-efagadB.
Fig. 4 is that different buffers extract CBM-DnaB-GAD fusion enzyme extract and CBM-DnaB-GAD merges enzyme
With the influence of the affine adsorption capacity of chitin；Wherein, PBS indicates disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution, Tris-HCl table
Show three (methylol) aminomethane-hydrochloride buffers.
Fig. 5 is to rapidly purify electrophorogram of the enterococcus faecium GAD native enzyme through SDS-PAGE electrophoresis being prepared；Its
In, swimming lane 1 is standard protein molecular weight, and swimming lane 2 is CBM-DnaB-GAD crude enzyme liquid, and swimming lane 3 and swimming lane 4 are enterococcus faecium
The pure enzyme of GAD native enzyme.
Fig. 6 is the relative migration distance of SDS-PAG electrophoresis Plays protein and the pure enzyme band of enterococcus faecium GAD native enzyme
With the linear relationship chart of protein molecular weight；Wherein, lg Mr indicates that the common logarithm of protein molecular weight, GAD indicate dung intestines ball
Bacterium GAD native enzyme；Y=-0.0104x+2.0752 is the linear equation of linearity curve；R2=0.9929 is the correlation of linear equation
Property coefficient.
Specific embodiment
Further illustrate the present invention below in conjunction with specific embodiment, but embodiment the present invention is not done it is any type of
It limits.Unless stated otherwise, the present invention uses reagent, method and apparatus for the art conventional reagent, method and apparatus,
Agents useful for same and material are commercially available.
The basic line of embodiment of the present invention is: with wild strain Enterococcusfaecium LNSF2 for GAD gene
The donor of gadB by cellulose binding module gene cbm3, includes peptide gene dnaB, gadB gene and voluntarily constructs
PRPOCDN expression vector carries out recombination to construct pRPOCDN-efagadB recombinant plasmid, and Transformed E .coli DH5 ɑ constructs engineering bacteria
Escherichia coli (DH5 α-LNSF2), culture expression CBM-DnaB-GAD merge enzyme, then with chitin to CBM-DnaB-
GAD merges the affine absorption of enzyme spcificity, is then handled using self cleavage buffer, makes DnaB that self cleavage occur and cut off GAD, from
And obtain the pure enzyme of enterococcus faecium GAD native enzyme.
One plant of basic line based on invention embodiment, design is de- for rapidly purifying preparation enterococcus faecium glutamic acid
The engineering bacteria of the pure enzyme of carboxylic acid native enzyme and the invention route schematic diagram of purification process are as shown in Figure 1.
In invention research, carried out through the GAD gene gadB to Enterococcusfaecium GDMCC No.60203
PCR amplification, gadB electrophoretogram such as Fig. 2 of acquisition, as it is clear from fig. 2 that gadB gene size is about 1.4kb, through gadB gene is sequenced
Length is 1401bp, and nucleotide sequence is shown in SEQ ID NO:7；The nucleotide of the recombinant plasmid pRPOCDN-efagadB of building
For sequence as shown in SEQ ID NO:13, structural schematic diagram is as shown in Figure 3；With recombinant plasmid pRPOCDN-efagadB conversion
E.coli DH5 ɑ competent cell, construct can high efficient expression CBM-DnaB-GAD fusion enzyme engineering bacteria Escherichia
Coli (DH5 α-LNSF2), the GAD vigor of the CBM-DnaB-GAD of LB culture medium fermentation expression reached (6041.82 ±
354.71)IU/L.The construction method detailed in Example 1 of engineering bacteria Escherichia coli (DH5 α-LNSF2).
In invention research, be respectively adopted 50mmol/LTris-HCl buffer, 100mmol/LPBS buffer,
20mmol/L PBS buffer solution, 10mmol/L PBS buffer solution merge zyme extract, warp and chitin as CBM-DnaB-GAD
Affine absorption measures enzyme activity after sufficiently washing, as a result as Fig. 4, Fig. 4 show the CBM-DnaB-GAD fusion that each buffer extracts
With chitin affine absorption can occur for enzyme to a certain extent, and wherein 20mmol/L PBS buffer solution effect is best, CBM-
DnaB-GAD merges enzyme and the affine adsorbance of chitin is most, and GAD vigor is maximum；It is extracted and is prepared with 20mmol/LPBS buffer
Chitin-CBM-DnaB-GAD vigor as 100%, 50mmol/LTris-HCl buffer, 100mmol/L PBS buffer
Chitin-the CBM-DnaB-GAD of liquid and 10mmol/LPBS buffer as CBM-DnaB-GAD fusion zyme extract preparation
Opposite enzyme activity is respectively (78.07 ± 6.3) %, (86.84 ± 1.82) % and (86.08 ± 7.72) %.
