CN110157653A - Recombinant escherichia coli for high-yield cyclic adenosine monophosphate and application of recombinant escherichia coli in cyclic adenosine monophosphate synthesis - Google Patents
Recombinant escherichia coli for high-yield cyclic adenosine monophosphate and application of recombinant escherichia coli in cyclic adenosine monophosphate synthesis Download PDFInfo
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- adenosine monophosphate
- cyclic adenosine
- recombination bacillus
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- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 title claims abstract description 35
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 25
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 12
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 11
- 108090000790 Enzymes Proteins 0.000 claims abstract description 32
- 102000004190 Enzymes Human genes 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 23
- 108060000200 adenylate cyclase Proteins 0.000 claims abstract description 15
- 239000007788 liquid Substances 0.000 claims abstract description 9
- 241000894006 Bacteria Species 0.000 claims abstract description 5
- 230000006798 recombination Effects 0.000 claims description 19
- 238000005215 recombination Methods 0.000 claims description 19
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 102000030621 adenylate cyclase Human genes 0.000 claims description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 108091008146 restriction endonucleases Proteins 0.000 claims description 7
- 238000006911 enzymatic reaction Methods 0.000 claims description 6
- 230000001939 inductive effect Effects 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 claims description 5
- 241000660147 Escherichia coli str. K-12 substr. MG1655 Species 0.000 claims description 5
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 239000011592 zinc chloride Substances 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 3
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 claims description 3
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims 2
- 230000002194 synthesizing effect Effects 0.000 claims 2
- 239000002126 C01EB10 - Adenosine Substances 0.000 claims 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 claims 1
- 101710095468 Cyclase Proteins 0.000 claims 1
- 239000007983 Tris buffer Substances 0.000 claims 1
- 229960005305 adenosine Drugs 0.000 claims 1
- 229950006790 adenosine phosphate Drugs 0.000 claims 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 10
- 238000004519 manufacturing process Methods 0.000 abstract description 4
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- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 abstract 2
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 abstract 2
- TTWYZDPBDWHJOR-IDIVVRGQSA-L adenosine triphosphate disodium Chemical compound [Na+].[Na+].C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O TTWYZDPBDWHJOR-IDIVVRGQSA-L 0.000 abstract 1
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- WJXICLCJFVEKKK-PWVLCOLPSA-N NC1=NC=NC2=C1N=CN2[C@@H]([C@@H]1O)O[C@H](COP(O)(O)=O)[C@H]1O.NC1=NC=NC2=C1N=CN2[C@@H]([C@@H]1O)O[C@H](COP(O)(O)=O)[C@H]1O.NC1=NC=NC2=C1N=CN2[C@@H]([C@@H]1O)O[C@H](COP(O)(O)=O)[C@H]1O.P.P Chemical compound NC1=NC=NC2=C1N=CN2[C@@H]([C@@H]1O)O[C@H](COP(O)(O)=O)[C@H]1O.NC1=NC=NC2=C1N=CN2[C@@H]([C@@H]1O)O[C@H](COP(O)(O)=O)[C@H]1O.NC1=NC=NC2=C1N=CN2[C@@H]([C@@H]1O)O[C@H](COP(O)(O)=O)[C@H]1O.P.P WJXICLCJFVEKKK-PWVLCOLPSA-N 0.000 description 1
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- CJGYSWNGNKCJSB-YVLZZHOMSA-N bucladesine Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](OC(=O)CCC)[C@@H]2N1C(N=CN=C2NC(=O)CCC)=C2N=C1 CJGYSWNGNKCJSB-YVLZZHOMSA-N 0.000 description 1
- 230000003491 cAMP production Effects 0.000 description 1
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- 230000003834 intracellular effect Effects 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y406/00—Phosphorus-oxygen lyases (4.6)
- C12Y406/01—Phosphorus-oxygen lyases (4.6.1)
- C12Y406/01001—Aodenylate cyclase (4.6.1.1)
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Abstract
The invention discloses a recombinant escherichia coli for high-yield cyclic adenosine monophosphate and application thereof in cyclic adenosine monophosphate synthesis. The adenylate cyclase gene cloned from cAMP producing strain has excellent catalytic activity and stability of the crude enzyme liquid obtained after crushing recombinant bacteria, and Adenosine Triphosphate (ATP) and Zn are added2+The method has the advantages of simple reaction system, mild condition, short period, few byproducts, cleanness, no pollution, simple, quick and efficient production path, and the conversion rate of ATP (adenosine triphosphate) as a substrate reaches more than 90 percent.
