CN1262644C - Nitrile hydratase and its coding gene and use - Google Patents
Nitrile hydratase and its coding gene and use Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses nitrile hydratase, coding genes thereof and an application thereof. The aim of the present invention is to provide nitrile hydratase, coding genes thereof, and an expressing method of the nitrile hydratase. The nitrile hydratase provided by the present invention is composed of an alpha sub-gene and a beta sub-gene, wherein the alpha sub-gene is provided with an amino acid residue sequence of SEQ ID NO. 3 in a sequence table; the beta sub-gene is provided with an amino acid residue sequence of SEQ ID NO. 2. The coding genes of the nitrile hydratase of the present invention can be expressed with high efficiency in recombinant escherichia coli. The activity and the stability of the obtained nitrile hydratase are high, and thereby, the nitrile hydratase lays the favorable foundation for the site-specific mutagenesis and the orthogenesis reconstruction of the nitrile hydratase genes and for the biotransformation of acrylamide, and the nitrile hydratase can perform an important function in the production of the acrylamide.
Description
Technical field
The present invention relates to a kind of Nitrile hydratase and encoding gene and application in the bioengineering field, the expression method of particularly a kind of Nitrile hydratase and encoding gene and this Nitrile hydratase.
Background technology
Utilize the various types of polyacrylamides of acrylamide synthetic, in industrial production such as tertiary oil recovery, water treatment, papermaking, mining, metallurgy, coal washing and manufacturing super absorbent resin, have very widely and use.
Discover, all containing with the Nitrile hydratase in rhodococcus (Rhodococcus), pseudomonas (Pseudomonas), Nocardia bacteria (Nocardia), genus bacillus multiple microbies such as (Bacillus) is the nitrile metabolic enzyme system of representative, they are the important component parts in the microorganism nitrogen metabolism network, these enzymes can toxicity is very strong and nitrile catalysis that be difficult to degrade become to acid amides or carboxylic acid and be used.The Nitrile hydratase that obtains of clone is by having SEQ ID № in the sequence table from rhodococcus: the α subunit of 1 amino acid residue sequence and have SEQ ID №: the β subunit of 2 amino acid residue sequence is formed; Its activation has SEQ ID № in the sequence table: 4 amino acid residue sequence.
Since nitrile metabolic enzyme system as biological catalyst have reaction conditions gentleness, productive rate height, by product autohemagglutination loss few, product is little, environmental pollution is little, cost is low, the incomparable superiority of chemical processes such as idea of development that meets green chemical industry is widely used in the synthetic of amino acid, acid amides, carboxylic acid and derivative thereof in recent years.Also make the production technology of acrylamide experience great change, develop into work simplification, the microbial method that operational condition and environmental pollution situation are greatly improved from traditional chemical method.About the structure and the physiological property of enzyme self, also deep day by day to the research of enzymic activity influence as component, sequestrant, sulfhydryl compound and aminocompound etc. in photolytic activity, thermolability, regioselectivity, stereoselectivity etc. and various factors such as pH value, the born of the same parents.
Though microbial method is produced acrylamide technology and is existed many advantages at present, the whole world surpasses 300 by the acrylamide that Nitrile hydratase is produced every year, 000 ton, but wherein the enzyme of ubiquity wild-type strain live poor stability, to the not high and intermittenceization production of tolerance of substrate and product, quality product such as stablizes inadequately at problem.In addition, in process of production, the product acrylamide has also reduced the productive rate and the purity of acrylamide to acrylic acid further conversion.These problems greatly affect the development that microbial method is produced this friendly process of acrylamide.
Along with molecular biological fast development, people recognize that genetic engineering technique is one of effective means that solves an above-mentioned difficult problem.Utilize gene recombination technology, structure has the active novel gene engineering strain of Nitrile hydratase, be expected to and solving the actual production problem in varying degrees, such as the expression level that improves enzyme, strengthen the thermostability of enzyme and to the tolerance of substrate and product, the generation of blocking-up side reaction, cooperate the production that reduces enzyme and separation costs or the like with follow-up novel process.
Since genetic engineering technique is applied to acrylamide production since the beginning of the nineties, Chinese scholars is constantly explored the various physics-chem characteristics of Nitrile hydratase on the one hand, be devoted to the clone of (also comprising nitrilase gene and amidase gene) of Nitrile hydratase gene in the different strains on the other hand always, sequence characteristic, the further investigation of gene expression regulation, disclosed the mechanism of enzyme reaction and regulation and control from molecular level, and on this basis by range gene engineering means, change the sequence of gene, configuration, in the hope of strengthening the characteristic of enzyme, function, the catalytic activity and the stability of raising enzyme.This friendly process for final realization acrylamide is significant.Because genetic engineering bacterium is expressed Nitrile hydratase the irreplaceable advantage of a lot of wild bacterium is arranged, such as three kinds of genes (nitrilase gene, Nitrile hydratase gene and amidase gene) clonal expression separately, some is degraded again when producing can not make acid amides, can not reduce the pH value of fermented liquid yet, and suppress the activity of Nitrile hydratase because of producing acid; The genetic background of genetic engineering bacterium is clear in addition, helps the further investigation of gene expression regulation mechanism, and its adaptability is strong simultaneously, fermentation period short, help realizing large scale culturing and suitability for industrialized production, and more wide application prospect is arranged.Therefore, the research about the reorganization Nitrile hydratase has more and more caused people's attention.
But, studies show that, in with the Nitrile hydratase gene expression system of intestinal bacteria as the host, because gene starts under new mechanism and regulates and control to be subjected to certain restriction, the albumen that gives expression to is easily assembled again and is formed inclusion body, so proteic overexpression and activity expression have run into sizable difficulty, make slow progress in more than ten years in the past.Ikehata 0. grade is at the Nitrile hydratase of escherichia coli expression rhodococcus, the result detects the activity less than enzyme, handle bacterium liquid with the 8M urea, again through dialysis, can obtain the activity ((Eur.J.Biochem. of 1U=1 μ mol acrylamide/min) of 0.282U/L bacterium liquid, 1989,181 (3): 563-570), Kobayashi M. etc. are at the Nitrile hydratase of expression in escherichia coli rhodococcus rhodochrous J1, expression level still extremely low (Biochim.Biophys.Acta., 1997,1129 (1): 23-33), calendar year 2001, Sylvain P. etc. are at the Nitrile hydratase of escherichia coli expression rhodococcus, and the enzyme work that obtains has only the dried bacterium of 2.8U/mg, are approximately active 10% (the FEMS Microbiol.Letters of wild strain that they adopt, 2001,204 (1): 155-161).Nitrile hydratase can't obtain highly active recombinant expressed all the time, the research that has restricted the research of enzyme reaction and Regulation Mechanism and utilized the various physical and chemical performances of method raising enzyme in application of transgenation.
The innovation and creation content
The purpose of this invention is to provide a kind of Nitrile hydratase and encoding gene thereof.
Nitrile hydratase provided by the present invention, name is called mNHBA, and by having SEQ ID № in the sequence table: the α subunit of 3 amino acid residue sequence and have SEQ ID №: the β subunit of 2 amino acid residue sequence is formed.
SEQ ID № in the sequence table: the 3rd, by the α subunit that 203 amino-acid residues are formed, its nitrogen end the 1st amino acids residue is a methionine(Met).
The β subunit that the amino acid residue sequence of sequence 2 is made up of 229 amino-acid residues in the sequence table.
Activation of described Nitrile hydratase has SEQ ID № in the sequence table: 4 amino acid residue sequence, this activation can improve the expression activity of Nitrile hydratase.
SEQ ID № in the sequence table: 4 are made up of 104 amino-acid residues.