According to step described in following 2~embodiments of embodiment 4 to engineering bacteria Escherichia coli (DH5 α-LNSF2)
The enterococcus faecium GAD native enzyme of expression carries out purifying preparation, examines through SDS-PAGE electrophoresis, as a result such as Fig. 5.From figure 5 it can be seen that pure
CBM-DnaB-GAD crude enzyme liquid kinds of protein before change is more, and passes through dung intestines ball prepared by chitin affinity purification and self cleavage
Single band is only presented in bacterium GAD native enzyme in electrophorogram, the results showed that the pure enzyme of enterococcus faecium GAD native enzyme of preparation it is pure
It is pure that degree has reached electrophoresis.Further, using standard protein and the pure enzyme of enterococcus faecium GAD native enzyme in SDS-PAGE electrophoresis
The logarithm of relative mobility and protein relative molecular weight carries out production standard curve, as a result such as Fig. 6, according to the linear side of Fig. 6
Journey and GAD relative mobility are calculated, and GAD relative molecular weight is 55.68kDa, basic with GAD theoretical molecular weight 53.71kDa
Unanimously, as a result illustrate that prepared pure enzyme is enterococcus faecium GAD native enzyme.
Specific embodiment example is as follows:
The building of 1 engineering bacteria Escherichia coli of embodiment (DH5 α-LNSF2)
S11. Enterococcusfaecium LNSF2 is accessed into PSB or MRS culture medium, in 37 DEG C of stationary culture 12h,
It takes culture solution 5mL to be centrifuged with 8500r/min, collects bacterial sediment, abandon supernatant, be that century bacterial genomes extract reagent according to health
Box specification extracts genomic DNA, is placed in -20 DEG C and saves backup；Design efa-1 and efa-2 primer, primer efa-1 and efa-2
Biological sequence is as shown in table 1, the synthesis of commission Sangon Biotech (Shanghai) Co., Ltd.；Using efa-1, efa-2 as primer, with
Enterococcusfaecium LNSF2 genomic DNA is template, is polymerize using full formula gold TransStart FastPfu DNA
Enzyme carries out PCR reaction；
PCR reaction condition are as follows: 95 DEG C of 2min；95 DEG C of 20s, 50 DEG C of 30s, 72 DEG C of 55s are recycled 33 times；72℃5min；
1 μ L Taq DNAPolymerase, 72 DEG C of reactions 30min, PCR is added after reaction, by 100 μ L volumes in PCR
As shown in fig. 2, it can be seen that the length of its product is 1401bp, i.e. gadB gene length is the electrophoresis result of product
1401bp, nucleotide sequence is as shown in SEQ ID NO:7；Then purification kit direct purification gadB gene is used, by the base
Because being connect with pMD19-T Simple Vector overnight, construction recombination plasmid pMD-efagadB, Transformed E .coli DH5 ɑ impression
State cell, send Sangon Biotech (Shanghai) Co., Ltd. to be sequenced.
S12. stress induced promoter PrpoS, cbm3 (family from Clostridium thermocellum are designed
3 cellulose binding domains) coded sequence, intein DnaB coded sequence dnaB, T7 terminator sequence, this auspicious biotechnology of trust money
Co., Ltd's synthesis；By pUC57 plasmid first with I/Hind of Nde, II digestion, PrpoS, cbm3, dnaB, T7 are then inserted into pUC57
Plasmid splicing building escherichia coli plasmid pRPOCDN；Wherein, pUC57 plasmid, stress induced promoter PrpoS, CBM coding
Sequence cbm3, intein DnaB coded sequence dnaB and T7 terminator nucleotide sequence respectively such as SEQ ID NO:8~SEQ
Shown in ID NO:12.
