CN109423469A - A kind of method producing glucuronic acid and its dedicated engineering bacteria - Google Patents
A kind of method producing glucuronic acid and its dedicated engineering bacteria Download PDFInfo
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Abstract
The invention discloses a kind of method for producing glucuronic acid and its dedicated engineering bacterias.The method (method first) of the first production glucuronic acid provided by the invention includes the following steps: using inositol as raw material, under the action of engineering bacteria, produces glucuronic acid；The engineering bacteria is the recombinant bacterium of expressive function albumen, is that the functional gene for encoding the functional protein is imported out what bacterium germination obtained.The method (method second) of second of production glucuronic acid provided by the invention includes the following steps: using inositol as raw material, under the action of functional protein, produces glucuronic acid.The functional protein is following (a1) or (a2) or (a3): protein shown in the sequence 3 of (a1) sequence table；(a2) fused protein obtained in N-terminal or/and C-terminal the connection label of (a)；(a3) protein shown in the sequence 7 of sequence table.The present invention will generate huge economic benefit for glucuronic acid production, have great application value.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of method for producing glucuronic acid and its special engineering
Bacterium.
Background technique
Glucuronic acid (D-glucuronic acid) is that glucose C-6 hydroxyls are oxidized to carboxyl and are formed by
Uronic acid, molecular formula C6H10O7, molecular weight 194.14.Glucuronic acid is white, needle-shaped crystals or powder, fusing point 155
DEG C -157 DEG C, structural schematic diagram is shown in Fig. 1.Glucuronic acid is widely present in plant, in animal, seldom exists in a free form
In nature.Glucuronic acid is not only traditional liver detoxification agent and immune function controlling agents, and is many medicine of synthesis
Important intermediate, be commonly used for the additive of functional beverage, slimming drugs, cosmetics etc..
Inositol (myo-inositol), also known as inositol (cyclohexanhexol), molecular formula C6H12O6, molecule
Amount is 180, and structural schematic diagram is shown in Fig. 2.Almost all creatures all inositols containing free state or reference state.The life of inositol at present
Production method has Hydrolyze method, microbial method, chemical synthesis and ion-exchange.Hydrolyze method is the production most important side of inositol
Method is mainly that raw material extract to obtain with rice bran, corn bran etc..
Currently, the production of glucuronic acid is mainly mainly the following method:
1. biological fermentation process: being fermented using relevant biological bacterial strain, thus synthesis of glucose aldehydic acid.In human amniotic membrane
There are UDP-glucose dehydrogenases, can be catalyzed UDP-glucose and generate UDP-glucose aldehydic acid, UDP-glucose aldehydic acid is in phase
Further synthesis of glucose aldehydic acid under the action of the enzyme of pass.
2. polysaccharide hydrolysis method: by hydrolyzing the polysaccharide containing uronic acid, to obtain glucuronic acid.Main includes hydrolyzing too
The hemicellulose of sun flower film, alkali process hydrolysis cotton and cellulose, the methods of water hot extraction's hemicellulose.
3. chemical oxidization method: the main method of production glucuronic acid at present mainly includes inorganic oxide method and catalysis oxygen
Two kinds of change method.The technique there are energy consumption height, poor selectivity, product yield is low, environmental pollution is serious the problems such as.
In conclusion there is an urgent need in the art to develop a kind of method of new efficient production glucuronic acid.
Summary of the invention
The object of the present invention is to provide a kind of method for producing glucuronic acid and its dedicated engineering bacterias.
It is provided by the invention the first production glucuronic acid method (method first), include the following steps: be with inositol
Raw material produces glucuronic acid under the action of engineering bacteria；The engineering bacteria is the recombinant bacterium of expressive function albumen, is that will compile
The functional gene of the code functional protein imports out what bacterium germination obtained.
The engineering bacteria is the recombinant bacterium of intracellular expression functional protein.
The bacterium germination out can be Escherichia coli, concretely Escherichia coli BW25113.
The functional gene imports out bacterium germination especially by recombinant plasmid.The recombinant plasmid can be by the functional gene
It is inserted into the recombinant plasmid that the carrier that sets out obtains.The carrier that sets out can be carrier pBAD/HisB.The recombinant plasmid is concretely
By DNA molecular second, (DNA molecular second is double chain DNA molecule, from upstream to downstream successively by 4 institute of nucleotide C and the sequence of sequence table
Show that nucleotide forms) it is inserted into the recombinant plasmid obtained between XhoI the and EcoRI restriction enzyme site of carrier pBAD/HisB.
In the method first, reaction temperature is 30 DEG C -40 DEG C, reaction pH is 8.0-9.0.
In the method first, reaction temperature is 37 DEG C, reaction pH is 8.5.
In the method first, the reaction time can be 8-12 hours.