Description
Technical field
The invention belongs to cyclic adenosine monophosphate preparation fields, and in particular to a kind of recombination bacillus coli of high yield cyclic adenosine monophosphate
And its application in synthesis cyclic adenosine monophosphate.
Background technique
Cyclic adenosine monophosphate (Cyclic adenosine monophosphate, CAMP) is that a kind of important nucleic acid is derivative
Object has different physiological roles, is widely used in medicine and animal husbandry field.In medical applications, CAMP is that human body is thin
Second messenger intracellular participates in adjusting glycometabolism, lipid metaboli, the synthesis of nucleic acid, the synthesis of protein.Studies have shown that including hat
The generation of more than 40 disease such as heart trouble, myocardial infarction, cancer is all related with the metabolic disorder of CAMP.Exogenous CAMP has
Diastole smooth muscle, improves myocardial anoxia, improves the physiological functions such as liver function expansion blood vessel.Therefore, it is clinically treated using CAMP
Angina pectoris, acute myocardial infarction AMI, cardiogenic shock and respiratory disease, disease in the liver and gallbladder etc..In addition, CAMP is also used as
Intermediate, synthesizes that fat-soluble effect is good, pharmacological action is stronger, work faster dibutyryl cyclic adenosine monophosphate and cyclic adenosine monophosphate Portugal
The drugs such as formic acid.In animal husbandry field, CAMP can be used as feed addictive, simulates the effect of growth hormone, promotes livestock and poultry
Growth-weight gain enhances the constitution of livestock and poultry, improves disease-resistant, cold tolerance, increases the yield of high-quality livestock-poultry.Most of state-owned enterprise, state
Industry produces CAMP using chemical method, and synthetic method includes alkali hydrolysis method, DCC evaporation, active rouge method, phosphorus oxychloride method etc..It is few
Number enterprise extracts CAMP from the natural products such as jujube.But existing chemical synthesis is at high cost, seriously polluted, raw material is malicious
Property is big.And the complex process extracted from natural products, the CAMP purity of acquisition are low, generally only use as health care product.These
Deficiency limits the large-scale production and extensive use of CAMP.Based on this status, it is efficient, pollution-free and suitable to be badly in need of exploitation
The novel C AMP production method of industrialization.Fermentation method and enzyme process are because its environmental-friendly feature is increasingly taken seriously.Fermentation
Method is to be realized by screening and cultivating the recombinant bacterial strain of the bacterial strain or building expression adenyl cyclase that produce CAMP in full cell
Catalytic production CAMP.However there is fermentation method reaction process uncertain, cell membrane to lead to permeability, the by-product of substrate or enzyme
Degradation and the accumulation of by-product of the product of cause etc. all hinder its popularization industrially.
As patent of invention CN 102433292 disclose it is a kind of using gene recombined escherichia coli be catalyzed generate CAMP
Method.Patent of invention CN 102899372 discloses a plant height and produces CAMP arthrobacterium and its fermentation process, and this method obtains CAMP
Yield be 7.52g/L.Patent of invention CN 104342468 discloses adenyl cyclase gene cloning and enters bacillus subtilis
A strain gene engineering bacterium is obtained, the fermentation level of 12.1g/L CAMP can be obtained by cultivating highest, be also unable to satisfy big rule
The industrial requirement of mould.Enzyme process is to generate CAMP by using adenyl cyclase catalysis ATP.Compared with fermentation method, enzyme process
Equally has the characteristics that mild condition, pollution-free.The advantages of enzyme process, is that the product reacted is more single-minded, and process is easily controllable,
Without containing impurity such as microorganism, toxic source chemicals in reaction system, it is very beneficial for subsequent isolate and purify.Currently, enzyme
The report that method produces CAMP is less, and reason is that the preparation of enzyme is complicated.Therefore, it is necessary to develop efficient adenyl cyclase
The bottleneck that preparation method, simple enzymatic reaction system, breakthrough.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of recombination large intestine bars of high yield cyclic adenosine monophosphate
Bacterium and its application in synthesis cyclic adenosine monophosphate, it is short that this method catalyzes and synthesizes the period, at low cost, is suitble to large-scale production ring phosphorus
Adenosine monophosphate.
