CN100575487C - A kind of method of production 5 '-flavour nucleotide - Google Patents
A kind of method of production 5 '-flavour nucleotide Download PDFInfo
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- CN100575487C CN100575487C CN200710038635A CN200710038635A CN100575487C CN 100575487 C CN100575487 C CN 100575487C CN 200710038635 A CN200710038635 A CN 200710038635A CN 200710038635 A CN200710038635 A CN 200710038635A CN 100575487 C CN100575487 C CN 100575487C
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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
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention belongs to the biochemical engineering technical field, be specifically related to a kind ofly make up genetic engineering bacterium, this genetic engineering bacterium is carried out screen mutation, and utilize the high reactivity genetic engineering bacterium to produce the method for 5 '-Nucleotide with the DNA recombinant technology.Comprise the steps: that 1. utilize recombinant DNA technology, make up the acid phosphatase expression vector, and transformed into escherichia coli, obtain efficiently expressing the genetic engineering bacterium of acid phosphatase.2. the sudden change of acid phosphatase gene and the screening that nucleosides is had than the acid phosphatase of high-affinity.3. with acid phosphatase enzyme catalysis nucleosides and the synthetic 5 '-Nucleotide of phosphate group donor.Utilize method catalysis of the present invention to synthesize 5 '-Nucleotide, have simply, efficient, the cycle is short, low cost, and characteristics such as compliance with environmental protection requirements are suitable for suitability for industrialized production.
Description
Technical field
The invention belongs to the biochemical engineering technical field, be specifically related to a kind of method of utilizing the high reactivity genetic engineering bacterium to produce 5 '-flavour nucleotide.
Background technology
5 '-Nucleotide is often used as foodstuff additive and medicinal intermediates, wherein 5 '-t-inosinic acid (Inosine-5 '-monophosphate, abbreviation 5 '-IMP) and guanylic acid (Guanosine-5 ' monophosphate, be called for short 5 '-GMP) and be the flavor agent and can mix the generation synergistic effect, make freshness improve several times to tens times with monosodium glutamate (Sodium Glutamate MSG) as a kind of; They have synergism to sweet taste, meat flavour in addition, and salty, sour, bitter taste and raw meat, the smell of burning are had restraining effect, therefore more and more come into one's own in foodstuffs industry is produced and welcome.
The method of suitability for industrialized production 5 '-Nucleotide mainly contains Yeast Nucleic Acid (RNA) enzymolysis process, is converted into 5 '-t-inosinic acid with chemical method or microbial enzyme method after utilizing microorganism direct fermentations such as glutamate-producing strain, Brevibacterium ammoniagenes, L-glutamic acid bacillus and utilizing fermentation inosines such as Bacillus subtilus, bacillus pumilus, Brevibacterium ammoniagenes.These methods or reactant are toxic or reaction substrate is expensive or by product is many to human body, so can not be used for efficient and cheap production 5 '-Nucleotide.
Three former Kang Bo etc. have reported a kind of acid phosphatase (acidphosphatase that utilizes, EC3.1.3.2) be the method that phosphodonor is produced 5 '-Nucleotide with the tetra-sodium sodium salt, with the genetic engineering bacterium after the sudden change is the enzyme source, react 12 hours IMP semi-invariants and reach 120.5g/l, inosine transformation efficiency about 76.5%.Yet this method still exists the production cycle long, the not high shortcoming of transformation efficiency.
Summary of the invention
The purpose of this invention is to provide a kind of enzyme, the gene of this enzyme of coding contains the recombinant DNA of this gene, the microorganism that can be used for producing 5 '-flavour nucleotide;
Another object of the present invention provides a kind of efficient cheap method of producing 5 '-flavour nucleotide.
The technical solution adopted in the present invention is as follows:
1. utilize recombinant DNA technology, make up the acid phosphatase expression vector, and transformed into escherichia coli (Escherichia coli), obtain efficiently expressing the genetic engineering bacterium of acid phosphatase.
The source of acid phosphatase does not limit, as long as energy corresponding nucleosides of catalysis and phosphate donor generate corresponding 5 '-Nucleotide in acidic buffer, for example enteroaerogen (Enterobacter aerogenes), carrot soft rot Erwinia (Erwinia carotovora) etc., in the scheme of special recommendation, the present invention uses the acid phosphatase of enteroaerogen (Enterobacteraerogenes W8401).
The carrier of construction recombination plasmid does not limit yet, as long as can efficiently express the acid phosphatase of external source, in the scheme of special recommendation, the expression vector pBV220 of use temperature induction type of the present invention, this carrier need not use chemical inducer, need only improve temperature to 42 ℃ induces 4h can induce the great expression of foreign protein, for usually used expression plasmid (as carry by IPTG inductive lac promotor plasmid), can save great amount of cost by the chemical inducer abduction delivering.