S13. design and entrust Sangon Biotech's synthetic primer Infu-22 and Infu-27, primer
The nucleotide sequence of Infu-22 and Infu-27 is as shown in table 1, using pRPOCDN as template, uses full formula gold TransStart
FastPfu archaeal dna polymerase carries out PCR reaction；
PCR reaction condition are as follows: 95 DEG C of 2min；95 DEG C of 20s, 54 DEG C of 30s, 72 DEG C of 1min 45s are recycled 33 times, 72 DEG C
5min；
After reaction, electrophoresis cuts glue to PCR, recycles linearized vector pRPOCDN with DNA plastic recovery kit, cuts DNA
Band, recovery purifying target fragment；Design and commission Sangon Biotech's synthetic primer Infu-13 and
The nucleotide sequence of Infu-24, primer I nfu-13 and Infu-24 are as shown in table 1, using pMD-efagadB as template amplification gadB
Gene, electrophoresis cut glue, recovery purifying gadB gene；After purification, by gadB gene and linearized vector pRPOCDN In-
Fusion HD Cloning Kit gene cloning kit is stitched together, and reaction system is 2 μ L of In-fusion enzyme, gadB
1 μ L of gene, 2 μ L of linearized vector, ddH25 μ L of O amounts to 10 μ L, is centrifuged 30s, and 50 DEG C of connection 15min are assembled into recombinant plasmid
The nucleotide sequence of pRPOCDN-efagadB, recombinant plasmid pRPOCDN-efagadB as shown in SEQ ID NO:13, show by structure
It is intended to as shown in Figure 3.
S14. by recombinant plasmid pRPOCDN-efagadB Transformed E .coli DH5 ɑ competent cell, constructing can efficient table
Up to the engineering bacteria Escherichia coli (DH5 α-LNSF2) of CBM-DnaB-GAD fusion enzyme.
Table 1 constructs primer used in engineering bacteria Escherichia coli (DH5 α-LNSF2)
The composition of PSB culture medium described in step S11 are as follows: peptone 15g/L, beef extract 10g/L, sucrose 12.5g/L, sodium acetate
6.0g/L, one sodium 10g/L of Pidolidone, Tween 80 1.0g/L, pH 6.8~7.0；
The composition of MRS culture medium described in step S11 are as follows: peptone 15g/L, beef extract 12.5g/L, sucrose 12.5g/L, lemon
Sour diammonium 2.0g/L, sodium acetate 5.0g/L, K2HPO42.0g/L、CaCl22.0g/L, Tween801.0mL/L, pH 6.8~7.0.
Embodiment 2 rapidly purifies the preparation pure enzyme of enterococcus faecium GAD native enzyme
S1., embodiment 1 is constructed to obtained Escherichia coli (DH5 α-LNSF2) and accesses LB culture medium, in 37
DEG C, 120r/min shaken cultivation for 24 hours, high efficient expression CBM-DnaB-GAD merge enzyme.
S2. Escherichia coli (DH5 α-LNSF2) fermentation liquid is centrifuged 10min in 4 DEG C, 8500r/m, collects bacterium
Body is added physiological saline, is dispersed with stirring washing thalline, thalline were collected by centrifugation again, according to wet thallus weight (g) and buffering liquid
Product (mL) ratio is that pH 8.0,20mmol/L disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution is added in 1 ︰ 15, and 400W is super in ice-water bath
Sonication cell (work 5s, cooling 5s, whole 50min), by clasmatosis liquid in 8500r/min, 4 DEG C of centrifugation 15min, takes
Supernatant, as CBM-DnaB-GAD merge enzyme crude enzyme liquid.
S3. chitin is impregnated into 12h, centrifugation or filtering removal residual moisture with water, CBM-DnaB-GAD fusion enzyme is thick
Enzyme solution volume (mL) is that 4 ︰ 1 mix 15min with chitin quality (g) ratio being filtered dry, and intermittent stirring melts CBM-DnaB-GAD
Synthase and chitin sufficiently occur affine absorption and form chitin-CBM-DnaB-GAD, are centrifuged or are collected by filtration chitin-CBM-
DnaB-GAD, with 6 times of chitin quality of disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution (20mmol/L, pH 8.0) point 5 times
Chitin-CBM-DnaB-GAD is washed, 3 times of self cleavage buffer of chitin quality, 30 DEG C, 100r/min item is then added
12h is sheared under part, filters out enzyme solution, can be obtained the pure enzyme of enterococcus faecium GAD native enzyme.
Wherein, LB culture medium described in step S1 forms are as follows: tryptone 10g/L, yeast powder 5g/L, NaCl10g/L, L- paddy
One sodium 10g/L, pH 6.0 of propylhomoserin；It is sub-packed in specification 250mL triangular flask, every bottle of 120mL, 121 DEG C of sterilizing 20min；Face after cooling
With preceding, 240 μ L 50mg/mL ampicillins are added, shake up；
Self cleavage buffer group described in step S3 becomes final concentration of containing NaCl final concentration of 0.5mol/L, EDTA
PH 6.5,0.2mol/L disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of 1mmol/L.
