CN107287172A - A kind of method that utilization Escherichia coli fermentation produces thymus gland phosphorylase - Google Patents
A kind of method that utilization Escherichia coli fermentation produces thymus gland phosphorylase Download PDFInfo
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- CN107287172A CN107287172A CN201610227380.1A CN201610227380A CN107287172A CN 107287172 A CN107287172 A CN 107287172A CN 201610227380 A CN201610227380 A CN 201610227380A CN 107287172 A CN107287172 A CN 107287172A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
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- C12N9/1077—Pentosyltransferases (2.4.2)
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- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/02—Pentosyltransferases (2.4.2)
- C12Y204/02004—Thymidine phosphorylase (2.4.2.4)
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Abstract
The present invention relates to a kind of method that utilization Escherichia coli fermentation produces thymus gland phosphorylase, belong to bioengineering field.The present invention can effectively improve the total enzyme activity of unit thalline, can effectively reduce the production cost of AIDS drug intermediate β thymidines by expressing thymidine phosphorylase mutant.As long as general fermentation plant（Such as amino acid, vitamin workshop）It can be put into production, be not required to purchase special installation, it is easy to popularization and application.
Description
Technical field
The present invention relates to a kind of method that utilization Escherichia coli fermentation produces thymus gland phosphorylase, belong to bioengineering neck
Domain.
Background technology
Thymidine is a kind of important medical material intermediate and biochemical reagents, in the research and production of biological chemical field
There is quite varied application.Thymidine is to prepare AIDS resisting medicine --- the important source material of retrovir (AZT), but require that it contains
Amount must more than 98%, and 5-methyl-uridin therein content no more than 0.3%.The preparation of current thymidine mainly has
Two methods of chemical synthesis and DNA enzymatic solution：Contain grease, pigment, 5- first in the thymidine crude obtained by chemical synthesis
The thymidine derivatives such as base uridine, and there is optical isomer and be difficult to remove；Also being needed to use in technical process a large amount of poisonous has
Harmful organic solvent, environmental pollution is also dangerous.It is resulting and enzymatic isolation method is by hydrolyzing the natural materials such as RNA or DNA
Contain desoxyadenossine (dA), deoxycytidine (dC), deoxidation crow glycosides (dG), the impurity such as deoxyinosine (dI), separation in thymidine crude
Extract sufficiently complex.
Thymidine phosphorylase plays an important roll in biosynthesis nucleotide medicine.The nucleotide medicine synthesized extensively should
For field of medicaments, such as neat Fu Duoding of hiv reverse transcriptase inhibitor (AZT) for the treatment of AIDS, dideoxyinosine (ddl), bundle
His western shore (ddC) etc..
Thymidine phosphorylase can specific effect in beta-thymidine, the reversible phosphorylation reaction of catalysis beta-thymidine is given birth to by beta-thymidine
Into deoxyribose-1-phosphate and thymidine, also beta-thymidine can be generated by deoxyribose-1-phosphate and thymidine.
The content of the invention
The goal of the invention of the present invention is to obtain a kind of utilization Escherichia coli fermentation production thymus gland for being suitable for industrialized production
The method of phosphorylase, comprises the following steps：
(1) the Escherichia coli single bacterium colony that picking contains SEQ ID NO.3 expression vectors is inoculated in the one-level culture after autoclaving
In base, 30 DEG C, 250rpm, incubated overnight；
(2) product for obtaining step (1) is according to 1:100 inoculative proportion is linked into the secondary medium after autoclaving,
In being cultivated in 30 DEG C to thalline OD values 5-6, it is placed in 25 DEG C, shakes immediately, 250rpm is cultivated 1 hour；
(3) IPTG is added in the product obtained to step (2) to its final concentration of 0.1mM, and in 25 DEG C, 250rpm continues to cultivate
16 hours；
(4) by nutrient solution under the conditions of 4 DEG C, centrifuged 20 minutes under 12000g, collect wet thallus, then further sent out using thalline
Ferment produces thymidine phosphorylase..