In the method first, the reaction time can be 10 hours.
Engineering bacteria in the method first is the engineering bacteria carried out after Fiber differentiation.The Fiber differentiation be using L- I
Primary sugar is induced and is cultivated.
The method first includes the following steps: to cultivate the engineering bacteria and carry out L-arabinose during the cultivation process to lure
It leads, is then centrifuged for collecting thallus；In buffer system, the thallus and inositol is added, is reacted, glucuronic acid is obtained.
The quality proportioning of thallus and inositol are as follows: 10:18.In reaction system, the initial concentration of each component is as follows: thallus 10g/L, inositol
18g/L.Thallus quality is weight in wet base.The buffer system is the borate buffer solution of 50mM.
The method first specifically comprises the following steps: in the LB liquid medium containing 50 μ g/mL streptomysins described in culture
Engineering bacteria is to OD600nmThen=0.6-0.8 is added L-arabinose and makes its concentration 0.2g/ in cultivating system
100mL, 30 DEG C, 200rpm shaken cultivation 12 hours are then centrifuged for collecting thallus；In buffer system, the thallus, flesh is added
Pure and mild TritonX-100, is reacted, and glucuronic acid is obtained.The quality proportioning of thallus and inositol are as follows: 10:18.Reaction system
In, the initial concentration of each component is as follows: thallus 10g/L, inositol 18g/L, TritonX-100 0.1% (volumn concentration).
Thallus quality is weight in wet base.The buffer system is the borate buffer solution of 50mM.
It is provided by the invention second production glucuronic acid method (method second), include the following steps: be with inositol
Raw material produces glucuronic acid under the action of functional protein.
In the method second, reaction temperature is 30 DEG C -40 DEG C, reaction pH is 8.0-9.0.
In the method second, reaction temperature is 37 DEG C, reaction pH is 8.0.
In the method second, the reaction time can be 20-40min.
In the method second, the reaction time can be 30min.
The method second includes the following steps: that functional protein, L-cysteine, inositol and divalent are added in buffer system
Iron compound is reacted, and glucuronic acid is obtained.The proportion of functional protein and inositol are as follows: 1 × 105μ g functional protein:
20mmol inositol.Functional protein, L-cysteine, the proportion of inositol and ferro-compound are as follows: 1 × 105μ g functional protein:
2mmolL- cysteine: 20mmol inositol: 1mmol ferro-compound.In reaction system, the initial concentration of each component is as follows:
Functional protein 100 μ g/mL, L-cysteine 2mmol/L, inositol 20mmol/L, Fe2+1mmol/L.The ferro-compound tool
Body is ferrous sulfate.The buffer system is the Tris-HCl buffer of pH8.0,50mM.
The present invention also protects a kind of recombinant bacterium, is that recombinant plasmid is imported out to what bacterium germination obtained；The recombinant plasmid is will
The functional gene importing of encoding function albumen sets out what carrier obtained；The carrier that sets out is pBAD/HisB carrier；It is described to set out
Bacterium is Escherichia coli BW25113.Concretely by DNA molecular second, (DNA molecular second is double chain DNA molecule to the recombinant plasmid, certainly
Successively the nucleotide shown in nucleotide C and the sequence of sequence table 4 forms in upstream to downstream) it is inserted into the XhoI of carrier pBAD/HisB
The recombinant plasmid obtained between EcoRI restriction enzyme site.
The present invention also protects the recombinant bacterium preparing the application in glucuronic acid.It is original with inositol in the application
Material.
The present invention goes back defencive function albumen and is preparing the application in glucuronic acid.In the application, using inositol as raw material.
Any description above functional protein is following (a1) or (a2) or (a3):
(a1) protein shown in the sequence 3 of sequence table；
(a2) fused protein obtained in N-terminal or/and C-terminal the connection label of (a)；
(a3) protein shown in the sequence 7 of sequence table.
Any description above functional gene is following (b1) or (b2) or (b3) or (b4):
(b1) code area DNA molecular as shown in sequence 4 in sequence table；
(b2) code area DNA molecular as shown in sequence 8 in sequence table；
(b3) hybridize under strict conditions with (b1) or (b2) DNA sequence dna limited and encode to have and convert Portugal for inositol
The DNA molecular of the protein of the ability of grape uronic acid；
(b4) DNA sequence dna limited with (b1) or (b2) has 90% or more homology and coding is with converting inositol to
The DNA molecular of the protein of the ability of glucuronic acid.
Above-mentioned stringent condition can be for 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS be miscellaneous in DNA or RNA
It hands over and hybridizes at 65 DEG C in experiment and wash film.
The present invention provides the new applications of functional protein, produce glucuronic acid by raw material of inositol.Correspondingly, of the invention
The recombinant bacterium for expressing the functional protein is provided, which can produce Portugal by raw material of inositol in a manner of full cell
Grape uronic acid.The present invention will generate huge economic benefit for glucuronic acid production, have great popularization and application valence
Value.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of glucuronic acid.