The substratum of culturing micro-organisms is not particularly limited, for obtaining to contain general carbon source, nitrogenous source, the ordinary culture medium of mineral ion and optional organic nutritive substance, in the scheme of special recommendation, what the present invention used is the LB substratum, and it is as follows to fill a prescription: peptone 10g, yeast extract 5g and NaCl10g, using 10mol/LNaOH to regulate pH is 7.2, is settled to 1L.Solid medium only needs to add in the liquid medium within 2% agar and gets final product.
The condition of culturing micro-organisms is not restriction especially also, for example can under aerobic conditions cultivate 12-48 hour, simultaneously the pH value is controlled at 5-8, and temperature is controlled at 25-40 ℃ scope.Cultivation for the microorganism that needs the abduction delivering exogenous protein, can after above-mentioned cultivation, select corresponding abduction delivering condition according to corresponding expression vectors again, when for example using the pBV220 expression vector, can 30 ℃ cultivate 16 hours after, temperature brought up to 42 ℃ method abduction delivering exogenous protein.
2. the sudden change of acid phosphatase gene has acid phosphatase than high-affinity with screening to nucleosides.
The method of the sudden change of acid phosphatase gene is unrestricted, as long as can make acid phosphatase produce sudden change.Such as handling the microorganism that contains this enzyme gene, also can directly suddenly change, perhaps the method orthomutation of DNA reorganization (DNA shuffling) with the method for rite-directed mutagenesis with ultraviolet radiation or with artificial mutagenic compound.
Screening does not limit the method that nucleosides has than the acid phosphatase of high-affinity, as long as can screen expression fast and efficiently nucleosides is had microorganism than the acid phosphatase of high-affinity.
3. with acid phosphatase enzyme catalysis nucleosides and the synthetic 5 '-Nucleotide of phosphate group donor.
The source of the acid phosphatase that uses does not limit, as long as can catalysis nucleosides and the synthetic corresponding 5 '-Nucleotide of phosphate group donor.Such as being, also can be to be derived from the intact cell of non-propagation of bacterium as the enzyme source through preliminary extractive thick enzyme.In the scheme of special recommendation, the present invention uses the genetic engineering bacterium of expressing the external source acid phosphatase as the enzyme source.
The nucleosides that uses does not limit, comprise that purine nucleoside is (such as inosine, guanosine, adenosine, xanthosine, 6-methyl purine nucleosides, 6-methoxyl group purine nucleoside, 2,6-diamino purine nucleoside, 6-fluoropurine nucleosides, 6-thio-purine nucleosides, TGR, the amino 6-thio-purine of 2-nucleosides, pectinose adenosine etc.), pyrimidine nucleoside (such as uridine, cytidine, 5-aminouridine, 5-hydroxyuridine, 5 broxuridines, 6-aza uridine, pectinose cytidine etc.).
The phosphate group donor that uses does not limit yet, comprise Tripyrophosphoric acid and salt thereof (as tetra-sodium, tripolyphosphate, three metaphosphoric acids, four metaphosphoric acids, hexa metaphosphoric acid, their mixture, its sodium salt, sylvite and composition thereof), benzenephosphonic acid and salt thereof are (as benzenephosphonic acid disodium, benzenephosphonic acid dipotassium, neighbour, neighbour-hexichol phosphoric anhydride and composition thereof), carbamyl phosphate and salt thereof (as carbamyl phosphate disodium, carbamyl phosphate dipotassium, carbamyl phosphate two ammoniums, carbamyl phosphate two lithiums and composition thereof), acetylphosphate and salt thereof (as acetylphosphate lithium potassium etc.).
The pH value of the damping fluid of reaction is between the 2.5-6.
The temperature of reaction is 20-50 ℃.
Nucleosides concentration is 10-200mM in the reaction substrate, and phosphate group donor concentration is its 1-10 times.
Reaction can be carried out under the immobilized condition, also can carry out under suitable stirring.
Reaction times is between 1-20 hour.
After reaction is finished, can use the method for synthetic resins absorption, use method or other conventional collections and separation method of precipitation agent, from mixture, collect and separates 5 '-Nucleotide of generation thus.
Utilize method catalysis of the present invention to synthesize 5 '-Nucleotide, have simply, efficient, the cycle is short, low cost, and characteristics such as compliance with environmental protection requirements are suitable for suitability for industrialized production.
Characteristics of the present invention are:
1. introduce sudden change S89N, A90F, G92D, its NPT activity of acid phosphatase behind the I171T improves greatly, when the acid phosphatase after the sudden change is substrate at the trisodium phosphate with the inosine of 50g/l and 300g/l, react 2 hours 5 '-IMP semi-invariants and reach 90.6g/L, transformation efficiency reaches 92%; When being substrate with the trisodium phosphate of the inosine of 100g/l and 300g/l, react that 5 '-IMP semi-invariant reaches 153.7g/L after 7 hours, transformation efficiency reaches 78%, compares with the technology of three former Kang Bo etc., and the reaction times has shortened, and 5 '-t-inosinic acid semi-invariant also is improved.