Wherein first cell culture medium is by following material composition：Tryptone 10g/L, yeast extract 5g/L, disodium hydrogen phosphate
3.55g/L, potassium dihydrogen phosphate 3.4g/L, ammonium chloride 2.68g/L, sodium sulphate 0.71g/L, epsom salt 0.493g/L, six water
Iron chloride 0.027g/L, glycerine 5g/L, glucose 0.8g/L, addition kanamycins to 50mg/L.
Secondary medium is by following material composition：Tryptone 10g/L, yeast extract 5g/L, disodium hydrogen phosphate
3.55g/L, potassium dihydrogen phosphate 3.4g/L, ammonium chloride 2.68g/L, sodium sulphate 0.71g/L, epsom salt 0.493g/L, six water
Iron chloride 0.027g/L, glycerine 5g/L, glucose 0.3g/L, addition kanamycins to 50mg/L.
Refer to 12-16 hours overnight in foregoing description.
Obtain after wet thallus, can also comprise the following steps：Wet thallus precipitation is cleaned twice with distilled water, bacterium is collected
Body.The thalline obtained in the present invention is in -70 DEG C of preservations.
Wherein, the amino acid sequence coded by this artificial sequence of SEQ ID NO.3 is SEQ ID NO：4, it is a kind of chest
Glycosides phosphorylated zymoprotein mutant.
This mutant is set out with Escherichia coli MG1655 thymidine phosphorylase albumen, by by its 10th by
Leu sport Ala, the 209th by Ala sport Gly, the 210th by Phe sport Gly, the 221st sported by Ala
Val, the 413rd Asp is sported by Ala and obtains a kind of new thymus gland phosphorylase.
The method disclosed in the present can effectively improve total enzyme of unit thalline by expressing thymidine phosphorylase mutant
It is living, it can effectively reduce the production cost of AIDS drug intermediate beta-thymidine.As long as general fermentation plant (such as amino acid,
Vitamin workshop) it can be put into production, it is not required to purchase special installation, it is easy to popularization and application.
Brief description of the drawings
Fig. 1 is thymidine phosphorylase mutant Seq ID NO.3 expression vector collection of illustrative plates.
Embodiment
In order to preferably explain the present invention, with reference to embodiment, the present invention will be further elaborated.In this implementation
Used instrument, reagent, are commercially available prod unless there are specified otherwise in example.
Embodiment 1
Escherichia coli MG1655 is placed in LB culture mediums and cultivated, it is 37.0 DEG C to control temperature, and 180rpm is cultivated 1 day, and centrifugation is received
Collection precipitation, with DNA extraction purifications kit QiAamp Kit (Qiagen, Germany) extraction purification Escherichia coli MG1655 bases
Because of a group DNA.
Enter performing PCR to Escherichia coli MG1655 genomic DNAs with Pfu high-fidelities enzyme to expand, the primer is
1655TP-F 5'ATGTTTCTCGCACAAGAAATTATTCG 3'
1655TP-R 5'TTATTCGCTGATACGGCGATAGA 3'
Due to Escherichia coli MG1655 DNA G/C content close to 50%, therefore directly expanded using Pfu high-fidelities enzyme.Afterwards will
The fragment of amplification is handled 10 minutes for 72 DEG C with Taq polymerase, in DNA 3' ends addition base A.PMD19T- is connected to afterwards
In simple (Takara treasured biotech firm, Beijing) cloning vector, picking monoclonal is delivered to Nanjing Jin Sirui biologies and is sequenced.
Sequencing obtains DNA sequence dna for SEQ ID NO.1, and corresponding amino acid sequence is SEQ ID NO.2.