A kind of recombination bacillus coli of high yield cyclic adenosine monophosphate replicates adenylate ring using Escherichia coli MG1655 as source
Change enzyme gene, and is building up in e. coli bl21 (DE3) to get recombination bacillus coli CYA446；The adenyl cyclase
The nucleotide sequence of gene is as shown in SCN NO.1.
It is to choose primer as improved, the adenyl cyclase gene on Escherichia coli MG1655 is replicated by PCR,
It is connect again with carrier pET22b, and is transferred to cloning vector Trans1-T1, after LB plate primary dcreening operation, chosen a progress bacterium colony PCR and test
It is sequenced after card.
It is that the primer middle and upper reaches primer has Nde I restriction enzyme site as improved, downstream primer has Xho I
Restriction enzyme site.Two sites of this selection are single restriction enzyme site in MG1655 gene and carrier PET222b, and price is just
Preferably.Facilitating industrialization reduces cost.
A kind of application of the recombination bacillus coli of above-mentioned high yield cyclic adenosine monophosphate in synthesis cyclic adenosine monophosphate.
The step of above-mentioned application, is as follows, culture recombination bacillus coli to OD600When to 0.5 ~ 0.7, IPTG is added to dense eventually
0.5 ‰ ~ 1 ‰ are spent, is crushed after 18 ~ 20h of inducing expression at 20 ~ 25 DEG C, supernatant, that is, crude enzyme liquid is collected and surveys protein concentration；To egg
Atriphos, the 10 ~ 20mM that concentration is 10 ~ 50mM are added in the adenyl cyclase crude enzyme liquid that white concentration is 4 ~ 8g/L
ZnCl2, TrisHCl, after mixing evenly under the conditions of pH7.0 ~ 9.0,30 ~ 35 DEG C react 4 ~ 6h, complete enzymatic reaction synthesis
Cyclic adenosine monophosphate.
It is to select recombination bacillus coli into LB/Amp culture medium as improved, under the conditions of 37 DEG C, 200rpm
Then oscillation is forwarded in fresh LB/Amp culture medium to OD600 ≈ 1 and continues culture to OD600 ≈ 0.5 ~ 0.7.
Be as improved be added into the adenyl cyclase crude enzyme liquid that protein concentration is 4 ~ 8g/L concentration be 10 ~
Atriphos, 10 ~ 20mM ZnCl2, the TrisHCl (pH8.0) of 50mM, after mixing evenly in pH7.0 ~ 9.0,30 ~ 35
4 ~ 6h is reacted under the conditions of DEG C, is completed enzymatic reaction and is synthesized cyclic adenosine monophosphate.
The utility model has the advantages that
1, currently reported cAMP production level reaches as high as 14.93 g/L, and uses the method production level reachable
To 20.12g/L, large-scale industrial production requirement can be met.
It 2, is Mg with active ion in popular response system2+Unlike, active ion is Zn in this reaction system2+, from
Sub- concentration is that 10mM enzyme activity can reach 60mM Mg2+Twice.
3, recombinant strains separate supernatant after crushing, directly carry out catalysis reaction using expression supernatant, reduce
The operating procedures such as purifying, the separation of enzyme greatly simplifie technique, save the cost.
Detailed description of the invention
Fig. 1 is the generation of HPLC detection catalysis cAMP in the present invention, wherein (a)-ATP standard items.(b)-cAMP standard
Product, (c)-embodiment cAMP；
Fig. 2 is the plate morphology of recombinant bacterial strain BL21 (DE3)-PET22b-CYA446；
Enzyme activity situation under inducing expression under the conditions of Fig. 3 is different OD；
Fig. 4 is the enzyme activity situation under various concentration IPTG induction；
Fig. 5 is the enzyme activity situation under different temperatures under inducing expression.
Fig. 6 is various concentration ZnCl2Enzyme activity situation under catalysis；
Fig. 7 is the enzyme activity situation under different pH catalysis；
Fig. 8 is the enzyme activity situation under different temperatures catalysis.