A kind of Nitrile hydratase encoding gene has one of following nucleotide sequences:
1) SEQ ID № in the sequence table: 5 dna sequence dna;
2) SEQ ID № in the sequence table: 6 dna sequence dna;
3) SEQ ID № in the code sequence tabulation: 2 and SEQ ID №: the polynucleotide of 3 protein sequences;
4) with sequence table in SEQ ID №: 5 or SEQ ID №: 6 dna sequence dnas that limit have 95% above homology, and the identical function protein DNA sequence of encoding.
Wherein, SEQ ID № in the sequence table: 5 derive from Nocardia bacteria (Nocardia sp.), and by 1714 based compositions, name is called NHBA, from the β subunit of 5 ' end the 268th to the 957th bit base encoding nitrile hydratase; Hold the 971st to the 1582nd bit base coding to have the Nitrile hydratase α subunit of the amino acid residue sequence of sequence 1 in the sequence table from 5 '.
In order to improve expression activity, (SEQ ID №: 5) have SEQ ID № in the sequence table through being connected with after modifying: the activation subsequence of 7 dna sequence dna obtains having SEQ ID № in the sequence table: 8 dna sequence dna to the Nitrile hydratase encoding gene.
SEQ ID № in the sequence table: 7 by 315 based compositions, SEQ ID № in the code sequence tabulation: activation shown in 4.SEQ ID № in the sequence table: 8 by 1626 based compositions, from the β subunit of 5 ' end the 1st to the 690th bit base encoding nitrile hydratase; From 5 ' end the 704th to the 1315th bit base encoding nitrile hydratase α subunit, activate son from 5 ' end the 1312nd to the 1626th bit base coding.
SEQ ID № in the sequence table: 6 by SEQ ID №: 5 obtain SEQID № from 5 ' end the 971st bit base rite-directed mutagenesis: 6 by 1714 based compositions, and name is called mNHBA, from the β subunit of 5 ' end the 268th to the 957th bit base encoding nitrile hydratase; Hold the 971st to the 1582nd bit base coding to have the Nitrile hydratase α subunit of the amino acid residue sequence of sequence 3 in the sequence table from 5 '.
In order to improve expression activity, (SEQ ID №: 6) have SEQ ID № in the sequence table through being connected with after modifying: the activation subsequence of 7 dna sequence dna obtains having SEQ ID № in the sequence table: 9 dna sequence dna to the Nitrile hydratase encoding gene.
SEQ ID № in the sequence table: 9 by 1626 based compositions, from the β subunit of 5 ' end the 1st to the 690th bit base encoding nitrile hydratase; Have the Nitrile hydratase α subunit of the amino acid residue sequence of sequence 3 in the sequence table from 5 ' end the 704th to the 1315th bit base coding, activate son from 5 ' end the 1312nd to the 1626th bit base coding.
Contain expression carrier of the present invention and clone and all belong to protection scope of the present invention.
Second purpose of the present invention provides a kind of method of expressing Nitrile hydratase.
The method of expression Nitrile hydratase provided by the present invention is that the recombinant expression vector that will contain above-mentioned Nitrile hydratase encoding gene imports expressive host, expresses obtaining Nitrile hydratase.
Wherein, described host can be intestinal bacteria, yeast, mammalian cell, insect cell, Bacillus subtilus, lactobacillus etc., is preferably intestinal bacteria, especially is preferably E.coli BL21 (DE3).
In present method, the expression vector that is used for inserting above-mentioned Nitrile hydratase encoding gene can be the carrier of expressing above-mentioned host, as can be at pET, the pUC of expression in escherichia coli, the pPIC9K that can express in Pichia yeast.The recombinant expression vector that contains above-mentioned Nitrile hydratase encoding gene can make up according to ordinary method.
Experiment shows, the encoding gene of Nitrile hydratase of the present invention can efficiently express in recombination bacillus coli, the Nitrile hydratase of gained is active and stability is high, thereby, will in the production of acrylamide, play an important role for carrying out the rite-directed mutagenesis of Nitrile hydratase gene and good basis has been established in the bio-transformation of orthogenesis transformation and acrylamide.
Description of drawings
Fig. 1 is (pET32a-NHBAX) the SDS-PAGE electrophoretogram of full cell of genetic engineering bacterium E.coli BL21 (DE3)
Fig. 2 is genetic engineering bacterium E.coli XL1-Blue (pUC18-NHase
M) the SDS-PAGE electrophoretogram of full cell
Embodiment
The clone of embodiment 1, Nitrile hydratase structure gene NHBA
(1) carries out the cultivation of Nocardia bacteria (Nocardia sp.) and the chromosomal DNA of extraction thalline
From the 9mm culture dish with aseptic transfering loop picking Nocardia bacteria list colony inoculation in the 500ml triangular flask that the 50mL liquid fermentation medium is housed.Consisting of of this fermention medium: glucose 10g/L; Yeast extract paste 5g/L; Urea 2.5g/L; Na
2HPO
40.5g/L; Na
2HPO
40.5g/L; MgCl
26H
2O 0.5g/L; Sodium Glutamate 0.25g/L; CoCl
260ppm; PH7.0.In temperature is that 28 ℃, rotating speed are to cultivate 72 hours in the shaking table of 200rpm.The centrifugal collection thalline of 10000rpm * 5min, the 10ml sterilized water is resuspended, the centrifugal collection thalline of 10000rpm * 5min, with the 40mg/ml N,O-Diacetylmuramidase to the processing of spending the night of gained cell, Wizard Genomic DNAPurification Kit with Promega company extracts and purified chromosomal dna, and is standby.
(2) the utilization round pcr is from chromosomal DNA amplification purpose Nitrile hydratase enzyme gene fragment
Design and synthesize the upstream and the downstream primer of Nitrile hydratase enzyme gene.Four kinds of involved Nucleotide are respectively: triphosphoric acid deoxyadenylic acid (being designated as A), triphosphoric acid deoxy-guanine triphosphoric acid (being designated as G), triphosphoric acid deoxycytidylic acid (being designated as C), triphosphoric acid deoxythymidine acid (being designated as T), institute's synthetic primer one, form by 21 nucleotide residues, be followed successively by: 5 '-phosphoric acid-C-A-G-A-A-T-T-C-A-C-G-T-C-C-C-C-G-T-A-G-T-phosphoric acid-3 '; Institute's synthetic primer two is made up of 20 nucleotide residues, is followed successively by: 5 '-phosphoric acid-A-A-G-G-A-T-C-C-G-A-A-A-G-C-G-C-G-A-T-G-phosphoric acid-3 '.Primer is synthetic by match Parkson, Beijing bio-engineering corporation.After primer is synthetic, is dissolved to concentration with sterilized water and is 50 μ mol/L, standby.The utilization round pcr is from chromosomal DNA amplification purpose Nitrile hydratase gene DNA fragment, and used polysaccharase and corresponding amplification buffer, four kinds of deoxynucleoside acid solutions are buied by precious biotechnology (Dalian) company limited.In a sterilized 0.2mL PCR thin-walled tube, add the chromosomal DNA solution, the EX Taq archaeal dna polymerase of 0.5 μ L of step (1) preparation of primer two, the 8 μ L of primer one, the 1 μ L of four kinds of deoxynucleotides, the 1 μ L of amplification buffer, the 5 μ L of 18uL sterilized water, 5 μ L successively, complementing to cumulative volume with sterilized water is 50 μ L; Centrifuge tube is placed temperature varying system, be warming up to 94 ℃, kept 5 minutes, then by be warming up to 94 ℃ and keep 1 minute, be cooled to 50 ℃ and keep 1.5 minutes, be warming up to 72 ℃ and keep 3 minutes variation sequential loop 30 times, kept the end amplified reaction 10 minutes in 72 ℃ at last.
(3) Nitrile hydratase gene DNA fragment and the plasmid pUC18 that the previous step amplification is obtained adopts restriction endonuclease cutting respectively.