Embodiment 2
The method synthesized by full genome, secondary structure and codon preference to gene are adjusted, to realize big
High expression in enterobacteria.Meanwhile, the five amino acid mutation of following position is introduced in synthesis:L10A,A209G,F210G,
A221V, A413D, utilize Primer Premier (http://primer3.ut.ee/) and OPTIMIZER (http://
Genomes.urv.es/OPTIMIZER/) it is designed, and ensures the control of Tm differences within 3 DEG C, primer length control exists
Within 50base, following primer is obtained：
Above-mentioned primer is synthesized, and the primer of acquisition is added after distilled water dissolving, is added in following reaction system so that each primer
Final concentration of 30nM, final concentration of 0.6 μM of head and the tail primer.
| 2mM dNTP mix(2mM each dNTP) | 5μl |
| 10×Pfu buffer | 5μl |
| Pfu DNA polymerase(10U/μl) | 0.5μl |
| ddH2O | So that reaction system cumulative volume is to 50 μ l |
The PCR reaction systems prepared are placed in rich day XP cycler gene-amplificative instraments, expanded by following procedure
Increase：98 DEG C of 30s, 65 DEG C of 45s, 72 DEG C of 120s, 35x.The DNA fragmentation that PCR is obtained carries out cutting glue purification, utilizes homologous recombination
Method clones the NdeI/XhoI sites into pET30a.Picking monoclonal is sequenced.Successful DNA sequence dna is sequenced for SEQ ID
NO.3, corresponding amino acid sequence is SEQ ID NO.4.
Embodiment 3
The Escherichia coli single bacterium colony that picking contains SEQ ID NO.3 expression vectors is inoculated in the culture medium after 10ml autoclavings
In：Tryptone 10g/L, yeast extract 5g/L, disodium hydrogen phosphate 3.55g/L, potassium dihydrogen phosphate 3.4g/L, ammonium chloride
2.68g/L, sodium sulphate 0.71g/L, epsom salt 0.493g/L, Iron trichloride hexahydrate 0.027g/L, glycerine 5g/L, glucose
0.8g/L, addition kanamycins to 50mg/L.30 DEG C, 250rpm incubated overnights.Next day takes 1L triangular flasks, by 1：100 inoculation
Ratio is linked into the culture medium after 100ml autoclavings：Tryptone 10g/L, yeast extract 5g/L, disodium hydrogen phosphate
3.55g/L, potassium dihydrogen phosphate 3.4g/L, ammonium chloride 2.68g/L, sodium sulphate 0.71g/L, epsom salt 0.493g/L, six water
Iron chloride 0.027g/L, glycerine 5g/L, glucose 0.3g/L, addition kanamycins to 50mg/L.In being cultivated in 30 DEG C to thalline
Triangular flask, is placed in 25 DEG C of shaking tables by OD 5-6 at once, and 250rpm is cultivated 1 hour.Plus IPTG is to final concentration 0.1mM, and in 25
DEG C, 250rpm continues to cultivate 16 hours.After culture terminates, by nutrient solution in 4 DEG C, centrifugation collects wet bacterium in 20 minutes under 12000g
Body.Then bacterial sediment is cleaned twice with distilled water, collects thalline, -70 DEG C of preservations.
Embodiment 4
Activity determination is carried out to the thymidine phosphorylase of acquisition by following system construction standard enzyme reaction solution：0.2M potassium phosphates delay
Fliud flushing (pH7.4), 25mM thymidine, lmM EDTA.
1Oml standard enzyme reaction solution is filled with 50ml centrifuge tubes, fermentation thalli is added by 5% wet thallus amount；Will be above-mentioned
Reaction solution oscillating reactions in water bath with thermostatic control, reaction condition is reacts 3 hours under 25 DEG C of water-baths, after reaction terminates, by reaction solution
10 minutes terminating reactions in boiling water.Reaction solution is transferred in centrifuge tube, precipitation is removed in 12000r/min centrifugations, takes supernatant
100 times are diluted with NaOH weak solutions.With ultraviolet specrophotometer, the change of the supernatant light absorption value at 290nm diluted is measured
Change.
With blank solution as control, identical processing is carried out according to foregoing experimental group processing mode.