Specific embodiment
The following describes the present invention further through the description of specific embodiments, but it is to limit of the invention that this, which is not,
System, those skilled in the art's basic thought according to the present invention can make various modifications or improvements, but without departing from this
The basic thought of invention, is all within the scope of the present invention.
The building of 1 BL21 of embodiment (DE3)-PET22b-CYA446 expression bacterial strain
Using Escherichia coli MG1655 full-length genome as template, pass through 1-446 core of standard PCR amplification adenyl cyclase albumen
Thuja acid coded sequence.
Upstream primer used has Nde I restriction enzyme site, sequence are as follows: GGAATTCCATATGatgTACCTCTATATTGA
GACTCTGAAAC。
Downstream primer has Xho I restriction enzyme site, sequence CCGCTCGAGTTCCGAGAGATCGGGTGAA.
Reaction condition are as follows: 95 DEG C of 2min, 95 DEG C of 20s, 50 DEG C of 20s, 72 DEG C of 50s, totally 30 recycle；72℃
5min.Obtained sequence recycles respective segments after 1% agarose gel electrophoresis.The sequence and expression vector pET22b are used
Nde I and Xho the I digestion of Takara company, endonuclease reaction system are as follows: 10 × buffer H, 2 μ l, Nde I, 0.5 μ l,
0.5 μ l of Xho I, genetic fragment or pET22b carrier 3 μ l, H2O14μl.Digestion system is reacted 2 hours under the conditions of 37 DEG C.It will
Digestion products connection, reaction system are as follows: 10 × Ligase buffer 1 μ l, T4 DNA Ligase (Takara) 1 μ l, gene piece
Section 7 μ l, 1 μ l of carrier.25 ° are connected to react 3 hours.Connection product is converted into Escherichia coli Trans1-T1.PCR screening is positive
Bacterial strain Trans1-T1-PET22b-CYA446 simultaneously carries out DNA sequencing, and verifying construction of recombinant plasmid is correct.
Positive strain is seeded to 5ml LB/Amp fluid nutrient medium, LB/Amp fluid nutrient medium group becomes peptone
10g/L, yeast powder 5g/L, sodium chloride 5g/L, shaken cultivation is stayed overnight under the conditions of 37 DEG C, 200rpm.According to Tiangeng after 24 hours
Plasmid extraction reagent kit operating instruction extracts plasmid pET22b-CYA446.1 μ l pET22b-CYA446 plasmid is taken to convert Escherichia coli
BL21(DE3).PCR screens positive strain BL21 (DE3)-PET22b-CYA446., bacterial strain flat board form is as shown in Figure 2.
The inducing expression and bacterial cell disruption of embodiment 2 BL21 (DE3)-PET22b-CYA446
1, the inducing expression of BL21 (DE3)-PET22b-CYA446
Positive strain BL21 (DE3)-PET22b-CYA446 is seeded to 100mlLB/Amp fluid nutrient medium, 37 DEG C, 200rpm
Under the conditions of shaken cultivation to OD600 ≈ 1.The fresh LB/Amp fluid nutrient medium of 500mL is inoculated in the ratio of 10:100,37
DEG C, under the conditions of 200rpm shaken cultivation to 0.5 ~ 0.7(of OD600 ≈ as shown in figure 3, its enzyme activity is all preferable between 0.5 ~ 0.7, but
It is induced as can be seen from Figure when 0D is 0.5, the enzyme activity of producing enzyme is also most strong).IPTG is added to final concentration 0.5 ‰ ~ 1 ‰
(as shown in figure 4, concentration is that enzyme activity is preferable between 0.5 ‰ ~ 1 ‰, but the IPTG effect of 1 ‰ concentration is best)；At 20 ~ 25 DEG C
18 ~ 20h of shaken cultivation under the conditions of (as shown in figure 5, enzyme activity is preferable between 20 ~ 25 DEG C, 20 DEG C best), 200rpm.6000rpm
It is centrifuged 10min, collects thallus.
2, bacterial cell disruption
The thallus buffer of 100mM TrisHcl8.0 is resuspended, with high pressure homogenization crusher machine, condition 12000psi, 4
DEG C, four circulations.6000rpm is centrifuged 10min and collects supernatant, that is, crude enzyme liquid, and Bradford method measures protein concentration.