Embodiment 3 rapidly purifies the preparation pure enzyme of enterococcus faecium GAD native enzyme
The present embodiment rapidly purifies the process of the preparation pure enzyme of enterococcus faecium GAD native enzyme, step S1~S2 according to embodiment 2
Identical, step S3~S4 is as follows:
S3. chitin and 3 times of chitin quality of distilled water are sufficiently mixed and are prepared into Chitin pulp, while stirring slowly
50 times of chitin quality of ice-cold concentrated phosphoric acid (> 85%) is added, mixes to pellucidity, ice bath 1h (is stirred) frequently, then side is stirred
Mix the ice water (avoid chitin occur blocking) that side is slowly added to 300 times of chitin quality, 4 DEG C, 8500r/min be centrifuged 15min,
Supernatant is removed, chitin precipitating is collected, is repeated 4 times, to remove phosphoric acid, is added 200 times of chitin quality in chitin precipitating
Distilled water, using 2mol/L Na2CO3Solution adjusts pH to 6~7, neutralizes remaining phosphoric acid, stirs and evenly mixs, and filters and removes liquid,
Then the 3 cleaning chitins of distillation moisture for using 300 times of chitin quality, prepare modified chitin.
S4. it is with modified chitin quality (g) ratio being filtered dry by CBM-DnaB-GAD fusion enzyme crude enzyme liquid volume (mL)
The ratio of 4 ︰ 1 mixes 15min, and intermittent stirring makes CBM-DnaB-GAD fusion enzyme and chitin that affine absorption sufficiently occur and formed
Chitin-CBM-DnaB-GAD is centrifuged or is collected by filtration chitin-CBM-DnaB-GAD, with 6 times of phosphoric acid of chitin quality
5 washing chitin-CBM-DnaB-GAD of disodium hydrogen-phosphate sodium dihydrogen buffer solution (20mmol/L, pH 8.0) point, are then added
3 times of self cleavage buffer of chitin quality, filters out enzyme solution, can be obtained dung by 30 DEG C, shear 12h under the conditions of 100r/min
The pure enzyme of enterococcus GAD native enzyme.
Self cleavage buffer group described in step S4 becomes final concentration of containing NaCl final concentration of 0.5mol/L, EDTA
PH 6.5,0.2mol/L disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of 1mmol/L.
Embodiment 4 rapidly purifies the preparation pure enzyme of enterococcus faecium GAD native enzyme
The present embodiment rapidly purifies the process of the preparation pure enzyme of enterococcus faecium GAD native enzyme, step S1~S2 according to embodiment 2
Identical, step S3~S4 is as follows:
S3. chitin and 25 times of chitin quality of NaOH/ urea mixed solution are mixed, -30 DEG C of freezing 4h, room temperature solution
Freeze and quickly stir, then then at -30 DEG C of freezing 4h, thaw at RT is simultaneously quickly stirred, and freeze thawing is repeated, until chitin is dissolved into
Transparence, then it is slowly added to 300 times of chitin quality of ice water (avoiding chitin from occurring blocking), 4 DEG C, 8500r/ while stirring
Min is centrifuged 15min, removes supernatant, collects chitin precipitating, washes repeatedly 4 times, to remove NaOH and urea, precipitates in chitin
The middle distilled water for being added 200 times of chitin quality adjusts pH to 6~7 using 1mol/L HCl solution, neutralizes remaining NaOH,
It stirs and evenly mixs, filters and remove liquid, then use 300 times of chitin quality 3 cleaning chitins of distillation moisture, prepare modification
Chitin.
S4. it is with modified chitin quality (g) ratio being filtered dry by CBM-DnaB-GAD fusion enzyme crude enzyme liquid volume (mL)
4 ︰ 1 mix 15min, and intermittent stirring makes CBM-DnaB-GAD fusion enzyme and chitin that affine absorption sufficiently occur and forms chitin-
CBM-DnaB-GAD is centrifuged or is collected by filtration chitin-CBM-DnaB-GAD, with 6 times of disodium hydrogen phosphate-of chitin quality
5 washing chitin-CBM-DnaB-GAD of phosphate sodium dihydrogen buffer solution (20mmol/L, pH 8.0) point, are then added chitin
3 times of self cleavage buffer of quality, filters out enzyme solution, can be obtained enterococcus faecium by 30 DEG C, shear 12h under the conditions of 100r/min
The pure enzyme of GAD native enzyme.
Wherein, self cleavage buffer group described in step S4, which becomes, contains final concentration of 0.5mol/L, EDTA final concentration of NaCl
For pH 6.5,0.2mol/L disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of 1mmol/L.