Enzyme-activity unit is defined as：In pH 7.4, at 25 DEG C, the wet thallus in 1min required for OD290nm changes 0.01
Amount is defined as an enzyme activity unit；
As a result it is 3.60U/mg to show the thymidine phosphorylase vigor containing five mutational sites, and before being mutated is only 0.32U/mg
Wet thallus, about 10 times or so of enzyme activity increase.
SEQUENCE LISTING
<110>Nanjing Nuoyun Biological Technology Co., Ltd.
<120>A kind of method that utilization Escherichia coli fermentation produces thymidine phosphorylase
<130> 2016
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1323
<212> DNA
<213> Escherichia coli MG1655
<400> 1
atgtttctcg cacaagaaat tattcgtaaa aaacgtgatg gtcatgcgct gagcgatgaa 60
gaaattcgtt tctttatcaa cggtattcgc gacaacacta tctccgaagg gcagattgcc 120
gccctcgcga tgaccatttt cttccacgat atgacaatgc ctgagcgtgt ctcgctgacc 180
atggcgatgc gagattcagg aaccgttctc gactggaaaa gcctgcatct gaatggcccg 240
attgttgata aacactccac cggtggcgtc ggcgatgtga cttcgctgat gttggggccg 300
atggtcgcag cctgcggcgg ctatattccg atgatctctg gtcgcggcct cggtcatact 360
ggcggtacgc tcgacaaact ggaatccatc cctggcttcg acattttccc ggatgacaac 420
cgtttccgcg aaattattaa agacgtcggc gtggcgatta tcggtcagac cagttcactg 480
gctccggctg ataaacgttt ctacgcgacc cgtgatatta ccgcaaccgt ggactccatc 540
ccgctgatca ccgcctctat tctggcgaag aaacttgcgg aaggtctgga cgcgctggtg 600
atggacgtga aagtgggtag cggcgcgttt atgccgacct acgaactctc tgaagccctt 660
gccgaagcga ttgttggcgt ggctaacggc gctggcgtgc gcaccaccgc gctgctcacc 720
gacatgaatc aggtactggc ctccagtgca ggtaacgcgg ttgaagttcg tgaagcggtg 780
cagttcctga cgggtgaata tcgtaacccg cgtctgtttg atgtcacgat ggcgctgtgc 840
gtggagatgc tgatctccgg caaactggcg aaagatgacg ccgaagcgcg cgcgaaattg 900
caggcggtgc tggacaacgg taaagcggca gaagtctttg gtcgtatggt agcggcacaa 960
aaaggcccga ccgacttcgt tgagaactac gcgaagtatc tgccgacagc gatgctgacg 1020
aaagcagtct atgctgatac cgaaggtttt gtcagtgaaa tggatacccg cgcgctgggg 1080
atggcagtgg ttgcaatggg cggcggacgc cgtcaggcat ctgacaccat cgattacagc 1140
gtcggcttta ctgatatggc gcgtctgggc gaccaggtag acggtcagcg tccgctggcg 1200
gttatccacg cgaaagacga aaacaactgg caggaagcgg cgaaagcggt gaaagcggca 1260
attaaacttg ccgataaagc accggaaagc acaccaactg tctatcgccg tatcagcgaa 1320
taa 1323
<210> 2
<211> 440
<212> PRT
<213> Escherichia coli str. K-12 substr. MG1655
<400> 2
Met Phe Leu Ala Gln Glu Ile Ile Arg Lys Lys Arg Asp Gly His Ala
1 5 10 15
Leu Ser Asp Glu Glu Ile Arg Phe Phe Ile Asn Gly Ile Arg Asp Asn
20 25 30
Thr Ile Ser Glu Gly Gln Ile Ala Ala Leu Ala Met Thr Ile Phe Phe
35 40 45
His Asp Met Thr Met Pro Glu Arg Val Ser Leu Thr Met Ala Met Arg
50 55 60
Asp Ser Gly Thr Val Leu Asp Trp Lys Ser Leu His Leu Asn Gly Pro
65 70 75 80