Fig. 2 is the structural schematic diagram of inositol.
Fig. 3 is the HPLC test map of inositol standard items and glucuronic acid standard items.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.Unless otherwise specified, the borate buffer solution in embodiment is the borate buffer solution of 50mM.Identical conditions are joined
It is identical as standard items retention time in sample to be tested (± 0.1min) for chromatogram under several, peak can regard as target peak.
Escherichia coli BW25113: it is purchased from NBRP E.coli strain, catalog number: ME9062 (https:// shigen.nig.ac.jp/ecoli/strain/resource/strainGeneMutant/list).Carrier pBAD/HisB:
Invitrogen company, catalog number V430-01.Inositol standard items: Aladdin company, catalog number I108336.Portugal
Grape uronic acid standard items: Aladdin company, catalog number G105701.
The building of embodiment 1, recombinant bacterium
1, by DNA molecular first, (DNA molecular first is double chain DNA molecule, from upstream to downstream successively by nucleotide C and sequence
The composition of nucleotide shown in the sequence 2 of table) it is inserted between XhoI the and EcoRI restriction enzyme site of carrier pBAD/HisB, obtain recombination matter
Grain first.According to sequencing result, structure is carried out to recombinant plasmid first and is described as follows: in XhoI the and EcoRI enzyme of carrier pBAD/HisB
DNA molecular first is inserted between enzyme site.Partial nucleotide in DNA molecular first and carrier forms fusion first, and expression is melted
Hop protein first.
2, by DNA molecular second, (DNA molecular second is double chain DNA molecule, from upstream to downstream successively by nucleotide C and sequence
The composition of nucleotide shown in the sequence 4 of table) it is inserted between XhoI the and EcoRI restriction enzyme site of carrier pBAD/HisB, obtain recombination matter
Grain second.According to sequencing result, structure is carried out to recombinant plasmid second and is described as follows: in XhoI the and EcoRI enzyme of carrier pBAD/HisB
DNA molecular second is inserted between enzyme site.Partial nucleotide in DNA molecular second and carrier forms fusion second, and expression is melted
Hop protein second.Fusion second is as shown in the sequence 8 of sequence table, and fusion protein second is as shown in the sequence 7 of sequence table.
3, by DNA molecular third, (DNA molecular third is double chain DNA molecule, from upstream to downstream successively by nucleotide C and sequence
The composition of nucleotide shown in the sequence 6 of table) it is inserted between XhoI the and EcoRI restriction enzyme site of carrier pBAD/HisB, obtain recombination matter
Grain third.According to sequencing result, structure is carried out to recombinant plasmid third and is described as follows: in XhoI the and EcoRI enzyme of carrier pBAD/HisB
DNA molecular third is inserted between enzyme site.Partial nucleotide on DNA molecular third and carrier forms fusion third, and expression is melted
Hop protein third.
4, the recombinant plasmid first for obtaining step 1 imports Escherichia coli BW25113, obtains recombinant bacterium I.
5, the recombinant plasmid second for obtaining step 2 imports Escherichia coli BW25113, obtains recombinant bacterium II.
6, the recombinant plasmid third for obtaining step 3 imports Escherichia coli BW25113, obtains recombinant bacterium III.
The preparation of embodiment 2, albumen
Test strain is respectively as follows: recombinant bacterium I, recombinant bacterium II or the recombinant bacterium III of the preparation of embodiment 1.
1, the monoclonal for taking test strain is seeded to the LB liquid medium containing 50 μ g/mL streptomysins, 37 DEG C, 220rpm
Shaken cultivation is to OD600nm=0.6~0.8.
2, after completing step 1, L-arabinose is added in cultivating system and makes its concentration 0.2g/100mL, 30 DEG C,
200rpm shaken cultivation 12 hours.
3, after completing step 2, it is rounded a cultivating system, 4 DEG C, 6000rpm centrifugation 15min collect bacterial sediment.
4, the bacterial sediment for taking step 3 to obtain is resuspended with buffer solution A and carries out ultrasonication, and then 10000rpm is centrifuged
30min collects supernatant.
Buffer solution A (pH8.0): Tris-HCl containing 50mmol/L and 100mmol/LNaCl, surplus are water.
5, the supernatant for taking step 4 to obtain collects filtrate with 0.45 μm of membrane filtration, carries out Ni-NTA column purification.
Pillar parameter: HisTrap HP pillar (1 × 5ml) is that the catalog number of GE Healthcare company is 17-
The product of 5248-01.