Plasmid pUC18 and restriction endonuclease and corresponding damping fluid are buied by precious biotechnology (Dalian) company limited.Get 10 μ l (2) step gained PCR product and 4 μ l pUC18 plasmid DNA and carry out enzyme respectively and cut, the composition of endonuclease reaction system is respectively:
PCR product 10 μ l pUC18 plasmid DNA 4 μ l
EcoRI(10U/μl) 1μl EcoRI(10U/μl) 1μl
BamHI(10U/μl) 1μl BamHI(10U/μl) 1μl
Damping fluid (10 * Buffer K) 2 μ l damping fluids (10 * Buffer K) 2 μ l
Sterilized water 6 μ l sterilized waters 12 μ l
Cumulative volume 20 μ l cumulative volumes 20 μ l
Reacted 4 hours down at 37 ℃, adopt conventional 0.7% agarose gel electrophoresis, obtaining length is that the enzyme that the enzyme about 2600bp is cut about back pUC18 plasmid DNA fragment and 1.7kb is cut back PCR product.
(4) the PCR product that step (3) is obtained is connected with pUC18 plasmid DNA endonuclease bamhi
Used T4DNA ligase enzyme and corresponding damping fluid are buied by Promega company.Connecting sample sets becomes:
Nitrile hydratase gene DNA fragment 6 μ l
PUC18 endonuclease bamhi (50ng/ μ l) 2.5 μ l
Connect damping fluid 1 μ l
T4 ligase enzyme (3U/ μ l) 0.5 μ l
Cumulative volume 10 μ l
Connecting sample reacted 8 hours down at 16 ℃.
(5) prepare colibacillary competent cell (Calcium Chloride Method)
Adopt Calcium Chloride Method to prepare colibacillary competent cell, used intestinal bacteria TOP-10F ' is buied by Invitrogen company.Picking list bacterium colony from the solid plate of thalline is inoculated in the 5mL liquid nutrient medium.This substratum is the LB substratum.Under 37 ℃ on 200 rev/mins shaking table culturing bacterium 10 hours, then get the 0.5mL nutrient solution and be inoculated in the 50mL LB liquid nutrient medium, be 0.4 on 200 rev/mins shaking table, being cultured to the light absorption value of nutrient solution under 37 ℃ at the 600nm place; Nutrient solution is changed in the centrifuge tube of sterilization of precooling, the ice bath cooling is after 10 minutes, and is centrifugal 10 minutes with 4000 rev/mins on 4 ℃ whizzer; Pour out supernatant liquor, under aseptic condition, will manage and be inverted 1 minute, so that residual supernatant liquor flows to end; In precipitation, add 10 milliliters of ice-cold 0.1mmol/L CaCl
2Solution, ice bath was placed 10 minutes; On 4 ℃ whizzer with 4000 rev/mins centrifugal 10 minutes, abandon supernatant; 0.1mmol/L CaCl with the precooling of 2mL ice
2The resuspended once more precipitation of solution is placed on ice and is promptly obtained competent cell a moment.
(6) gene clone of Nitrile hydratase gene DNA fragment
The gene clone agents useful for same isopropyl-of Nitrile hydratase gene DNA fragment (being called for short IPTG) and 5-bromo-4-chloro-3-indoles-β-D-galactoside (being called for short X-gal) are buied by precious biotechnology (Dalian) company limited.Get 200 μ L competence intestinal bacteria TOP-10F ' cells, add 5 μ L step (4) gained ligation things, placed 30 minutes on ice, immediately pipe is transferred in 42 ℃ the circulator bath, placed 90 seconds, immediately pipe is transferred in the ice bath, placed 2 minutes, every pipe adds the LB liquid nutrient medium of 0.8mL, placed 45 minutes down at 37 ℃, under the room temperature centrifugal 2 minutes with 4000 rev/mins speed, abandon supernatant, stay about 100 μ L, coat after shaking up and contain 40 μ g/mL IPTG, on the LB solid medium of 40 μ g/mL X-gal and 15g/L agar powder, screen the white colony of no α-Hu Bu ability, picking colony inserts the LB substratum, extracts plasmid DNA according to ordinary method.
(7) plasmid DNA of step (6) results is carried out enzyme and cut checking, cut with EcoRI and BamHI, the product that enzyme is cut carries out 0.7% agarose gel electrophoresis again, the result obtains the band of a 1.7kb and the band of a 2.6kb, and its length is respectively (2) and goes on foot the length of the PCR product that obtains and the length of the linearizing pUC18 plasmid DNA that step (3) obtains.Illustrate that recombinant plasmid pUC18-NHBA successfully constructs.
(8) recombinant plasmid is carried out dna sequencing, sequencing result shows that gained gene NHBA has the polynucleotide sequence shown in the sequence 5 in the sequence table, SEQ ID № in the sequence table: 5 by 1714 based compositions, from the β subunit of 5 ' end the 268th to the 957th bit base encoding nitrile hydratase; Hold the 971st to the 1582nd bit base coding to have the Nitrile hydratase α subunit of the amino acid residue sequence of sequence 1 in the sequence table from 5 '.
Embodiment 2, the clone who carries the Nitrile hydratase gene NHBAx that activates subsequence
(1) carry out the chromosomal DNA that thalline was cultivated and extracted to Nocardia bacteria (Nocardia sp.), this method is with embodiment 1 (1).
(2) the utilization round pcr carries the Nitrile hydratase enzyme gene fragment that activates subsequence from the chromosomal DNA amplification
Design and synthesize the upstream and the downstream primer that carry the Nitrile hydratase enzyme gene that activates subsequence.Four kinds of involved Nucleotide are respectively: triphosphoric acid deoxyadenylic acid (being designated as A), triphosphoric acid deoxy-guanine triphosphoric acid (being designated as G), triphosphoric acid deoxycytidylic acid (being designated as C), triphosphoric acid deoxythymidine acid (being designated as T), institute's synthetic primer three, form by 29 nucleotide residues, be followed successively by: 5 '-phosphoric acid-A-A-T-G-G-A-T-C-C-A-T-G-G-A-T-G-G-T-A-T-C-C-A-C-G-A-C-A-C-phosphoric acid-3 '; Institute's synthetic primer four is made up of 28 nucleotide residues, is followed successively by: 5 '-phosphoric acid-C-G-G-A-A-T-T-C-T-C-A-G-T-C-G-A-T-G-A-T-G-G-C-C-A-T-C-G-phosphoric acid-3 '.Primer is synthetic by match Parkson, Beijing bio-engineering corporation.After primer is synthetic, is dissolved to concentration with sterilized water and is 50 μ mol/L, standby.The utilization round pcr is from chromosomal DNA amplification purpose Nitrile hydratase gene DNA fragment, and used polysaccharase and corresponding amplification buffer, four kinds of deoxynucleoside acid solutions are buied by precious biotechnology (Dalian) company limited.In a sterilized 0.2mL PCR thin-walled tube, add the chromosomal DNA solution, the EX Taq archaeal dna polymerase of 0.5 μ L of step (1) preparation of primer four, the 8 μ L of primer three, the 1 μ L of four kinds of deoxynucleotides, the 1 μ L of amplification buffer, the 5 μ L of 18 μ L sterilized waters, 5 μ L successively, complementing to cumulative volume with sterilized water is 50 μ L; Centrifuge tube is placed temperature varying system, be warming up to 94 ℃, kept 5 minutes, then by be warming up to 94 ℃ and keep 1 minute, be cooled to 61 ℃ and keep 1.5 minutes, be warming up to 72 ℃ and keep 2 minutes variation sequential loop 25 times, kept the end amplified reaction 10 minutes in 72 ℃ at last.
(3) carry the Nitrile hydratase gene DNA fragment and the plasmid pET32a that activate subsequence that the previous step amplification is obtained adopts restriction endonuclease cutting respectively.