Embodiment 3 catalyzes and synthesizes CAMP
Following reaction system is mixed in test tube: concentration is added into the adenyl cyclase crude enzyme liquid that protein concentration is 4 ~ 8g/L
Atriphos, 10 ~ 20mM ZnCl for 10 ~ 50mM2(as shown in fig. 6, enzyme activity is preferable between 10 ~ 20mM, 10 mM are most
It is good), TrisHCl (pH8.0), after mixing evenly in pH7.0 ~ 9.0(as shown in fig. 7, enzyme activity is preferable between pH7.0 ~ 9.0,
PH8.0 is best), react 4 ~ 6h under the conditions of 30 ~ 35 DEG C (as shown in figure 8, enzyme activity is preferable between 30 ~ 35 DEG C, 30 DEG C best), complete
Enzymatic reaction synthesizes cyclic adenosine monophosphate.With the generation of cAMP in Agilent HC-C18 chromatography post detection supernatant, mobile phase is phosphorus
Acid+triethylamine (6mL+18.4mL is settled to 1L): methanol=75:25, flow velocity 0.4ml/min, Detection wavelength 254nm.
Sequence table
<110>Nanjing University of Technology
<120>a kind of recombination bacillus coli of high yield cyclic adenosine monophosphate and its application in synthesis cyclic adenosine monophosphate
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1338
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
ttgtacctct atattgagac tctgaaacag agactggatg ccataaatca attgcgtgtg 60
gatcgcgcgc ttgctgctat ggggcctgca ttccaacagg tctacagtct actgccgaca 120
ttgttgcact atcaccatcc gctaatgccg ggttaccttg atggtaacgt tcccaaaggc 180
atttgccttt acacgcctga tgaaactcaa cgccactacc tgaacgagct tgaactgtat 240
cgtggaatgt cagtacagga tccgccgaaa ggtgagcttc caattactgg tgtatacacc 300
atgggcagca cctcgtccgt agggcaaagt tgttcctctg acctggatat ctgggtctgt 360
catcaatcct ggctcgatag cgaagagcgc caattgctac aacgtaaatg tagcctgctg 420
gaaaactggg ccgcctcgct gggtgtggaa gtcagcttct tcctgattga tgaaaaccgc 480
ttccgtcata atgaaagcgg cagcctgggg ggcgaagatt gtggctccac ccagcatata 540
ctgctgcttg acgaatttta tcgtaccgcc gtgcgtctcg ccggtaagcg tattctgtgg 600
aatatggtgc cgtgcgacga agaagagcat tacgacgact atgtgatgac gctttacgcg 660
cagggcgtgc tgacgccaaa tgaatggctg gatctcggtg gcttaagctc gctttctgct 720
gaagagtact ttggtgccag cctttggcag ctctacaaga gtatcgattc cccatacaaa 780
gcggtactga aaacactgct gctggaagcc tattcctggg aatacccgaa cccacgtctg 840
ctggcgaaag atatcaaaca gcgtttgcac gacggcgaga ttgtatcgtt tggtctcgat 900
ccatactgca tgatgctgga gcgtgttact gaatacctga cggcgattga agattttacc 960
cgtctggatt tagtacgtcg ctgcttctat ttaaaagtgt gcgaaaagct cagccgtgaa 1020
cgcgcctgcg taggctggcg tcgcgcagtg ttgagccagt tagtgagcga gtggggttgg 1080
gacgaagctc gtctggcaat gctcgataac cgcgctaact ggaagattga tcaggtgcgt 1140
gaggcgcaca acgagttgct cgacgcgatg atgcagagct accgtaatct gatccgcttt 1200
gcgcgtcgca ataaccttag cgtctccgcc agtccgcagg atatcggcgt gctgacgcgt 1260
aagctgtatg ccgcgtttga agcattacca ggtaaagtga cgctggtaaa cccgcagatt 1320
tcacccgatc tctcggaa 1338
<210> 2
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggaattccat atgatgtacc tctatattga gactctgaaa c 41
<210> 3
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ccgctcgagt tccgagagat cgggtgaa 28
Claims (7)
1. a kind of recombination bacillus coli of high yield cyclic adenosine monophosphate, which is characterized in that using Escherichia coli MG1655 as source, duplication
Adenyl cyclase gene, and be building up in e. coli bl21 (DE3) to get recombination bacillus coli CYA446；The adenosine
The nucleotide sequence of cyclase of acid gene is as shown in SCN NO.1.