Ile Val Asp Lys His Ser Thr Gly Gly Val Gly Asp Val Thr Ser Leu
85 90 95
Met Leu Gly Pro Met Val Ala Ala Cys Gly Gly Tyr Ile Pro Met Ile
100 105 110
Ser Gly Arg Gly Leu Gly His Thr Gly Gly Thr Leu Asp Lys Leu Glu
115 120 125
Ser Ile Pro Gly Phe Asp Ile Phe Pro Asp Asp Asn Arg Phe Arg Glu
130 135 140
Ile Ile Lys Asp Val Gly Val Ala Ile Ile Gly Gln Thr Ser Ser Leu
145 150 155 160
Ala Pro Ala Asp Lys Arg Phe Tyr Ala Thr Arg Asp Ile Thr Ala Thr
165 170 175
Val Asp Ser Ile Pro Leu Ile Thr Ala Ser Ile Leu Ala Lys Lys Leu
180 185 190
Ala Glu Gly Leu Asp Ala Leu Val Met Asp Val Lys Val Gly Ser Gly
195 200 205
Ala Phe Met Pro Thr Tyr Glu Leu Ser Glu Ala Leu Ala Glu Ala Ile
210 215 220
Val Gly Val Ala Asn Gly Ala Gly Val Arg Thr Thr Ala Leu Leu Thr
225 230 235 240
Asp Met Asn Gln Val Leu Ala Ser Ser Ala Gly Asn Ala Val Glu Val
245 250 255
Arg Glu Ala Val Gln Phe Leu Thr Gly Glu Tyr Arg Asn Pro Arg Leu
260 265 270
Phe Asp Val Thr Met Ala Leu Cys Val Glu Met Leu Ile Ser Gly Lys
275 280 285
Leu Ala Lys Asp Asp Ala Glu Ala Arg Ala Lys Leu Gln Ala Val Leu
290 295 300
Asp Asn Gly Lys Ala Ala Glu Val Phe Gly Arg Met Val Ala Ala Gln
305 310 315 320
Lys Gly Pro Thr Asp Phe Val Glu Asn Tyr Ala Lys Tyr Leu Pro Thr
325 330 335
Ala Met Leu Thr Lys Ala Val Tyr Ala Asp Thr Glu Gly Phe Val Ser
340 345 350
Glu Met Asp Thr Arg Ala Leu Gly Met Ala Val Val Ala Met Gly Gly
355 360 365
Gly Arg Arg Gln Ala Ser Asp Thr Ile Asp Tyr Ser Val Gly Phe Thr
370 375 380
Asp Met Ala Arg Leu Gly Asp Gln Val Asp Gly Gln Arg Pro Leu Ala
385 390 395 400
Val Ile His Ala Lys Asp Glu Asn Asn Trp Gln Glu Ala Ala Lys Ala
405 410 415
Val Lys Ala Ala Ile Lys Leu Ala Asp Lys Ala Pro Glu Ser Thr Pro
420 425 430
Thr Val Tyr Arg Arg Ile Ser Glu
435 440
<210> 3
<211> 1323
<212> DNA
<213>Artificial sequence
<400> 3
atgttcctgg ctcaggaaat catccgtaaa aaacgtgacg gtcacgctct gtctgacgaa 60
gaaatccgtt tcttcatcaa cggtatccgt gacaacacca tctctgaagg tcagatcgct 120
gctctggcta tgaccatctt cttccacgac atgaccatgc cggaacgtgt ttctctgacc 180
atggctatgc gtgactctgg taccgttgct gactggaaat ctctgcacct gaacggtccg 240
atcgttgaca aacactctac cggtggtgtt ggtgacgtta cctctctgat gctgggtccg 300
atggttgctg cttgcggtgg ttacatcccg atgatctctg gtcgtggtct gggtcacacc 360
ggtggtaccc tggacaaact ggaatctatc ccgggtttcg acatcttccc ggacgacaac 420
cgtttccgtg aaatcatcaa agacgttggt gttgctatca tcggtcagac ctcttctctg 480
gctccggctg acaaacgttt ctacgctacc cgtgacatca ccgctaccgt tgactctatc 540
ccgctgatca ccgcttctat cctggctaaa aaactggctg aaggtctgga cgctctggtt 600
atggacgtta aagttggttc tggtggtggt atgccgacct acgaactgtc tgaagctctg 660
gttgaagcta tcgttggtgt tgctaacggt gctggtgttc gtaccaccgc tctgctgacc 720
gacatgaacc aggttctggc ttcttctgct ggtaacgctg ttgaagttcg tgaagctgtt 780