Plasmid pET32a and restriction endonuclease and corresponding damping fluid are buied by precious biotechnology (Dalian) company limited.Get 10 μ l (2) step gained PCR product and 6 μ l pET32a plasmid DNA and carry out enzyme respectively and cut, the composition of endonuclease reaction system is respectively:
PCR product 10 μ l pUC18 plasmid DNA 6 μ l
EcoRI(10U/μl) 1μl EcoRI(10U/μl) 1μl
BamHI(10U/ul) 1μl BamHI(10U/μl) 1μl
Damping fluid (10 * Buffer K) 2 μ l damping fluids (10 * Buffer K) 2 μ l
Sterilized water 6 μ l sterilized waters 10 μ l
Cumulative volume 20 μ l cumulative volumes 20 μ l
Reacted 4 hours down at 37 ℃, adopt conventional 0.7% agarose gel electrophoresis, obtaining length is that the enzyme that the enzyme about 5900bp is cut about back pET32a plasmid DNA fragment and 1.6kb is cut back PCR product.
(4) the PCR product that step (3) is obtained is connected with pUC18 plasmid DNA endonuclease bamhi
Used T4DNA ligase enzyme and corresponding damping fluid are buied by Promega company.Connecting sample sets becomes:
Nitrile hydratase gene DNA fragment 5 μ l
PET32a endonuclease bamhi (20ng/ μ l) 3.5 μ l
Connect damping fluid 1 μ l
T4 ligase enzyme (3U/ μ l) 0.5 μ l
Cumulative volume 10 μ l
Connecting sample reacted 8 hours down at 16 ℃.
(5) method according to embodiment 1 (5) prepares colibacillary competent cell.
(6) carry the clone of the Nitrile hydratase gene that activates subsequence
The agents useful for same sodium ampicillin is buied by Beijing ancient cooking vessel state biotechnology limited liability company.Get 200 μ L competence intestinal bacteria TOP-10F ' cells, add 5 μ L step (4) gained ligation things, placed 30 minutes on ice, immediately pipe is transferred in 42 ℃ the circulator bath, placed 90 seconds, immediately pipe is transferred in the ice bath, placed 2 minutes, every pipe adds the LB liquid nutrient medium of 0.8mL, placed 45 minutes down at 37 ℃, under the room temperature centrifugal 2 minutes with 4000 rev/mins speed, abandon supernatant, stay about 100 μ L, coat after shaking up on the LB solid medium that contains 100 μ g/mL sodium ampicillins and 20g/L agar powder, 37 ℃ of incubations are after 12 hours, and picking colony inserts the LB substratum, extract plasmid DNA according to ordinary method.
(7) plasmid DNA of (6) results is carried out enzyme and cut checking, cut with EcoRI and BamHI, the product that enzyme is cut carries out 0.7% agarose gel electrophoresis again, the result obtains the band of a 1.6kb and the band of a 5.9kb, and its length is respectively the length that (2) length and (3) that goes on foot the PCR product that obtains goes on foot the linearizing pET32a plasmid DNA that obtains.Illustrate that recombinant plasmid pET32a-NHBAX successfully constructs.
(8) recombinant plasmid is carried out dna sequencing, sequencing result shows that gained gene NHBAX has the polynucleotide sequence shown in the sequence 8 in the sequence table, SEQ ID № in the sequence table: 8 by 1626 based compositions, from the β subunit of 5 ' end the 1st to the 690th bit base encoding nitrile hydratase; Have the Nitrile hydratase α subunit of the amino acid residue sequence of sequence 3 in the sequence table from 5 ' end the 704th to the 1315th bit base coding, activate son from 5 ' end the 1312nd to the 1626th bit base coding.
The expression of embodiment 3. Nitrile hydratases
1, the structure of recombinant plasmid and recombinant bacterial strain
The recombinant plasmid pUC18-NHBA that makes up respectively among embodiment 1 and the embodiment 2 and pET32a-NHBAX according to Calcium Chloride Method transformed host cell E.coli BL21 (DE3), are obtained genetic engineering bacterium E.coli JM105 (pUC18-NHBA) and E.coli BL21 (DE3) respectively (pET32a-NHBAX).
2, the abduction delivering of Nitrile hydratase in the genetic engineering bacterium
(1) genetic engineering bacterium is expressed the cultivation and the inductive condition of Nitrile hydratase
The genetic engineering bacterium that obtains in the step 1 is inoculated in respectively in the LB liquid nutrient medium (consisting of of LB substratum: yeast powder 0.5%, Tryptones 1%, NaCl 1%, pH7.0), 37 ℃ of 200rpm shaking table overnight incubation.Switching 30mL bacterium liquid shakes in the bottle to the 300mL of the LB substratum that contains 50 μ g/mL penbritins, adds IPTG, the 10 μ L CoCl of 0.5mmol/L simultaneously
2Induce, 30 ℃ are continued to cultivate about 15h, centrifugal collection thalline down.The thalline of collecting is carried out full bacterium SDS-PAGE electrophoresis, show that genetic engineering bacterium E.coli BL21 (DE3) (pET32a-NHBAX) band all occurred at the β of wild Nocardia bacteria Nitrile hydratase and the corresponding position of α subunit, the band of β subunit is obvious, the band of α subunit is weak (as shown in Figure 1, among the figure, swimming lane 1 is (pET32a-NHBAX) the SDS-PAGE electrophoresis of full cell of genetic engineering bacterium E.coli BL21 (DE3), swimming lane 2 is protein molecular weight marker, and swimming lane 3 is the SDS-PAGE electrophoresis of the full cell of wild Nocardia bacteria sample); And on the SDS-PAGE electrophoretogram of genetic engineering bacterium E.coli JM105 (pUC18-NHBA), two bands are all very weak.
(2) genetic engineering bacterium catalyzing propone nitrile generates acrylamide
The genetic engineering bacterium liquid of abduction delivering in the step (1) is centrifugal respectively, collect thalline.(pH7.2) is resuspended in the 50mL triangular flask with the 0.05mol/L phosphate buffered saline buffer, and adding final concentration is 0.5% vinyl cyanide liquid, and 28 ℃, 200rpm shaking table accurate response 10min.And adding 1ml hydrochloric acid stopped reaction, utilize GC-9AM gas chromatograph detection reaction product to measure the activity that genetic engineering bacterium catalyzing propone nitrile generates acrylamide, the result shows that the activity of Nitrile hydratase NHBA is 0, and the activity of NHBAX is the dried bacterium of 0.04-0.06U/mg (1U refers to that 1 minute endoenzyme can react generation 1 μ mol acrylamide).
Clone and the expression of embodiment 4, Nitrile hydratase gene mNHBA
(1) designs and synthesizes upstream and the downstream primer that on Nitrile hydratase α subunit, to undergo mutation
Four kinds of involved Nucleotide are respectively: triphosphoric acid deoxyadenylic acid (being designated as A), triphosphoric acid deoxy-guanine triphosphoric acid (being designated as G), triphosphoric acid deoxycytidylic acid (being designated as C), triphosphoric acid deoxythymidine acid (being designated as T), institute's synthetic primer five, form by 24 nucleotide residues, be followed successively by: 5 '-phosphoric acid-A-T-G-A-G-C-G-A-G-C-A-C-G-T-C-A-A-T-A-A-G-T-A-C-phosphoric acid-3 '; Institute's synthetic primer six is made up of 24 nucleotide residues, is followed successively by: 3 '-phosphoric acid-T-A-G-A-G-A-C-G-C-A-C-T-T-T-C-C-T-T-A-T-G-C-T-Ap-phosphoric acid-5 '.Wherein, 5 ' of primer six terminal base A carries out phosphorylation.Primer is synthetic by match Parkson, Beijing bio-engineering corporation.After primer is synthetic, is dissolved to concentration with sterilized water and is 50 μ mol/L, standby.
(2) carry out the operation of Nitrile hydratase site-directed point mutation according to the method for test kit.