2. select for use the expression vector of temperature-induced type to express foreign protein, save great amount of cost.
3. directly change the nucleosides reaction, need not the broken cell enzyme purification with the catalysis of genetic engineering bacterium thalline.
Embodiment
The invention is further illustrated by the following examples, but the present invention is not limited only to these examples.
Embodiment 1
1. the structure of genetic engineering bacterium
Phoc gene order design primer according to the Enterobacter aerogenes that provides among the GeneBank is as follows:
Primer1:5 '-c
GggatccCatgaaaaagcgcgttctcgccctctg-3 ' (BamH I) (being designated as SEQ.ID.NO.5),
Primer2:5 '-cc
GctcgagCgatgacgttacttctgcgttttggcg-3 ' (Xhol I) (being designated as SEQ.ID.NO.6),
Chromosomal DNA with Enterobacter aerogenes is that template is PCR:95 ℃ of 5min, 30 * (95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 60s), and 72 ℃ of 10min.Obtain the PCR product of the about 760bp of length, behind restriction enzyme digestion, be connected respectively, Transformed E .coli DH5 α competent cell with the T4DNA ligase enzyme with the pBV220 carrier.Identify that with pBV220 carrier universal primer PCR and restriction enzyme digestion transformant obtains genetic engineering bacterium pBV220-phoc.The dna sequencing result is SEQ.ID.NO.1:
atgaaaaagcgcgttctcgccctctgcctggccagtttcttctccgttaacgcctttgctctggttccccccgggaacgatgtcaccaccaagcccgatctctactatctgaccaatgcccaggccatcgacagcctggcgctgttgccaccaccgccggcggtgggcagtatcgcatttttaaacgatcaggcgatgtatgagcaaggacgtctgctgcgcaataccgagcgcgggaagctggcggcagaggatgctaacctcagcgcgggcggcgtggccaacgccttctccagcgcctttggttcgccgattaccgagaaagacgcgccgcagcttcacaaactgctgaccaatatgattgaagacgccggcgacctggcgacccgcagcgcgaaagagaaatacatgcgcattcgcccgtttgcgttctatggcgtctccacctgcaacaccaccgaacaggacaagctggcgaaaaacggctcttacccgtccgggcatacctctatcggctgggccaccgccctggtgctggcggagatcaacccgcagcggcaaaacgaaattttgaagcgcggctatgagctcggcgagagccgggtgatctgcggctatcactggcagagcgatgtcgatgcggcgcgcatcgtcggctccgcggtggtcgctacgctgcacaccaacccggccttccagcagcagttgcagaaagccaaagatgaattcgccaaaacgcagaagtaa
Its aminoacid sequence is SEQ.ID.NO.2:
mkkrvlalclasffsvnafalvppgndvttkpdlyyltnaqaidslallppppavgsiaflndqamyeqgrllrntergklaaedanlsaggvanafssafgspitekdapqlhklltnmiedagdlatrsakekymrirpfafygvstcntteqdklakngsypsghtsigwatalvlaeinpqrqneilkrgyelgesrvicgyhwqsdvdaarivgsavvatlhtnpafqqqlqkakdefaktqk
The amino acid sequence homology that provides with GeneBank is 97.2%, has following the change: L13F, A24P, A28V, K38T, E54A, K69Q, S159A.
2. the preparation of wet thallus.
Be inoculated in the LB substratum that contains 100 μ g/mL penbritins after the activation of reorganization bacterium, cultivate OD for 30 ℃
6000.6 time is warming up to 42 ℃ immediately and cultivates 4h, the thalline after centrifugal is with the washing of 10mmol/L pH7.0 phosphate buffered saline buffer, centrifugal again wet thallus.
3. 5 '-IMP is synthesized in enzyme catalysis.
Reaction system is as follows:
Damping fluid: the acetate buffer of 100mmol/L pH3.5
Substrate: 10mg/ml inosine and 250mg/ml Na
4P
2O
710H
2O
Biomass: 2% (weight in wet base)
React on 30 ℃ of shaking baths, shake fast 150r/min, reaction finishes back 100 ℃ and boils 5min, and 12000r/min is centrifugal, and 5min gets supernatant liquor.
Get 2.5 μ l sample TLC and detect its 5 '-IMP concentration, method is as follows: with the silica GF254 bed board, TLC moving phase is n-propyl alcohol/ammonia/water (20/15/4), dry behind the chromatography, scrape the pairing point of 5 '-IMP under the ultraviolet, added the 2ml water dissolution 1 hour, centrifugal back ultraviolet spectrophotometer is measured its OD
249, with 5 ' of different concns-IMP OD
249The mark of being done
Directrix curve comes quantitatively.Calculate transformation efficiency according to following formula.
React 5 hours 5 '-IMP concentration and reach 8.29g/L, transformation efficiency reaches 42.1%.