The preferred embodiment that the above specific embodiment is of the invention for ease of understanding and illustrates, but the invention is not limited to
Above-described embodiment does not mean that the present invention must rely on above-described embodiment and could implement.Person of ordinary skill in the field
It is the addition of equivalence replacement and auxiliary element to raw material selected by the present invention, specific it will be clearly understood that any improvement in the present invention
The selection etc. of mode, all of which fall within the scope of protection and disclosure of the present invention.
Sequence table
<110>south of the Five Ridges college of education
<120>one plants of engineering bacterias and purifying side for rapidly purifying the preparation pure enzyme of enterococcus faecium glutamate decarboxylase native enzyme
Method
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atgttatacg gaaaagataa tcaag 25
<210> 2
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ttagtgagta aagccgtacg ttttc 25
<210> 3
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gaattcctcg agggctcttc cag 23
<210> 4
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ggatcccggt tctttacccc aaacc 25
<210> 5
<211> 52
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gtttggggta aagaaccggg atccatgtta tacggaaaag ataatcaaga ag 52
<210> 6
<211> 42
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gccctcgagg aattcttagt gagtaaagcc gtacgttttc ac 42
<210> 7
<211> 1401
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
atgttatacg gaaaagataa tcaagaagaa aaaaactatt tggaaccaat ttttggctct 60
gcaagtgagg atgttgactt gcctaaatat aagttaaaca aagaatccat tgaaccacga 120
attgcttatc aattagtaca agacgagatg ttggatgaag gaaatgcgcg attaaactta 180
gctacttttt gtcaaacgta tatggaacct gaagcagtga aattgatgac ccaaacgtta 240
gaaaaaaatg caattgataa atcagaatac ccacgaacaa cggaaattga aaaccgctgt 300
gtaaatatga ttgctgattt atggcatgct ccaaataatg aaaaattcat gggaacttca 360
acgatcggct cttcagaagc ctgcatgctg ggtggtatgg ccatgaaatt tgcttggcgt 420
aaacgtgctg aaaaattagg tcttgatatt caagcaaaaa aacctaacct ggtgatctct 480
tctggttacc aagtttgttg ggaaaaattc tgtgtatatt gggatgtgga actgagagaa 540
gtcccaatgg atgaaaaaca tatgtcaatt aatctagata ctgtcatgga ttatgtggat 600
gagtacacaa ttggtattgt aggtattatg ggtattactt acactggtcg ttatgatgat 660
atcaagggtc tgaatgattt agttgaagct cacaataaac aaactgacta taaagtatac 720
attcatgttg acgctgcatc gggtggcttt tatgcaccat ttactgaacc tgatctagtt 780
tgggattttc aattgaaaaa tgttatctca attaattctt caggtcacaa atatggtttg 840
gtatatccag gtgtgggttg ggtcttatgg cgtgaccaac aatacttacc agaagaatta 900
gtatttaaag ttagttactt aggtggagaa atgccaacta tggctatcaa cttctctcat 960
agtgcagcac aactaattgg gcaatactac aactttgtac gctatggctt tgatggttat 1020
cgtgatattc accaaagaac tcatgatgtt gctgtttatt tagccaaaga gatcgaaaaa 1080
actggtattt ttgaaatcat taatgatgga tcagaattgc cagttgtgtg ctataagctg 1140
aaagaagatc ccaatcgcga atggacacta tatgatttat ctgatcgtct gttaatgaag 1200
ggatggcaag tcccagccta cccattacct aaagacttgg atcaattaat tattcaacgc 1260
ttagttgttc gagcagactt tggaatgaac atggctggtg attatgtaca agatatgaac 1320
caagcaattg aagagttgaa taaagctcat attgtttatc ataaaaaaca ggatgtgaaa 1380
acgtacggct ttactcacta a 1401
<210> 8
<211> 2710
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420
tgcatctaga tatcggatcc cgggcccgtc gactgcagag gcctgcatgc aagcttggcg 480
taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 540
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 600
ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 660
taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 720
tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 780
aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 840
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 900
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 960
acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 1020
ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 1080
tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 1140
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 1200
gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 1260
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 1320
tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 1380
agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 1440
tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1500
acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1560
tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1620
agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1680
tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1740
acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1800
tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1860
ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1920
agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1980
tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 2040
acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 2100
agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 2160
actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 2220
tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 2280
gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 2340
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 2400
tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 2460
aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 2520
tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2580
tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2640
gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg 2700
ccctttcgtc 2710
<210> 9
<211> 743
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ccatacgcgc tgaacgttgg tcagaccttg caggtgggta atgcttccgg tacgccaatc 60
actggcggaa atgccattac ccaggccgac gcagcagagc aaggagttgt gatcaagcct 120
gcacaaaatt ccaccgttgc tgttgcgtcg caaccgacaa ttacgtattc tgagtcttcg 180
ggtgaacaga gtgctaacaa aatgttgccg aacaacaagc caactgcgac cacggtcaca 240
gcgcctgtaa cggtaccaac agcaagcaca accgagccga ctgtcagcag tacatcaacc 300
agtacgccta tctccacctg gcgctggccg actgagggca aagtgatcga aacctttggc 360
gcttctgagg ggggcaacaa ggggattgat atcgcaggca gcaaaggaca ggcaattatc 420
gcgaccgcag atggccgcgt tgtttatgct ggtaacgcgc tgcgcggcta cggtaatctg 480
attatcatca aacataatga tgattacctg agtgcctacg cccataacga cacaatgctg 540
gtccgggaac aacaagaagt taaggcgggg caaaaaatag cgaccatggg tagcaccgga 600
accagttcaa cacgcttgca ttttgaaatt cgttacaagg ggaaatccgt aaacccgctg 660
cgttatttgc cgcagcgata aatcggcgga accaggcttt tgcttgaatg ttccgtcaag 720
ggatcacggg taggagccac ctt 743
<210> 10
<211> 480
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
atgccggtgt ctggcaacct gaaagtggaa ttttacaaca gcaatccgag cgatacgacc 60
aatagcatca acccgcagtt taaagtgacc aacacgggta gctctgcaat tgatctgtct 120
aaactgaccc tgcgttatta ctatacggtt gatggccaga aagaccaaac cttttggtgc 180
gaccatgcgg ccattatcgg ctctaacggt agttacaatg gtatcacctc gaatgtcaaa 240
ggcacgttcg tgaaaatgag ttcctcaacc aacaatgccg atacgtatct ggaaattagc 300
tttaccggcg gtacgctgga accgggtgca cacgtccaga tccaaggccg tttcgctaaa 360
aacgattggt caaattacac ccagtccaac gactattcat ttaaatcggc gagccagttc 420
gttgaatggg atcaagtcac cgcctacctg aatggcgtgc tggtttgggg taaagaaccg 480
<210> 11
<211> 522
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
aacaacggta acaacggtct cgaactgcgc gagtccggag ctatctctgg cgatagtctg 60
atcagcctgg ctagcacagg aaaaagagtt tctattaaag atttgttaga tgaaaaagat 120
tttgaaatat gggcaattaa tgaacagacg atgaagctag aatcagctaa agttagtcgt 180
gtattttgta ctggcaaaaa gctagtttat attctaaaaa ctcgactagg tagaactatc 240
aaggcaacag caaatcatag atttttaact attgatggtt ggaaaagatt agatgagcta 300
tctttaaaag agcatattgc tctaccccgt aaactagaaa gctcctcttt acaattgtca 360
ccagaaatag aaaagttgtc tcagagtgat atttactggg actccatcgt ttctattacg 420
gagactggag tcgaagaggt ttttgatttg actgtgccag gaccacataa ctttgtcgcg 480
aatgacatca ttgtacacaa cggaagagcc atgggcggcc gc 522
<210> 12
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
ccccttgggg cctctaaacg ggtcttgagg ggttttttg 39
<210> 13
<211> 5652
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatggt cgacccatac gcgctgaacg ttggtcagac cttgcaggtg ggtaatgctt 240
ccggtacgcc aatcactggc ggaaatgcca ttacccaggc cgacgcagca gagcaaggag 300
ttgtgatcaa gcctgcacaa aattccaccg ttgctgttgc gtcgcaaccg acaattacgt 360
attctgagtc ttcgggtgaa cagagtgcta acaaaatgtt gccgaacaac aagccaactg 420
cgaccacggt cacagcgcct gtaacggtac caacagcaag cacaaccgag ccgactgtca 480
gcagtacatc aaccagtacg cctatctcca cctggcgctg gccgactgag ggcaaagtga 540
tcgaaacctt tggcgcttct gaggggggca acaaggggat tgatatcgca ggcagcaaag 600
gacaggcaat tatcgcgacc gcagatggcc gcgttgttta tgctggtaac gcgctgcgcg 660
gctacggtaa tctgattatc atcaaacata atgatgatta cctgagtgcc tacgcccata 720
acgacacaat gctggtccgg gaacaacaag aagttaaggc ggggcaaaaa atagcgacca 780
tgggtagcac cggaaccagt tcaacacgct tgcattttga aattcgttac aaggggaaat 840
ccgtaaaccc gctgcgttat ttgccgcagc gataaatcgg cggaaccagg cttttgcttg 900
aatgttccgt caagggatca cgggtaggag ccaccttatg ccggtgtctg gcaacctgaa 960
agtggaattt tacaacagca atccgagcga tacgaccaat agcatcaacc cgcagtttaa 1020
agtgaccaac acgggtagct ctgcaattga tctgtctaaa ctgaccctgc gttattacta 1080
tacggttgat ggccagaaag accaaacctt ttggtgcgac catgcggcca ttatcggctc 1140
taacggtagt tacaatggta tcacctcgaa tgtcaaaggc acgttcgtga aaatgagttc 1200
ctcaaccaac aatgccgata cgtatctgga aattagcttt accggcggta cgctggaacc 1260
gggtgcacac gtccagatcc aaggccgttt cgctaaaaac gattggtcaa attacaccca 1320
gtccaacgac tattcattta aatcggcgag ccagttcgtt gaatgggatc aagtcaccgc 1380
ctacctgaat ggcgtgctgg tttggggtaa agaaccggga tccaacaacg gtaacaacgg 1440
tctcgaactg cgcgagtccg gagctatctc tggcgatagt ctgatcagcc tggctagcac 1500
aggaaaaaga gtttctatta aagatttgtt agatgaaaaa gattttgaaa tatgggcaat 1560