The rite-directed mutagenesis test kit ExSite that adopts
TMPCR-Based Site-Directed Mutagenesis Kit is available from Stratagene company.Bacterial strain E.coli JM105 (pUC18-NHase) with structure among the embodiment 3 is the template of sudden change, utilize step (1) design synthetic primer, operation instructions in strict accordance with the sudden change test kit is carried out PCR reaction, enzyme is cut, connects, transformed, and obtains genetic engineering bacterium E.coli XL1-Blue (pUC18-NHase
M), and check order, the result obtains having the mNHBA of the polynucleotide sequence shown in the sequence 6 in the sequence table, the β subunit from 5 ' end the 268th to the 957th bit base encoding nitrile hydratase of sequence 6; Hold the 971st to the 1582nd bit base coding to have the Nitrile hydratase α subunit of the amino acid residue sequence of sequence 3 in the sequence table from 5 '.
(3) with genetic engineering bacterium E.coli XL1-Blue (pUC18-NHase
M) on the LB solid medium of 100 μ g/mL sodium ampicillins and 20g/L agar powder, 37 ℃ of incubations are after 12 hours, picking colony inserts the LB substratum, under condition similarly to Example 3, cultivate and induce, the result shows before the Nitrile hydratase specific activity of expression is suddenlyd change tangible increase, and its activity is 51U/mg.The thalline of collecting is carried out full bacterium SDS-PAGE electrophoresis, and the result shows genetic engineering bacterium E.coli XL1-Blue (pUC18-NHase as shown in Figure 2
M) at genetic engineering bacterium E.coli BL21 (DE3) (pET32a-NHBAX) on the corresponding position of α subunit that expression amount is extremely low significantly band has appearred.Among Fig. 2, swimming lane 1 is protein molecular weight marker, and swimming lane 2 is wild Nocardia bacteria sample, and swimming lane 3 is E.coli XL1-Blue (pUC18-NHase
M), swimming lane 4 be genetic engineering bacterium E.coli BL21 (DE3) (pET32a-NHBAX).
Embodiment 5, carry the Nitrile hydratase gene mNHBAX clone who activates subsequence and express
(1) carries the Nitrile hydratase gene mNHBAX clone who activates subsequence
The rite-directed mutagenesis test kit ExSite that adopts
TMPCR-Based Site-Directed Mutagenesis Kit is available from Stratagene company.Bacterial strain E.coli BL21 (DE3) with structure among the embodiment 2 is the template of sudden change (pET32a-NHBAX), utilize embodiment 4 steps (1) design synthetic primer, operation instructions in strict accordance with the sudden change test kit is carried out PCR reaction, enzyme is cut, connects, transformed, and obtains genetic engineering bacterium E.coli XL1-Blue (pUC18-NHBAX
M), and check order, sequencing result shows that gained gene mAHBAX has the polynucleotide sequence shown in the sequence 9 in the sequence table, SEQ ID № in the sequence table: 9 by 1626 based compositions, from the β subunit of 5 ' end the 1st to the 690th bit base encoding nitrile hydratase; Have the Nitrile hydratase α subunit of the amino acid residue sequence of sequence 3 in the sequence table from 5 ' end the 704th to the 1315th bit base coding, activate son from 5 ' end the 1312nd to the 1626th bit base coding.
(2) carry the expression of the Nitrile hydratase gene mNHBAX that activates subsequence
With genetic engineering bacterium E.coli XL1-Blue (pUC18-NHBAX
M) on the LB solid medium of 100 μ g/mL sodium ampicillins and 20g/L agar powder, 37 ℃ of incubations are after 12 hours, picking colony inserts the LB substratum, under condition similarly to Example 3, cultivate and induce, the result shows before the Nitrile hydratase specific activity of expression is suddenlyd change tangible increase, and its activity is 8U/mg.
Sequence table
<160>9
<210>1
<211>203
<212>PRT
＜213〉Nocardia bacteria (Nocardia sp.)
<400>1
Val?Ser?Glu?His?Val?Asn?Lys?Tyr?Thr?Glu?Tyr?Glu?Ala?Arg?Thr?Lys
1 5 10 15
Ala?Ile?Glu?Thr?Leu?Leu?Tyr?Glu?Arg?Gly?Leu?Ile?Thr?Pro?Ala?Ala
202 5 30
Val?Asp?Arg?Val?Val?Ser?Tyr?Tyr?Glu?Asn?Glu?Ile?Gly?Pro?Met?Gly
35 40 45
Gly?Ala?Lys?Val?Val?Ala?Lys?Ser?Trp?Val?Asp?Pro?Glu?Tyr?Arg?Lys
50 55 60
Trp?Leu?Glu?Glu?Asp?Ala?Thr?Ala?Ala?Met?Ala?Ser?Leu?Gly?Tyr?Ala
65 70 75 80
Gly?Glu?Gln?Ala?His?Gln?Ile?Ser?Ala?Val?Phe?Asn?Asp?Ser?Gln?Thr
85 90 95
His?His?Val?Val?Val?Cys?Thr?Leu?Cys?Ser?Cys?Tyr?Pro?Trp?Pro?Val
100 105 110
Leu?Gly?Leu?Pro?Pro?Ala?Trp?Tyr?Lys?Ser?Met?Glu?Tyr?Arg?Ser?Arg
115 120 125
Val?Val?Ala?Asp?Pro?Arg?Gly?Val?Leu?Lys?Arg?Asp?Phe?Gly?Phe?Asp
130 135 140
Ile?Pro?Asp?Glu?Val?Glu?Val?Arg?Val?Trp?Asp?Ser?Ser?Ser?Glu?Ile
145 150 155 160
Arg?Tyr?Ile?Val?Ile?Pro?Glu?Arg?Pro?Ala?Gly?Thr?Asp?Gly?Trp?Ser
165 170 175
Glu?Asp?Glu?Leu?Thr?Lys?Leu?Val?Ser?Arg?Asp?Ser?Met?Ile?Gly?Val
180 185 190
Ser?Asn?Ala?Leu?Thr?Pro?Gln?Glu?Val?Ile?Val
195 200
<210>2
<211>229
<212>PRT
＜213〉Nocardia bacteria (Nocardia sp.)