Embodiment 2
1.phoc sudden change
Design following primer:
Primer3:5 '-ctaacctcagcttcggcgacgtggc-3 ' (being designated as SEQ.ID.NO.7),
Primer4:5 '-gccacgtcgccgaagctgaggttag-3 ' (being designated as SEQ.ID.NO.8),
Primer5:5 '-gcatacctctaccggctgggccac-3 ' (being designated as SEQ.ID.NO.9),
Primer6:5 '-gtggcccagccggtagaggtatgc-3 ' (being designated as SEQ.ID.NO.10),
With primer1/primer4, primer3/primer6, primer5/primer2 are template as primer with the pBV220-phoc recombinant plasmid respectively, PCR, and condition is as follows: 95 ℃ of 5min, 30 * (95 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 60s), 72 ℃ of 10min.Obtain the about 200bp of length respectively, 200bp, the PCR product of 300bp, reclaiming the back is template with it, is that primer carries out second and takes turns PCR with primer1/primer2, condition is 95 ℃ of 5min, 30 * (95 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 60s), 72 ℃ of 10min.Obtain the PCR product of the about 760bp of length, behind restriction enzyme digestion, be connected respectively, Transformed E .coli DH5 α competent cell with the T4DNA ligase enzyme with the pBV220 carrier.With pBV220 carrier universal primer PCR screening positive clone.
2. the screening of enzymatic activity high bacterium
Reaction system is as follows:
Damping fluid: the acetate buffer of 100mmol/L pH3.5
Substrate: 10mg/ml Ir and 250mg/ml Na
4P
2O
710H
2O
Biomass: 2% (weight in wet base)
React on 30 ℃ of shaking baths, shake fast 150r/min, react after 30 minutes 100 ℃ and boil 5min, 12000r/min is centrifugal, and 5min gets supernatant liquor, and TLC detects 5 '-IMP concentration.Finally obtain the active bacterium a8265 of a plant height, sequencing result is SEQ.ID.NO.3:
atgaaaaagcgcgttctcgccctctgcctggccagtttcttctccgttaacgcctttgctctggttccccccgggaacgatgtcaccaccaagcccgatctctactatctgaccaatgcccaggccatcgatagcctggcgctgttgccaccaccgccggcggtgggcagtatcgcatttttaaacgatcaggcgatgtatgagcaaggacgtctgctgcgcaataccgagcgcgggaagctggcggcagaggatgctaacctcaacttcggcgacgtggccaacgccttctccagcgcctttggttcgccgattaccgagaaagacgcgccgcagcttcacaaactgctgaccaatatgattgaagacgccggcgacctggcgacccgcagcgcgaaagagaaatacatgcgcattcgcccgtttgcgttctatggcgtctccacctgcaacaccaccgaacaggacaagctggcgaaaaacggctcttacccgtccgggcatacctctaccggctgggccaccgccctggtgctggcggagatcaacccgcagcggcaaaacgaaattttgaagcgcggctatgagctcggcgagagccgggtgatctgcggctatcactggcagagcgatgtcgatgcggcgcgcatcgtcggctccgcggtggtcgctacgctgcacaccaacccggccttccagcagcagttgcagaaagccaaagatgaattcgccaaaacgcagaagtaa
Its aminoacid sequence is SEQ.ID.NO.4:
mkkrvlalclasffsvnafalvppgndvttkpdlyyltnaqaidslallppppavgsiaflndqamyeqgrllrntergklaaedanlnfgdvanafssafgspitekdapqlhklltnmiedagdlatrsakekymrirpfafygvstcntteqdklakngsypsghtstgwatalvlaeinpqrqneilkrgyelgesrvicgyhwqsdvdaarivgsavvatlhtnpafqqqlqkakdefaktqk
There is following sudden change: S89N, A90F, G92D, I171T.
3. 5 '-IMP is synthesized in enzyme catalysis.
3.1, synthesize 5 '-IMP with following system enzyme catalysis behind the preparation a8265 wet thallus with embodiment one step 2:
Damping fluid: the acetate buffer of 100mmol/L pH3.5
Substrate: 50mg/ml Ir and 300mg/ml Na
4P
2O
710H
2O
Biomass: 2% (weight in wet base)
React 2 hours 5 '-IMP concentration and reach 90.6g/L, transformation efficiency reaches 92%.
3.2, synthesize 5 '-IMP with following system enzyme catalysis behind the preparation a8265 wet thallus with embodiment one step 2:
Damping fluid: the acetate buffer of 100mmol/L pH3.5
Substrate: 100mg/ml Ir and 300mg/ml Na
4P
2O
710H
2O
Biomass: 2% (weight in wet base)
React that 5 '-IMP concentration reaches 153.7g/L after 7 hours, transformation efficiency reaches 78%.
3.3, synthesize 5 '-IMP with following system enzyme catalysis behind the preparation a8265 wet thallus with embodiment one step 2:
Damping fluid: the acetate buffer of 100mmol/L pH3.5
Substrate: 100mg/ml Ir and 300mg/ml Na
4P
2O
710H
2O
Biomass: 1% (weight in wet base)
React that 5 '-IMP concentration reaches 151.7g/L after 16 hours, transformation efficiency reaches 77%.