taatgaacag acgatgaagc tagaatcagc taaagttagt cgtgtatttt gtactggcaa 1620
aaagctagtt tatattctaa aaactcgact aggtagaact atcaaggcaa cagcaaatca 1680
tagattttta actattgatg gttggaaaag attagatgag ctatctttaa aagagcatat 1740
tgctctaccc cgtaaactag aaagctcctc tttacaattg tcaccagaaa tagaaaagtt 1800
gtctcagagt gatatttact gggactccat cgtttctatt acggagactg gagtcgaaga 1860
ggtttttgat ttgactgtgc caggaccaca taactttgtc gcgaatgaca tcattgtaca 1920
caacggaaga gccatgggcg gccgcatgtt atacggaaaa gataatcaag aagaaaaaaa 1980
ctatttggaa ccaatttttg gctctgcaag tgaggatgtt gacttgccta aatataagtt 2040
aaacaaagaa tccattgaac cacgaattgc ttatcaatta gtacaagacg agatgttgga 2100
tgaaggaaat gcgcgattaa acttagctac tttttgtcaa acgtatatgg aacctgaagc 2160
agtgaaattg atgacccaaa cgttagaaaa aaatgcaatt gataaatcag aatacccacg 2220
aacaacggaa attgaaaacc gctgtgtaaa tatgattgct gatttatggc atgctccaaa 2280
taatgaaaaa ttcatgggaa cttcaacgat cggctcttca gaagcctgca tgctgggtgg 2340
tatggccatg aaatttgctt ggcgtaaacg tgctgaaaaa ttaggtcttg atattcaagc 2400
aaaaaaacct aacctggtga tctcttctgg ttaccaagtt tgttgggaaa aattctgtgt 2460
atattgggat gtggaactga gagaagtccc aatggatgaa aaacatatgt caattaatct 2520
agatactgtc atggattatg tggatgagta cacaattggt attgtaggta ttatgggtat 2580
tacttacact ggtcgttatg atgatatcaa gggtctgaat gatttagttg aagctcacaa 2640
taaacaaact gactataaag tatacattca tgttgacgct gcatcgggtg gcttttatgc 2700
accatttact gaacctgatc tagtttggga ttttcaattg aaaaatgtta tctcaattaa 2760
ttcttcaggt cacaaatatg gtttggtata tccaggtgtg ggttgggtct tatggcgtga 2820
ccaacaatac ttaccagaag aattagtatt taaagttagt tacttaggtg gagaaatgcc 2880
aactatggct atcaacttct ctcatagtgc agcacaacta attgggcaat actacaactt 2940
tgtacgctat ggctttgatg gttatcgtga tattcaccaa agaactcatg atgttgctgt 3000
ttatttagcc aaagagatcg aaaaaactgg tatttttgaa atcattaatg atggatcaga 3060
attgccagtt gtgtgctata agctgaaaga agatcccaat cgcgaatgga cactatatga 3120
tttatctgat cgtctgttaa tgaagggatg gcaagtccca gcctacccat tacctaaaga 3180
cttggatcaa ttaattattc aacgcttagt tgttcgagca gactttggaa tgaacatggc 3240
tggtgattat gtacaagata tgaaccaagc aattgaagag ttgaataaag ctcatattgt 3300
ttatcataaa aaacaggatg tgaaaacgta cggctttact cactaagaat tcctcgaggg 3360
ctcttccaga tctccccttg gggcctctaa acgggtcttg aggggttttt tgaagcttgg 3420
cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 3480
acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 3540
cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 3600
attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 3660
cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 3720
caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 3780
caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 3840
ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 3900
cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 3960
ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 4020
tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 4080
gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 4140
ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 4200
ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 4260
gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 4320
aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 4380
tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 4440
ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 4500
tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 4560
aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 4620
tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 4680
ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 4740
gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 4800
gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 4860
taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 4920
tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 4980
ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 5040
tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 5100
ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 5160
tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 5220
ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 5280
aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 5340
actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 5400
aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 5460
tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 5520
aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 5580
ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga 5640
ggccctttcg tc 5652
Claims (10)
1. one plant for rapidly purifying the engineering bacteria of the preparation pure enzyme of enterococcus faecium GAD native enzyme, which is characterized in that the engineering bacteria
ForEscherichia coli(DH5 α-LNSF2), the engineering bacteria were preserved in Guangdong Province microorganism fungus kind on September 18th, 2018
Collection, deposit number are as follows: GDMCC No.60446.