2. a kind of recombination bacillus coli of high yield cyclic adenosine monophosphate according to claim 1, it is characterised in that: selection is drawn
Object replicates the adenyl cyclase gene on Escherichia coli MG1655 by PCR, then connect with carrier pET22b, and is transferred to gram
Grand carrier Trans1-T1 is sequenced after choosing a progress bacterium colony PCR verifying after LB plate primary dcreening operation.
3. a kind of recombination bacillus coli of high yield cyclic adenosine monophosphate according to claim 2, it is characterised in that: the primer
Middle and upper reaches primer has Nde I restriction enzyme site, and downstream primer has Xho I restriction enzyme site.
4. a kind of application of recombination bacillus coli of high yield cyclic adenosine monophosphate in synthesis cyclic adenosine monophosphate.
5. the recombination bacillus coli of high yield cyclic adenosine monophosphate according to claim 4 is on catalyzing and synthesizing cyclic adenosine monophosphate
Using, which is characterized in that culture recombination bacillus coli to OD600When to 0.5 ~ 0.7, IPTG is added to final concentration 0.5 ‰ ~ 1 ‰,
It is crushed after 18 ~ 20h of inducing expression at 20 ~ 25 DEG C, collects supernatant, that is, crude enzyme liquid and surveys protein concentration；To protein concentration be 4 ~
Atriphos, the 10 ~ 20mM ZnCl that concentration is 10 ~ 50mM are added in the adenyl cyclase crude enzyme liquid of 8g/L2、Tris·
HCl reacts 4 ~ 6h under the conditions of pH7.0 ~ 9.0,30 ~ 35 DEG C after mixing evenly, completes enzymatic reaction and synthesizes cyclic adenosine monophosphate.
6. the recombination bacillus coli of high yield cyclic adenosine monophosphate according to claim 5 is on catalyzing and synthesizing cyclic adenosine monophosphate
Application, which is characterized in that select recombination bacillus coli into LB/Amp culture medium, vibrated under the conditions of 37 DEG C, 200rpm
To OD600 ≈ 1, then it is forwarded in fresh LB/Amp culture medium and continues culture to OD600 ≈ 0.5 ~ 0.7.
7. the side that the recombination bacillus coli of high yield cyclic adenosine monophosphate according to claim 5 catalyzes and synthesizes cyclic adenosine monophosphate
Method, which is characterized in that it is the three of 10 ~ 50mM that concentration, which is added, into the adenyl cyclase crude enzyme liquid that protein concentration is 4 ~ 8g/L
Adenosine phosphate, 10 ~ 20mM ZnCl2, TrisHCl (pH8.0), it is anti-under the conditions of pH7.0 ~ 9.0,30 ~ 35 DEG C after mixing evenly
4 ~ 6h is answered, enzymatic reaction is completed and synthesizes cyclic adenosine monophosphate.
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| CN112725323A (en) * | 2021-02-05 | 2021-04-30 | 南京工业大学 | Recombinant salt-tolerant adenylate cyclase and coding gene and application thereof |
| CN116064493A (en) * | 2022-07-14 | 2023-05-05 | 美亚药业海安有限公司 | Adenylate cyclase mutant and application thereof in preparation of cyclic adenosine monophosphate |
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| CN111269870A (en) * | 2020-03-06 | 2020-06-12 | 南京工业大学 | Recombinant escherichia coli with high cytidylic acid yield and application thereof |
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| CN112725323A (en) * | 2021-02-05 | 2021-04-30 | 南京工业大学 | Recombinant salt-tolerant adenylate cyclase and coding gene and application thereof |
| CN116064493A (en) * | 2022-07-14 | 2023-05-05 | 美亚药业海安有限公司 | Adenylate cyclase mutant and application thereof in preparation of cyclic adenosine monophosphate |
| CN116064493B (en) * | 2022-07-14 | 2023-12-01 | 美亚药业海安有限公司 | Adenylate cyclase mutant and application thereof in preparation of cyclic adenosine monophosphate |
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