<400>2
Met?Asp?Gly?Ile?His?Asp?Thr?Gly?Gly?Met?Thr?Gly?Tyr?Gly?Pro?Val
1 5 10 15
Pro?Tyr?Gln?Lys?Asp?Glu?Pro?Phe?Phe?His?Tyr?Glu?Trp?Glu?Gly?Arg
20 25 30
Thr?Leu?Ser?Ile?Leu?Thr?Trp?Met?His?Leu?Lys?Gly?Met?Ser?Trp?Trp
35 40 45
Asp?Lys?Ser?Arg?Phe?Phe?Arg?Glu?Ser?Met?Gly?Asn?Glu?Asn?Tyr?Val
50 55 60
Asn?Glu?Ile?Arg?Asn?Ser?Tyr?Tyr?Thr?His?Trp?Leu?Ser?Ala?Ala?Glu
65 70 75 80
Arg?Ile?Leu?Val?Ala?Asp?Lys?Ile?Ile?Thr?Glu?Glu?Glu?Arg?Lys?His
85 90 95
Arg?Val?Gln?Glu?Ile?Leu?Glu?Gly?Arg?Tyr?Thr?Asp?Arg?Asn?Pro?Ser
100 105 110
Arg?Lys?Phe?Asp?Pro?Ala?Glu?Ile?Glu?Lys?Ala?Ile?Glu?Arg?Leu?His
115 120 125
Glu?Pro?His?Ser?Leu?Ala?Leu?Pro?Gly?Ala?Glu?Pro?Ser?Phe?Ser?Leu
130 135 140
Gly?Asp?Lys?Val?Lys?Val?Lys?Asn?Met?Asn?Pro?Leu?Gly?His?Thr?Arg
145 150 155 160
Cys?Pro?Lys?Tyr?Val?Arg?Asn?Lys?Ile?Gly?Glu?Ile?Val?Thr?Ser?His
165 170 175
Gly?Cys?Gln?Ile?Tyr?Pro?Glu?Ser?Ser?Ser?Ala?Gly?Leu?Gly?Asp?Asp
180 185 190
Pro?Arg?Pro?Leu?Tyr?Thr?Val?Ala?Phe?Ser?Ala?Gln?Glu?Leu?Trp?Gly
195 200 205
Asp?Asp?Gly?Asn?Gly?Lys?Asp?Val?Val?Cys?Val?Asp?Leu?Trp?Glu?Pro
210 215 220
Tyr?Leu?Ile?Ser?Ala
225
<210>3
<211>203
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>3
Met?Ser?Glu?His?Val?Asn?Lys?Tyr?Thr?Glu?Tyr?Glu?Ala?Arg?Thr?Lys
1 5 10 15
Ala?Ile?Glu?Thr?Leu?Leu?Tyr?Glu?Arg?Gly?Leu?Ile?Thr?Pro?Ala?Ala
20 25 30
Val?Asp?Arg?Val?Val?Ser?Tyr?Tyr?Glu?Asn?Glu?Ile?Gly?Pro?Met?Gly
35 40 45
Gly?Ala?Lys?Val?Val?Ala?Lys?Ser?Trp?Val?Asp?Pro?Glu?Tyr?Arg?Lys
50 55 60
Trp?Leu?Glu?Glu?Asp?Ala?Thr?Ala?Ala?Met?Ala?Ser?Leu?Gly?Tyr?Ala
65 70 75 80
Gly?Glu?Gln?Ala?His?Gln?Ile?Ser?Ala?Val?Phe?Asn?Asp?Ser?Gln?Thr
85 90 95
His?His?Val?Val?Val?Cys?Thr?Leu?Cys?Ser?Cys?Tyr?Pro?Trp?Pro?Val
100 105 110
Leu?Gly?Leu?Pro?Pro?Ala?Trp?Tyr?Lys?Ser?Met?Glu?Tyr?Arg?Ser?Arg
115 120 125
Val?Val?Ala?Asp?Pro?Arg?Gly?Val?Leu?Lys?Arg?Asp?Phe?Gly?Phe?Asp
130 135 140
Ile?Pro?Asp?Glu?Val?Glu?Val?Arg?Val?Trp?Asp?Ser?Ser?Ser?Glu?Ile
145 150 155 160
Arg?Tyr?Ile?Val?Ile?Pro?Glu?Arg?Pro?Ala?Gly?Thr?Asp?Gly?Trp?Ser
165 170 175
Glu?Asp?Glu?Leu?Thr?Lys?Leu?Val?Ser?Arg?Asp?Ser?Met?Ile?Gly?Val
180 185 190
Ser?Asn?Ala?Leu?Thr?Pro?Gln?Glu?Val?Ile?Val
195 200
<210>4
<211>104
<212>PRT
＜213〉Nocardia bacteria (Nocardia sp.)
<400>4
Met?Ser?Glu?Asp?Thr?Leu?Thr?Asp?Arg?Leu?Pro?Ala?Thr?Gly?Thr?Ala
1 5 10 15
Ala?Pro?Pro?Arg?Asp?Asn?Gly?Glu?Leu?Val?Phe?Thr?Glu?Pro?Trp?Glu
20 25 30
Ala?Thr?Ala?Phe?Gly?Val?Ala?Ile?Ala?Leu?Ser?Asp?Gln?Lys?Ser?Tyr
35 40 45
Glu?Trp?Glu?Phe?Phe?Arg?Gln?Arg?Leu?Ile?His?Ser?Ile?Ala?Glu?Ala
50 55 60
Asn?Gly?Cys?Glu?Ala?Tyr?Tyr?Glu?Ser?Trp?Thr?Lys?Ala?Leu?Glu?Ala
65 70 75 80
Ser?Val?Val?Asp?Ser?Gly?Leu?Ile?Ser?Glu?Asp?Glu?Ile?Arg?Glu?Arg
85 90 95
Met?Glu?Ser?Met?Ala?Ile?Ile?Asp
100
<210>5
<211>1714
<212>DNA
＜213〉Nocardia bacteria (Nocardia sp.)
<400>5
cagaattcac?gtccccgtag?tgtgcgggga?gagcgcccga?ccgcagggat?ggtatccatg 60
cgccccttct?cttttcgaac?gagaaccggc?cgctacagcc?gacccggaga?cactgtgacg 120
ccgttcaacg?attgttgtgc?tgtgaaggat?tcactcaagc?caactgatat?cgccattccg 180
ttgccggaac?atttgacacc?ttctccctac?gagtagaagc?cagctggacc?cctctttgag 240
cccagctccg?atgaaaggaa?tgaggaaatg?gatggtatcc?acgacacagg?cggcatgacc 300
ggatacggac?cggtccccta?tcagaaggac?gagcccttct?tccactacga?gtgggagggt 360
cggaccctgt?cgattctgac?ctggatgcat?ctcaagggca?tgtcgtggtg?ggacaagtcg 420
cggttcttcc?gggagtcgat?ggggaacgaa?aactacgtca?acgagattcg?caactcgtac 480
tacacccact?ggctgagtgc?ggcagaacgt?atcctcgtcg?ccgacaagat?catcaccgaa 540
gaagagcgaa?agcaccgtgt?gcaggagatc?ctcgagggtc?ggtacacgga?caggaacccg 600
tcgcggaagt?tcgatccggc?cgagatcgag?aaggcgatcg?aacggcttca?cgagccccac 660
tccctagcac?ttccaggagc?ggagccgagt?ttctccctcg?gtgacaaggt?caaagtgaag 720
aatatgaacc?cgctgggaca?cacacggtgc?ccgaaatatg?tgcggaacaa?gatcggggaa 780
atcgtcacct?cccacggctg?ccagatctat?cccgagagca?gctccgccgg?cctcggcgac 840
gatccccgcc?cgctctacac?ggtcgcgttt?tccgcccagg?aactgtgggg?cgacgacgga 900
aacgggaaag?acgtagtgtg?cgtcgatctc?tgggaaccgt?acctgatctc?tgcgtgaaag 960
gaatacgata?gtgagcgagc?acgtcaataa?gtacacggag?tacgaggcac?gtaccaaggc 1020
aatcgaaact?ttgctgtacg?agcgagggct?catcacgccc?gccgcggtcg?accgagtcgt 1080
ttcgtactac?gagaacgaga?tcggcccgat?gggcggtgcc?aaggtcgtgg?cgaagtcctg 1140
ggtggaccct?gagtaccgca?agtggctcga?agaggacgcg?acggccgcga?tggcgtcatt 1200
gggctatgcc?ggtgagcagg?cacaccaaat?ttcggcggtc?ttcaacgact?cccaaacgca 1260
tcacgtggtg?gtgtgcactc?tgtgttcgtg?ctatccgtgg?ccggtgcttg?gtctcccgcc 1320
cgcctggtac?aagagcatgg?agtaccggtc?ccgagtggta?gcggaccctc?gtggagtgct 1380
caagcgcgat?ttcggtttcg?acatccccga?tgaggtggag?gtcagggttt?gggacagcag 1440
ctccgaaatc?cgctacatcg?tcatcccgga?acggccggcc?ggcaccgacg?gttggtccga 1500
ggacgagctg?gcgaagctgg?tgagccggga?ctcgatgatc?ggtgtcagta?atgcgctcac 1560
accccaggaa?gtgatcgtat?gagtgaagac?acactcactg?atcggctccc?ggcgactggg 1620
accgccgcac?cgccccgcga?caatggcgag?cttgtattca?ccgagccttg?ggaagcaacg 1680
gcatacgggg?tcgccatcgc?gctttcggat?cctt 1714
<210>6
<211>1714
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>6
cagaattcac?gtccccgtag?tgtgcgggga?gagcgcccga?ccgcagggat?ggtatccatg 60
cgccccttct?cttttcgaac?gagaaccggc?cgctacagcc?gacccggaga?cactgtgacg 120
ccgttcaacg?attgttgtgc?tgtgaaggat?tcactcaagc?caactgatat?cgccattccg 180
ttgccggaac?atttgacacc?ttctccctac?gagtagaagc?cagctggacc?cctctttgag 240
cccagctccg?atgaaaggaa?tgaggaaatg?gatggtatcc?acgacacagg?cggcatgacc 300