Sequence table
＜110〉Fudan University
＜120〉method of a kind of High-efficient Production 5 '-flavour nucleotide
<160>10
<170>PatentIn?Version?2.1
<210>1
<211>747
<212>DNA
＜213〉enteroaerogen (Enterobacter aerogenes)
<220>
<221>CDS
<222>(1)...(747)
<400>1
atg?aaa?aag?cgc?gtt?ctc?gcc?ctc?tgc?ctg?gcc?agt?ttc?ttc?tcc?gtt?48
Met?Lys?Lys?Arg?Val?Leu?Ala?Leu?Cys?Leu?Ala?Ser?Phe?Phe?Ser?Val
1?5?10?15
aac?gcc?ttt?gct?ctg?gtt?ccc?ccc?ggg?aac?gat?gtc?acc?acc?aag?ccc?96
Asn?Ala?Phe?Ala?Leu?Val?Pro?Pro?Gly?Asn?Asp?Val?Thr?Thr?Lys?Pro
20?25?30
gat?ctc?tac?tat?ctg?acc?aat?gcc?cag?gcc?atc?gac?agc?ctg?gcg?ctg?144
Asp?Leu?Tyr?Tyr?Leu?Thr?Asn?Ala?Gln?Ala?Ile?Asp?Ser?Leu?Ala?Leu
35?40?45
ttg?cca?cca?ccg?ccg?gcg?gtg?ggc?agt?atc?gca?ttt?tta?aac?gat?cag?192
Leu?Pro?Pro?Pro?Pro?Ala?Val?Gly?Ser?Ile?Ala?Phe?Leu?Asn?Asp?Gln
50?55?60
gcg?atg?tat?gag?caa?gga?cgt?ctg?ctg?cgc?aat?acc?gag?cgc?ggg?aag?240
Ala?Met?Tyr?Glu?Gln?Gly?Arg?Leu?Leu?Arg?Asn?Thr?Glu?Arg?Gly?Lys
65?70?75?80
ctg?gcg?gca?gag?gat?gct?aac?ctc?agc?gcg?ggc?ggc?gtg?gcc?aac?gcc?288
Leu?Ala?Ala?Glu?Asp?Ala?Asn?Leu?Ser?Ala?Gly?Gly?Val?Ala?Asn?Ala
85?90?95
ttc?tcc?agc?gcc?ttt?ggt?tcg?ccg?att?acc?gag?aaa?gac?gcg?ccg?cag?336
Phe?Ser?Ser?Ala?Phe?Gly?Ser?Pro?Ile?Thr?Glu?Lys?Asp?Ala?Pro?Gln
100?105?110
ctt?cac?aaa?ctg?ctg?acc?aat?atg?att?gaa?gac?gcc?ggc?gac?ctg?gcg?384
Leu?His?Lys?Leu?Leu?Thr?Asn?Met?Ile?Glu?Asp?Ala?Gly?Asp?Leu?Ala
115?120?125
acc?cgc?agc?gcg?aaa?gag?aaa?tac?atg?cgc?att?cgc?ccg?ttt?gcg?ttc?432
Thr?Arg?Ser?Ala?Lys?Glu?Lys?Tyr?Met?Arg?Ile?Arg?Pro?Phe?Ala?Phe
130?135?140
tat?ggc?gtc?tcc?acc?tgc?aac?acc?acc?gaa?cag?gac?aag?ctg?gcg?aaa?480
Tyr?Gly?Val?Ser?Thr?Cys?Asn?Thr?Thr?Glu?Gln?Asp?Lys?Leu?Ala?Lys
145?150?155?160
aac?ggc?tct?tac?ccg?tcc?ggg?cat?acc?tct?atc?ggc?tgg?gcc?acc?gcc?528
Asn?Gly?Ser?Tyr?Pro?Ser?Gly?His?Thr?Ser?Ile?Gly?Trp?Ala?Thr?Ala
165?170?175
ctg?gtg?ctg?gcg?gag?atc?aac?ccg?cag?cgg?caa?aac?gaa?att?ttg?aag?576
Leu?Val?Leu?Ala?Glu?Ile?Asn?Pro?Gln?Arg?Gln?Asn?Glu?Ile?Leu?Lys
180?185?190
cgc?ggc?tat?gag?ctc?ggc?gag?agc?cgg?gtg?atc?tgc?ggc?tat?cac?tgg?624
Arg?Gly?Tyr?Glu?Leu?Gly?Glu?Ser?Arg?Val?Ile?Cys?Gly?Tyr?His?Trp
195?200?205
cag?agc?gat?gtc?gat?gcg?gcg?cgc?atc?gtc?ggc?tcc?gcg?gtg?gtc?gct?672
Gln?Ser?Asp?Val?Asp?Ala?Ala?Arg?Ile?Val?Gly?Ser?Ala?Val?Val?Ala
210?215?220
acg?ctg?cac?acc?aac?ccg?gcc?ttc?cag?cag?cag?ttg?cag?aaa?gcc?aaa?720
Thr?Leu?His?Thr?Asn?ProAla?Phe?Gln?Gln?Gln?Leu?Gln?Lys?Ala?Lys
225?230?235?240
gat?gaa?ttc?gcc?aaa?acg?cag?aag?taa?747
Asp?Glu?Phe?Ala?Lys?Thr?Gln?Lys
245
<210>2