cagttcctga ccggtgaata ccgtaacccg cgtctgttcg acgttaccat ggctctgtgc 840
gttgaaatgc tgatctctgg taaactggct aaagacgacg ctgaagctcg tgctaaactg 900
caggctgttc tggacaacgg taaagctgct gaagttttcg gtcgtatggt tgctgctcag 960
aaaggtccga ccgacttcgt tgaaaactac gctaaatacc tgccgaccgc tatgctgacc 1020
aaagctgttt acgctgacac cgaaggtttc gtttctgaaa tggacacccg tgctctgggt 1080
atggctgttg ttgctatggg tggtggtcgt cgtcaggctt ctgacaccat cgactactct 1140
gttggtttca ccgacatggc tcgtctgggt gaccaggttg acggtcagcg tccgctggct 1200
gttatccacg ctaaagacga aaacaactgg caggaagacg ctaaagctgt taaagctgct 1260
atcaaactgg ctgacaaagc tccggaatct accccgaccg tttaccgtcg tatctctgaa 1320
taa 1323
<210> 4
<211> 440
<212> PRT
<213>Artificial sequence
<400> 4
Met Phe Leu Ala Gln Glu Ile Ile Arg Lys Lys Arg Asp Gly His Ala
1 5 10 15
Leu Ser Asp Glu Glu Ile Arg Phe Phe Ile Asn Gly Ile Arg Asp Asn
20 25 30
Thr Ile Ser Glu Gly Gln Ile Ala Ala Leu Ala Met Thr Ile Phe Phe
35 40 45
His Asp Met Thr Met Pro Glu Arg Val Ser Leu Thr Met Ala Met Arg
50 55 60
Asp Ser Gly Thr Val Ala Asp Trp Lys Ser Leu His Leu Asn Gly Pro
65 70 75 80
Ile Val Asp Lys His Ser Thr Gly Gly Val Gly Asp Val Thr Ser Leu
85 90 95
Met Leu Gly Pro Met Val Ala Ala Cys Gly Gly Tyr Ile Pro Met Ile
100 105 110
Ser Gly Arg Gly Leu Gly His Thr Gly Gly Thr Leu Asp Lys Leu Glu
115 120 125
Ser Ile Pro Gly Phe Asp Ile Phe Pro Asp Asp Asn Arg Phe Arg Glu
130 135 140
Ile Ile Lys Asp Val Gly Val Ala Ile Ile Gly Gln Thr Ser Ser Leu
145 150 155 160
Ala Pro Ala Asp Lys Arg Phe Tyr Ala Thr Arg Asp Ile Thr Ala Thr
165 170 175
Val Asp Ser Ile Pro Leu Ile Thr Ala Ser Ile Leu Ala Lys Lys Leu
180 185 190
Ala Glu Gly Leu Asp Ala Leu Val Met Asp Val Lys Val Gly Ser Gly
195 200 205
Gly Gly Met Pro Thr Tyr Glu Leu Ser Glu Ala Leu Val Glu Ala Ile
210 215 220
Val Gly Val Ala Asn Gly Ala Gly Val Arg Thr Thr Ala Leu Leu Thr
225 230 235 240
Asp Met Asn Gln Val Leu Ala Ser Ser Ala Gly Asn Ala Val Glu Val
245 250 255
Arg Glu Ala Val Gln Phe Leu Thr Gly Glu Tyr Arg Asn Pro Arg Leu
260 265 270
Phe Asp Val Thr Met Ala Leu Cys Val Glu Met Leu Ile Ser Gly Lys
275 280 285
Leu Ala Lys Asp Asp Ala Glu Ala Arg Ala Lys Leu Gln Ala Val Leu
290 295 300
Asp Asn Gly Lys Ala Ala Glu Val Phe Gly Arg Met Val Ala Ala Gln
305 310 315 320
Lys Gly Pro Thr Asp Phe Val Glu Asn Tyr Ala Lys Tyr Leu Pro Thr
325 330 335
Ala Met Leu Thr Lys Ala Val Tyr Ala Asp Thr Glu Gly Phe Val Ser
340 345 350
Glu Met Asp Thr Arg Ala Leu Gly Met Ala Val Val Ala Met Gly Gly
355 360 365
Gly Arg Arg Gln Ala Ser Asp Thr Ile Asp Tyr Ser Val Gly Phe Thr
370 375 380
Asp Met Ala Arg Leu Gly Asp Gln Val Asp Gly Gln Arg Pro Leu Ala
385 390 395 400
Val Ile His Ala Lys Asp Glu Asn Asn Trp Gln Glu Asp Ala Lys Ala
405 410 415
Val Lys Ala Ala Ile Lys Leu Ala Asp Lys Ala Pro Glu Ser Thr Pro