ggatacggac?cggtccccta?tcagaaggac?gagcccttct?tccactacga?gtgggagggt 360
cggaccctgt?cgattctgac?ctggatgcat?ctcaagggca?tgtcgtggtg?ggacaagtcg 420
cggttcttcc?gggagtcgat?ggggaacgaa?aactacgtca?acgagattcg?caactcgtac 480
tacacccact?ggctgagtgc?ggcagaacgt?atcctcgtcg?ccgacaagat?catcaccgaa 540
gaagagcgaa?agcaccgtgt?gcaggagatc?ctcgagggtc?ggtacacgga?caggaacccg 600
tcgcggaagt?tcgatccggc?cgagatcgag?aaggcgatcg?aacggcttca?cgagccccac 660
tccctagcac?ttccaggagc?ggagccgagt?ttctccctcg?gtgacaaggt?caaagtgaag 720
aatatgaacc?cgctgggaca?cacacggtgc?ccgaaatatg?tgcggaacaa?gatcggggaa 780
atcgtcacct?cccacggctg?ccagatctat?cccgagagca?gctccgccgg?cctcggcgac 840
gatccccgcc?cgctctacac?ggtcgcgttt?tccgcccagg?aactgtgggg?cgacgacgga 900
aacgggaaag?acgtagtgtg?cgtcgatctc?tgggaaccgt?acctgatctc?tgcgtgaaag 960
gaatacgata?atgagcgagc?acgtcaataa?gtacacggag?tacgaggcac?gtaccaaggc 1020
aatcgaaact?ttgctgtacg?agcgagggct?catcacgccc?gccgcggtcg?accgagtcgt 1080
ttcgtactac?gagaacgaga?tcggcccgat?gggcggtgcc?aaggtcgtgg?cgaagtcctg 1140
ggtggaccct?gagtaccgca?agtggctcga?agaggacgcg?acggccgcga?tggcgtcatt 1200
gggctatgcc?ggtgagcagg?cacaccaaat?ttcggcggtc?ttcaacgact?cccaaacgca 1260
tcacgtggtg?gtgtgcactc?tgtgttcgtg?ctatccgtgg?ccggtgcttg?gtctcccgcc 1320
cgcctggtac?aagagcatgg?agtaccggtc?ccgagtggta?gcggaccctc?gtggagtgct 1380
caagcgcgat?ttcggtttcg?acatccccga?tgaggtggag?gtcagggttt?gggacagcag 1440
ctccgaaatc?cgctacatcg?tcatcccgga?acggccggcc?ggcaccgacg?gttggtccga 1500
ggacgagctg?gcgaagctgg?tgagccggga?ctcgatgatc?ggtgtcagta?atgcgctcac 1560
accccaggaa?gtgatcgtat?gagtgaagac?acactcactg?atcggctccc?ggcgactggg 1620
accgccgcac?cgccccgcga?caatggcgag?cttgtattca?ccgagccttg?ggaagcaacg 1680
gcatacgggg?tcgccatcgc?gctttcggat?cctt 1714
<210>7
<211>315
<212>DNA
＜213〉Nocardia bacteria (Nocardia sp.)
<400>7
atgagtgaag?acacactcac?tgatcggctc?ccggcgactg?ggaccgccgc?accgccccgc 60
gacaatggcg?agcttgtatt?caccgagcct?tgggaagcaa?cggcatacgg?ggtcgccatc 120
gcgctttcgg?atcagaagtc?gtacgaatgg?gagttcttcc?gacagcgtct?cattcactcc 180
atcgctgagg?ccaacggttg?cgaggcatac?tacgagagct?ggacaaaggc?gctcgaggcc 240
agcgtggtcg?actcggggct?gatcagcgaa?gatgagatcc?gcgagcgcat?ggaatcgatg 300
gccatcatcg?actga 315
<210>8
<211>1626
<212>DNA
＜213〉Nocardia bacteria (Nocardia sp.)
<400>8
atggatggta?tccacgacac?aggcggcatg?accggatacg?gaccggtccc?ctatcagaag 60
gacgagccct?tcttccacta?cgagtgggag?ggtcggaccc?tgtcgattct?gacctggatg 120
catctcaagg?gcatgtcgtg?gtgggacaag?tcgcggttct?tccgggagtc?gatggggaac 180
gaaaactacg?tcaacgagat?tcgcaactcg?tactacaccc?actggctgag?tgcggcagaa 240
cgtatcctcg?tcgccgacaa?gatcatcacc?gaagaagagc?gaaagcaccg?tgtgcaggag 300
atcctcgagg?gtcggtacac?ggacaggaac?ccgtcgcgga?agttcgatcc?ggccgagatc 360
gagaaggcga?tcgaacggct?tcacgagccc?cactccctag?cacttccagg?agcggagccg 420
agtttctccc?tcggtgacaa?ggtcaaagtg?aagaatatga?acccgctggg?acacacacgg 480
tgcccgaaat?atgtgcggaa?caagatcggg?gaaatcgtca?cctcccacgg?ctgccagatc 540
tatcccgaga?gcagctccgc?cggcctcggc?gacgatcccc?gcccgctcta?cacggtcgcg 600
ttttccgccc?aggaactgtg?gggcgacgac?ggaaacggga?aagacgtagt?gtgcgtcgat 660
ctctgggaac?cgtacctgat?ctctgcgtga?aaggaatacg?ataatgagcg?agcacgtcaa 720
taagtacacg?gagtacgagg?cacgtaccaa?ggcaatcgaa?actttgctgt?acgagcgagg 780
gctcatcacg?cccgccgcgg?tcgaccgagt?cgtttcgtac?tacgagaacg?agatcggccc 840
gatgggcggt?gccaaggtcg?tggcgaagtc?ctgggtggac?cctgagtacc?gcaagtggct 900
cgaagaggac?gcgacggccg?cgatggcgtc?attgggctat?gccggtgagc?aggcacacca 960
aatttcggcg?gtcttcaacg?actcccaaac?gcatcacgtg?gtggtgtgca?ctctgtgttc 1020
gtgctatccg?tggccggtgc?ttggtctccc?gcccgcctgg?tacaagagca?tggagtaccg 1080
gtcccgagtg?gtagcggacc?ctcgtggagt?gctcaagcgc?gatttcggtt?tcgacatccc 1140
cgatgaggtg?gaggtcaggg?tttgggacag?cagctccgaa?atccgctaca?tcgtcatccc 1200
ggaacggccg?gccggcaccg?acggttggtc?cgaggacgag?ctggcgaagc?tggtgagccg 1260
ggactcgatg?atcggtgtca?gtaatgcgct?cacaccccag?gaagtgatcg?tatgagtgaa 1320
gacacactca?ctgatcggct?cccggcgact?gggaccgccg?caccgccccg?cgacaatggc 1380
gagcttgtat?tcaccgagcc?ttgggaagca?acggcatacg?gggtcgccat?cgcgctttcg 1440
gatcagaagt?cgtacgaatg?ggagttcttc?cgacagcgtc?tcattcactc?catcgctgag 1500
gccaacggtt?gcgaggcata?ctacgagagc?tggacaaagg?cgctcgaggc?cagcgtggtc 1560
gactcggggc?tgatcagcga?agatgagatc?cgcgagcgca?tggaatcgat?ggccatcatc 1620
gactga 1626
<210>9
<211>1626
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>9
atggatggta?tccacgacac?aggcggcatg?accggatacg?gaccggtccc?ctatcagaag 60
gacgagccct?tcttccacta?cgagtgggag?ggtcggaccc?tgtcgattct?gacctggatg 120
catctcaagg?gcatgtcgtg?gtgggacaag?tcgcggttct?tccgggagtc?gatggggaac 180
gaaaactacg?tcaacgagat?tcgcaactcg?tactacaccc?actggctgag?tgcggcagaa 240
cgtatcctcg?tcgccgacaa?gatcatcacc?gaagaagagc?gaaagcaccg?tgtgcaggag 300
atcctcgagg?gtcggtacac?ggacaggaac?ccgtcgcgga?agttcgatcc?ggccgagatc 360
gagaaggcga?tcgaacggct?tcacgagccc?cactccctag?cacttccagg?agcggagccg 420
agtttctccc?tcggtgacaa?ggtcaaagtg?aagaatatga?acccgctggg?acacacacgg 480
tgcccgaaat?atgtgcggaa?caagatcggg?gaaatcgtca?cctcccacgg?ctgccagatc 540
tatcccgaga?gcagctccgc?cggcctcggc?gacgatcccc?gcccgctcta?cacggtcgcg 600
ttttccgccc?aggaactgtg?gggcgacgac?ggaaacggga?aagacgtagt?gtgcgtcgat 660
ctctgggaac?cgtacctgat?ctctgcgtga?aaggaatacg?ataatgagcg?agcacgtcaa 720
taagtacacg?gagtacgagg?cacgtaccaa?ggcaatcgaa?actttgctgt?acgagcgagg 780
gctcatcacg?cccgccgcgg?tcgaccgagt?cgtttcgtac?tacgagaacg?agatcggccc 840