<211>248
<212>PRT
＜213〉enteroaerogen (Enterobacter aerogenes)
<400>2
Met?Lys?Lys?Arg?Val?Leu?Ala?Leu?Cys?Leu?Ala?Ser?Phe?Phe?Ser?Val
1?5?10?15
Asn?Ala?Phe?Ala?Leu?Val?Pro?Pro?Gly?Asn?Asp?Val?Thr?Thr?Lys?Pro
20?25?30
Asp?Leu?Tyr?Tyr?Leu?Thr?Asn?Ala?Gln?Ala?Ile?Asp?Ser?Leu?Ala?Leu
35?40?45
Leu?Pro?Pro?Pro?Pro?Ala?Val?Gly?Ser?Ile?Ala?Phe?Leu?Asn?Asp?Gln
50?55?60
Ala?Met?Tyr?Glu?Gln?Gly?Arg?Leu?Leu?Arg?Asn?Thr?Glu?Arg?Gly?Lys
65?70?75?80
Leu?Ala?Ala?Glu?Asp?Ala?Asn?Leu?Ser?Ala?Gly?Gly?Val?Ala?Asn?Ala
85?90?95
Phe?Ser?Ser?Ala?Phe?Gly?Ser?Pro?Ile?Thr?Glu?Lys?Asp?Ala?Pro?Gln
100?105?110
Leu?His?Lys?Leu?Leu?Thr?Asn?Met?Ile?Glu?Asp?Ala?Gly?Asp?Leu?Ala
115?120?125
Thr?Arg?Ser?Ala?Lys?Glu?Lys?Tyr?Met?Arg?Ile?Arg?Pro?Phe?Ala?Phe
130?135?140
Tyr?Gly?Val?Ser?Thr?Cys?Asn?Thr?Thr?Glu?Gln?Asp?Lys?Leu?Ala?Lys
145?150?155?160
Asn?Gly?Ser?Tyr?Pro?Ser?Gly?His?Thr?Ser?Ile?Gly?Trp?Ala?Thr?Ala
165?170?175
Leu?Val?Leu?Ala?Glu?Ile?Asn?Pro?Gln?Arg?Gln?Asn?Glu?Ile?Leu?Lys
180?185?190
Arg?Gly?Tyr?Glu?Leu?Gly?Glu?Ser?Arg?Val?Ile?Cys?Gly?Tyr?His?Trp
195?200?205
Gln?Ser?Asp?Val?Asp?Ala?Ala?Arg?Ile?Val?Gly?Ser?Ala?Val?Val?Ala
210?215?220
Thr?Leu?His?Thr?Asn?Pro?Ala?Phe?Gln?Gln?Gln?Leu?Gln?Lys?Ala?Lys
225?230?235?240
Asp?Glu?Phe?Ala?Lys?Thr?Gln?Lys
245
<210>3
<211>747
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)...(747)
<400>3
atg?aaa?aag?cgc?gtt?ctc?gcc?ctc?tgc?ctg?gcc?agt?ttc?ttc?tcc?gtt?48
Met?Lys?Lys?Arg?Val?Leu?Ala?Leu?Cys?Leu?Ala?Ser?Phe?Phe?Ser?Val
1?5?10?15
aac?gcc?ttt?gct?ctg?gtt?ccc?ccc?ggg?aac?gat?gtc?acc?acc?aag?ccc?96
Asn?Ala?Phe?Ala?Leu?Val?Pro?Pro?Gly?Asn?Asp?Val?Thr?Thr?Lys?Pro
20?25?30
gat?ctc?tac?tat?ctg?acc?aat?gcc?cag?gcc?atc?gat?agc?ctg?gcg?ctg?144
Asp?Leu?Tyr?Tyr?Leu?Thr?Asn?Ala?Gln?Ala?Ile?Asp?Ser?Leu?Ala?Leu
35?40?45
ttg?cca?cca?ccg?ccg?gcg?gtg?ggc?agt?atc?gca?ttt?tta?aac?gat?cag?192
Leu?Pro?Pro?Pro?Pro?Ala?Val?Gly?Ser?Ile?Ala?Phe?Leu?Asn?Asp?Gln
50?55?60
gcg?atg?tat?gag?caa?gga?cgt?ctg?ctg?cgc?aat?acc?gag?cgc?ggg?aag?240
Ala?Met?Tyr?Glu?Gln?Gly?Arg?Leu?Leu?Arg?Asn?Thr?Glu?Arg?Gly?Lys
65?70?75?80
ctg?gcg?gca?gag?gat?gct?aac?ctc?aac?ttc?ggc?gac?gtg?gcc?aac?gcc?288
Leu?Ala?Ala?Glu?Asp?Ala?Asn?Leu?Asn?Phe?Gly?Asp?Val?Ala?Asn?Ala
85?90?95
ttc?tcc?agc?gcc?ttt?ggt?tcg?ccg?att?acc?gag?aaa?gac?gcg?ccg?cag?336
Phe?Ser?Ser?Ala?Phe?Gly?Ser?Pro?Ile?Thr?Glu?Lys?Asp?Ala?Pro?Gln
100?105?110
ctt?cac?aaa?ctg?ctg?acc?aat?atg?att?gaa?gac?gcc?ggc?gac?ctg?gcg?384
Leu?His?Lys?Leu?Leu?Thr?Asn?Met?Ile?Glu?Asp?Ala?Gly?Asp?Leu?Ala
115?120?125