2. engineering bacteria according to claim 1, which is characterized in that the engineering bacteria are as follows: containing by stress induced starting
SonPrpoS, CBM coded sequencecbm3, intein DnaB coded sequencednaB、Enterococcus faecium LNSF2's
GAD genegadBWith pRPOCDN- made of T7 terminator sequence recombination to constructefagadBRecombinant plasmid.
3. a kind of method for constructing engineering bacteria as claimed in claim 1 or 2, which comprises the following steps:
S11. it clonesEnterococcus faecium The GAD gene of LNSF2gadB, construction recombination plasmid pMD-efagadB；
S12. building includes stress induced promoterPrpoS, cellulose binding module CBM coded sequencecbm3, intein
DnaB coded sequencednaB, T7 terminator sequence pRPOCDN expression vector；
S13. with pMD-efagadBFor template amplificationgadBGene, using the carrier that pRPOCDN is linearized as template amplification, building
Recombinant plasmid pRPOCDN-efagadB；
S14. by recombinant plasmid pRPOCDN-efagadBConvert the competent cell of Escherichia coli, building expression CBM-DnaB-
The engineering bacteria of GAD fusion enzymeEscherichia coli(DH5 α-LNSF2)；
Wherein, CBM is cellulose binding module, and DnaB is intein, and GAD is glutamate decarboxylase.
4. engineering bacteria as claimed in claim 1 or 2Escherichia coli(DH5 α-LNSF2) is natural in preparation enterococcus faecium GAD
Application in the pure enzyme of enzyme.
5. a kind of method for rapidly purifying the preparation pure enzyme of enterococcus faecium GAD native enzyme using as claimed in claim 1 or 22 engineering bacterias,
Characterized by comprising the following steps:
S1. it under conditions of ice-water bath, is crushed in bufferEscherichia coli(DH5 α-LNSF2) thallus extracts
CBM-DnaB-GAD merges enzyme crude enzyme liquid；
S2. CBM-DnaB-GAD fusion enzyme crude enzyme liquid is mixed and stirred for the chitin being filtered dry, affine absorption occurs and forms first
Shell element-CBM-DnaB-GAD；
S3. chitin-CBM-DnaB-GAD is washed, self cleavage buffer is added and is sheared, filters, obtained filter liquor is i.e.
For the pure enzyme of enterococcus faecium GAD native enzyme.
6. according to the method described in claim 5, it is characterized in that, chitin described in step S2 is unmodified or through concentrated phosphoric acid
Modified or modified through NaOH/ urea mixed solution chitin.
7. according to the method described in claim 5, it is characterized in that, self cleavage buffer described in step S3 be containing NaCl and
Disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of EDTA.
8. the method according to the description of claim 7 is characterized in that final concentration of 0.5~0.7 mol/L of NaCl, EDTA's
Final concentration of 0.5~3 mmol/L.
9. according to the method described in claim 5, it is characterized in that, the temperature of shearing described in step S3 be 20~32 DEG C, the time
For 6~15 h.
10. according to the method described in claim 5, it is characterized in that, the concentration of self cleavage buffer described in step S3 be 0.2~
0.3 mol/L, pH 5.5~6.5；Preferably, buffer described in step S1 is disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution；Into
Preferably, the concentration of buffer described in step S1 is 10~100 mmol/L to one step.
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