gatgggcggt?gccaaggtcg?tggcgaagtc?ctgggtggac?cctgagtacc?gcaagtggct 900
cgaagaggac?gcgacggccg?cgatggcgtc?attgggctat?gccggtgagc?aggcacacca 960
aatttcggcg?gtcttcaacg?actcccaaac?gcatcacgtg?gtggtgtgca?ctctgtgttc 1020
gtgctatccg?tggccggtgc?ttggtctccc?gcccgcctgg?tacaagagca?tggagtaccg 1080
gtcccgagtg?gtagcggacc?ctcgtggagt?gctcaagcgc?gatttcggtt?tcgacatccc 1140
cgatgaggtg?gaggtcaggg?tttgggacag?cagctccgaa?atccgctaca?tcgtcatccc 1200
ggaacggccg?gccggcaccg?acggttggtc?cgaggacgag?ctggcgaagc?tggtgagccg 1260
ggactcgatg?atcggtgtca?gtaatgcgct?cacaccccag?gaagtgatcg?tatgagtgaa 1320
gacacactca?ctgatcggct?cccggcgact?gggaccgccg?caccgccccg?cgacaatggc 1380
gagcttgtat?tcaccgagcc?ttgggaagca?acggcatacg?gggtcgccat?cgcgctttcg 1440
gatcagaagt?cgtacgaatg?ggagttcttc?cgacagcgtc?tcattcactc?catcgctgag 1500
gccaacggtt?gcgaggcata?ctacgagagc?tggacaaagg?cgctcgaggc?cagcgtggtc 1560
gactcggggc?tgatcagcga?agatgagatc?cgcgagcgca?tggaatcgat?ggccatcatc 1620
gactga 1626
Claims (11)
1, a kind of Nitrile hydratase, by having SEQ ID № in the sequence table: the α subunit of 3 amino acid residue sequence and have SEQ ID №: the β subunit of 2 amino acid residue sequence is formed.
2, Nitrile hydratase according to claim 1 is characterized in that: described Nitrile hydratase also is connected with and activates son, and described activation is the SEQ ID № in the sequence table: 4 amino acid residue sequence.
3, the encoding gene of the described Nitrile hydratase of claim 1 is one of following nucleotide sequences:
1) SEQ ID № in the sequence table: 6 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: 2 and SEQ ID №: the polynucleotide of 3 protein sequences.
4, gene according to claim 3 is characterized in that: described Nitrile hydratase encoding gene is SEQ ID № in the sequence table: the dna sequence dna shown in 6.
5, according to claim 3 or 4 described genes, it is characterized in that: described Nitrile hydratase encoding gene also is connected with the encoding sequence that activates son, and the encoding sequence of described activation is SEQ ID № in the sequence table: 7 dna sequence dna.
6, contain described expression carrier of claim 3 and transgenic cell line.
7, a kind of method of expressing the described Nitrile hydratase of claim 1 is that the recombinant expression vector that will contain the Nitrile hydratase encoding gene imports expressive host, expresses obtaining Nitrile hydratase; Described Nitrile hydratase encoding gene has one of following nucleotide sequences: 1) SEQ ID № in the sequence table: 6 dna sequence dna; 2) SEQ ID № in the code sequence tabulation: 2 and SEQ ID №: the polynucleotide of 3 protein sequences.
8, method according to claim 7 is characterized in that: described host is intestinal bacteria, yeast, mammalian cell, insect cell, Bacillus subtilus or lactobacillus.
9, method according to claim 8 is characterized in that: described host is intestinal bacteria.
10, method according to claim 9 is characterized in that: described host is E.coli BL21 (DE3).
11, the application of the described Nitrile hydratase of claim 1 in acrylamide is produced.
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| CNB2004100425760A CN1262644C (en) | 2004-05-24 | 2004-05-24 | Nitrile hydratase and its coding gene and use |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186911B (en) * | 2007-09-20 | 2010-06-02 | 清华大学 | Method for constructing nitrile hydratase gene engineering bacterium, genetic engineering strain and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1861789B (en) * | 2005-05-13 | 2010-04-28 | 中国科学院沈阳应用生态研究所 | Integrated gene recombined process, recombined gene and coding protein obtained thereby |
| CN101215806B (en) * | 2008-01-04 | 2011-06-08 | 叶天降 | Solid lubricant for paper-making and preparing method thereof |
| CN102517271B (en) * | 2011-12-13 | 2013-04-03 | 清华大学 | Mutant nitrile hydratase |
| CN104450657B (en) * | 2014-11-06 | 2017-10-03 | 浙江大学 | Nitrile hydratase and its encoding gene and application |
| CN104561065A (en) * | 2014-12-30 | 2015-04-29 | 杭州师范大学 | Heat-resistant recombinant nitrile hydratase gene, encoded enzyme, engineering bacterium and application of gene engineering bacterium |
| CN104725476B (en) * | 2015-03-31 | 2018-10-16 | 清华大学 | A kind of hook formation oligopeptides and its application |
| CN104830747B (en) * | 2015-05-13 | 2018-01-16 | 江南大学 | A kind of genetic engineering bacterium of high efficient expression HMW type nitrile hydratase and its application |
| CN107177581B (en) * | 2017-06-16 | 2020-04-28 | 清华大学 | Modified nitrile hydratase and application thereof |
| WO2020147031A1 (en) * | 2019-01-16 | 2020-07-23 | 江南大学 | Nitrile hydratase mutant, genetically engineered bacterium containing same, and use thereof |
| CN111826357B (en) * | 2019-04-15 | 2021-11-19 | 中国科学院分子植物科学卓越创新中心 | Scopolia acutangula atypical III type polyketide synthase and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101186911B (en) * | 2007-09-20 | 2010-06-02 | 清华大学 | Method for constructing nitrile hydratase gene engineering bacterium, genetic engineering strain and application thereof |
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