acc?cgc?agc?gcg?aaa?gag?aaa?tac?atg?cgc?att?cgc?ccg?ttt?gcg?ttc?432
Thr?Arg?Ser?Ala?Lys?Glu?Lys?Tyr?Met?Arg?Ile?Arg?Pro?Phe?Ala?Phe
130?135?140
tat?ggc?gtc?tcc?acc?tgc?aac?acc?acc?gaa?cag?gac?aag?ctg?gcg?aaa?480
Tyr?Gly?Val?Ser?Thr?Cys?Asn?Thr?Thr?Glu?Gln?Asp?Lys?Leu?Ala?Lys
145?150?155?160
aac?ggc?tct?tac?ccg?tcc?ggg?cat?acc?tct?acc?ggc?tgg?gcc?acc?gcc?528
Asn?Gly?Ser?Tyr?Pro?Ser?Gly?His?Thr?Ser?Thr?Gly?Trp?Ala?Thr?Ala
165?170?175
ctg?gtg?ctg?gcg?gag?atc?aac?ccg?cag?cgg?caa?aac?gaa?att?ttg?aag?576
Leu?Val?Leu?Ala?Glu?ILe?Asn?Pro?Gln?Arg?Gln?Asn?Glu?Ile?Leu?Lys
180?185?190
cgc?ggc?tat?gag?ctc?ggc?gag?agc?cgg?gtg?atc?tgc?ggc?tat?cac?tgg?624
Arg?Gly?Tyr?Glu?Leu?Gly?Glu?Ser?Arg?Val?Ile?Cys?Gly?Tyr?His?Trp
195?200?205
cag?agc?gat?gtc?gat?gcg?gcg?cgc?atc?gtc?ggc?tcc?gcg?gtg?gtc?gct?672
Gln?Ser?Asp?Val?Asp?Ala?Ala?Arg?Ile?Val?Gly?Ser?Ala?Val?Val?Ala
210?215?220
acg?ctg?cac?acc?aac?ccg?gcc?ttc?cag?cag?cag?ttg?cag?aaa?gcc?aaa?720
Thr?Leu?His?Thr?Asn?Pro?Ala?Phe?Gln?Gln?Gln?Leu?Gln?Lys?Ala?Lys
225?230?235?240
gat?gaa?ttc?gcc?aaa?acg?cag?aag?taa?747
Asp?Glu?Phe?Ala?Lys?Thr?Gln?Lys
245
<210>4
<211>248
<212>PRT
＜213〉artificial sequence
<400>4
Met?Lys?Lys?Arg?Val?Leu?Ala?Leu?Cys?Leu?Ala?Ser?Phe?Phe?Ser?Val
1?5?10?15
Asn?Ala?Phe?Ala?Leu?Val?Pro?Pro?Gly?Asn?Asp?Val?Thr?Thr?Lys?Pro
20?25?30
Asp?Leu?Tyr?Tyr?Leu?Thr?Asn?Ala?Gln?Ala?Ile?Asp?Ser?Leu?Ala?Leu
35?40?45
Leu?Pro?Pro?Pro?Pro?Ala?Val?Gly?Ser?Ile?Ala?Phe?Leu?Asn?Asp?Gln
50?55?60
Ala?Met?Tyr?Glu?Gln?Gly?Arg?Leu?Leu?Arg?Asn?Thr?Glu?Arg?Gly?Lys
65?70?75?80
Leu?Ala?Ala?Glu?Asp?Ala?Asn?Leu?Asn?Phe?Gly?Asp?Val?Ala?Asn?Ala
85?90?95
Phe?Ser?Ser?Ala?Phe?Gly?Ser?Pro?Ile?Thr?Glu?Lys?Asp?Ala?Pro?Gln
100?105?110
Leu?His?Lys?Leu?Leu?Thr?Asn?Met?Ile?Glu?Asp?Ala?Gly?Asp?Leu?Ala
115?120?125
Thr?Arg?Ser?Ala?Lys?Glu?Lys?Tyr?Met?Arg?Ile?Arg?Pro?Phe?Ala?Phe
130?135?140
Tyr?Gly?Val?Ser?Thr?Cys?Asn?Thr?Thr?Glu?Gln?Asp?Lys?Leu?Ala?Lys
145?150?155?160
Asn?Gly?Ser?Tyr?Pro?Ser?Gly?His?Thr?Ser?Thr?Gly?Trp?Ala?Thr?Ala
165?170?175
Leu?Val?Leu?Ala?Glu?Ile?Asn?Pro?Gln?Arg?Gln?Asn?Glu?Ile?Leu?Lys
180?185?190
Arg?Gly?Tyr?Glu?Leu?Gly?Glu?Ser?Arg?Val?Ile?Cys?Gly?Tyr?His?Trp
195?200?205
Gln?Ser?Asp?Val?Asp?Ala?Ala?Arg?Ile?Val?Gly?Ser?Ala?Val?Val?Ala
210?215?220
Thr?Leu?His?Thr?Asn?Pro?Ala?Phe?Gln?Gln?Gln?Leu?Gln?Lys?Ala?Lys
225?230?235?240
Asp?Glu?Phe?Ala?Lys?Thr?Gln?Lys
245
<210>5
<211>35
<212>DNA
＜213〉artificial sequence
<400>5
cgggatccca?tgaaaaagcg?cgttctcgcc?ctctg?35
<210>7
<211>25
<212>DNA
＜213〉artificial sequence
<400>7
ctaacctcag?cttcggcgac?gtggc?25
<210>8