420 425 430
Thr Val Tyr Arg Arg Ile Ser Glu
435 440
Claims (4)
1. a kind of method that utilization Escherichia coli fermentation produces thymidine phosphorylase, it is characterized in that：Comprise the following steps：
（1）The Escherichia coli single bacterium colony that picking contains SEQ ID NO.3 expression vectors is inoculated in the one-level culture after autoclaving
In base, 30 DEG C, 250rpm, incubated overnight；
（2）By step（1）The product of acquisition is according to 1:100 inoculative proportion is linked into the secondary medium after autoclaving,
In being cultivated in 30 DEG C to thalline OD values 5-6, it is placed in 25 DEG C, shakes immediately, 250rpm is cultivated 1 hour；
（3）To step（2）IPTG is added in the product of acquisition to its final concentration of 0.1mM, and in 25 DEG C, 250rpm continues to cultivate
16 hours；
（4）By nutrient solution under the conditions of 4 DEG C, centrifuged 20 minutes under 12000g, collect wet thallus, then further sent out using thalline
Ferment produces thymidine phosphorylase.
2. according to the method described in claim 1, it is characterized in that：Wherein first cell culture medium is by following material composition：Tryptone
10 g/L, the g/L of yeast extract 5, the g/L of disodium hydrogen phosphate 3.55, the g/L of potassium dihydrogen phosphate 3.4, the g/L of ammonium chloride 2.68, sulphur
The sour g/L of sodium 0.71, the g/L of epsom salt 0.493, Iron trichloride hexahydrate 0.027 g/L, glycerine 5g/L, glucose 0.8g/L, add
Plus kanamycins is to 50mg/L.
3. according to the method described in claim 1, it is characterized in that：Secondary medium is by following material composition：Tryptone 10
G/L, the g/L of yeast extract 5, the g/L of disodium hydrogen phosphate 3.55, the g/L of potassium dihydrogen phosphate 3.4, the g/L of ammonium chloride 2.68, sulfuric acid
The g/L of sodium 0.71, the g/L of epsom salt 0.493, Iron trichloride hexahydrate 0.027 g/L, glycerine 5g/L, glucose 0.3g/L, addition
Kanamycins is to 50mg/L.
4. according to the method described in claim 1, it is characterized in that：Obtain after wet thallus, it is further comprising the steps of：Wet thallus is sunk
Shallow lake is cleaned twice with distilled water, collects thalline.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109735553A (en) * | 2018-12-29 | 2019-05-10 | 南京诺云生物科技有限公司 | A kind of preparation method of anti-AIDS drug atazanavir intermediate |
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| CN109943577B (en) * | 2018-12-29 | 2022-08-30 | 南京诺云生物科技有限公司 | Biotransformation method of anti-AIDS drug atazanavir intermediate |
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