<211>25
<212>DNA
＜213〉artificial sequence
<400>8
gccacgtcgc?cgaagctgag?gttag?25
<210>9
<211>24
<212>DNA
＜213〉artificial sequence
<400>9
gcatacctct?accggctggg?ccac?24
<210>10
<211>24
<212>DNA
＜213〉artificial sequence
<400>10
gtggcccagc?cgg?tagaggt?atgc?24
Claims (2)
1. a method of producing 5 '-flavour nucleotide is characterized in that comprising the steps:
1.1 utilize recombinant DNA technology, make up the acid phosphatase expression vector, and transformed into escherichia coli, obtain efficiently expressing the genetic engineering bacterium of acid phosphatase; Described acid phosphatase derives from enteroaerogen, and its nucleotides sequence is classified SEQ.ID.NO.1 as, and aminoacid sequence is SEQ.ID.NO.2;
1.2 the sudden change of acid phosphatase gene has acid phosphatase than high-affinity with screening to nucleosides; The nucleotides sequence of this mutant acid phosphatase is classified SEQ.ID.NO.3 as, and aminoacid sequence is SEQ.ID.NO.4;
1.3 nucleosides is had acid phosphatase enzyme catalysis nucleosides and the synthetic 5 '-flavour nucleotide of phosphate group donor than high-affinity;
Wherein, described nucleosides is an inosine, and described phosphate group donor is a pyrophosphate salt.
2. method according to claim 1 is characterized in that, temperature of reaction is 20-50 ℃ in the step 1.3, the pH value of damping fluid is 2.5-6, nucleosides concentration is 10-200mM in the reaction substrate, and phosphate group donor concentration is 1-10 times of nucleosides concentration, and the reaction times is 1-20 hour.
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| JP6893000B2 (en) * | 2018-08-17 | 2021-06-23 | ボンタック バイオエンジニアリング（シェンゼン） カンパニー リミテッド | Method for preparing acid phosphatase mutant and nicotinamide riboside |
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Non-Patent Citations (7)
| Title |
|---|
| . EMBL-EBI,AC:Q9F1U1. 2007 |
| . EMBL-EBI,AC:Q9F1U1. 2007;Acid Phosphatase/Phosphotransferases from Enteric Bacteria. YASUHIRO MIHARA et al.Journal of Bioscience and Bioengineering,Vol.92 No.1. 2001 * |
| Acid Phosphatase/Phosphotransferases from Enteric Bacteria. YASUHIRO MIHARA et al.Journal of Bioscience and Bioengineering,Vol.92 No.1. 2001 |
| 呈味核苷酸及其生产应用的研究. 赵珺.长春大学学报，第14卷第6期. 2004 |
| 呈味核苷酸及其生产应用的研究. 赵珺.长春大学学报，第14卷第6期. 2004 * |
| 嘌呤核苷酸磷酸化酶基因的克隆及原核表达载体的构建. 李晓晖，孙嘉康等.工业微生物，第36卷第3期. 2006 |
| 嘌呤核苷酸磷酸化酶基因的克隆及原核表达载体的构建. 李晓晖，孙嘉康等.工业微生物，第36卷第3期. 2006 * |
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