Purifying procedure: pillar is sufficiently 1. balanced with 50ml buffer solution A；2. loading 30ml filtrate；3. containing 50mmol/ with 50ml
The buffer solution A of L imidazoles is washed；4. being eluted with the buffer solution A of 15ml imidazoles containing 300mmol/L and collecting solution after column.
6, the solution after crossing column that step 5 obtains is taken, desalination is carried out and buffer system is replaced into buffer solution B, obtain albumen
Solution.
Buffer solution B (pH8.0): Tris-HCl containing 50mmol/L, surplus are water.
Recombinant bacterium I carries out the protein solution that above-mentioned steps obtain, and is named as protein solution I (containing fusion protein first).
Recombinant bacterium II carries out the protein solution that above-mentioned steps obtain, and is named as protein solution II (containing fusion protein second).
Recombinant bacterium III carries out the protein solution that above-mentioned steps obtain, and is named as protein solution III (containing fusion protein the third).
The enzyme activity determination of embodiment 3, albumen
Testing protein solution is protein solution I, protein solution II or protein solution III prepared by embodiment 2.
1, the protein concentration of testing protein solution is detected.
2, the enzyme activity of testing protein solution is detected
Reaction system (provides Fe by testing protein solution, L-cysteine, inositol, ferrous sulfate2+) and buffer composition.
Buffer is the Tris-HCl buffer of pH8.0,50mM.In reaction system, protein concentration be 100 μ g/mL (albumen it is unique
Source is testing protein solution), the concentration of L-cysteine is 2mmol/L, Fe2+Concentration be 1mmol/L, the concentration of inositol is
20mmol/L。
Reaction condition: 37 DEG C of standings react 30min.
After reaction, it samples, is diluted to 10 times of volumes with distilled water, containing for glucuronic acid is then detected by HPLC
Amount.
HPLC system: Agilent 1260；Chromatographic column: Aminex HPX-87H (300mm × 7.8mm)；
Mobile phase: aqueous sulfuric acid (5mmol/L).
Flow velocity: 0.5mL/min；
Temperature: 30 DEG C；
Detector: RID.
Inositol standard items and glucuronic acid standard items are subjected to above-mentioned HPLC detection, map is shown in Fig. 3.Glucuronic acid
Retention time is 9.193min, and the retention time of inositol is 10.163min.The standard curve of glucuronic acid are as follows: y=20192x
+ 817.7, R2=0.998 (wherein x is peak area, and y is the content of glucuronic acid, unit mmol/L).
Enzyme activity definition: 1 enzyme-activity unit (U) is defined as reacting per hour needed for generating 1 μm of ol product glucuronic acid
Enzyme amount.
3, the Rate activity that testing protein is calculated according to protein concentration and enzyme activity, the results are shown in Table 1.
Table 1
| After reaction, the glucuronic acid concentration (mmol/L) in reaction system | Rate activity (U/mg) | |
| Fusion protein first | 4.34±0.38 | 1.45±0.13 |
| Fusion protein second | 10.88±1.15 | 4.42±0.40 |
| Fusion protein third | 2.20±0.30 | 0.74±0.10 |
Embodiment 4 prepares glucuronic acid using recombinant bacterium
Test strain is respectively as follows: recombinant bacterium I, recombinant bacterium II or the recombinant bacterium III of the preparation of embodiment 1.
1, the monoclonal for taking test strain is seeded to the LB liquid medium containing 50 μ g/mL streptomysins, 37 DEG C, 220rpm
Shaken cultivation is to OD600nm=0.6~0.8.
2, after completing step 1, L-arabinose is added in cultivating system and makes its concentration 0.2g/100mL, 30 DEG C,
200rpm shaken cultivation 12 hours.
3, after completing step 2, it is rounded a cultivating system, 4 DEG C, 6000rpm centrifugation 15min collect bacterial sediment.
4, glucuronic acid is prepared
The composition of reaction system: bacterial sediment, inositol, TritonX-100 and the borate buffer solution that step 3 obtains.Instead
It answers in system, the initial concentration of each component is as follows: cell concentration 10g (weight in wet base)/L, inositol 18g/L, TritonX-1000.1%
(volumn concentration).
Reaction condition: 37 DEG C of standings are reacted 10 hours.
Use the pH of 5M NaOH aqueous solution control reaction system for 8.5 in reaction process.
After reaction, it samples, dilutes 10 times with distilled water, the content of glucuronic acid is then detected by HPLC.
HPLC detection method is shown in the step 2 of embodiment 3.
It the results are shown in Table 2.
Table 2
| After reaction, the glucuronic acid concentration in reaction system | Conversion ratio | |
| Recombinant bacterium I | 9.7g/L | 49.9% |
| Recombinant bacterium II | 17.7g/L | 91.1% |
| Recombinant bacterium III | 11.8g/L | 60.7% |
SEQUENCE LISTING
<110>Institute of Microorganism, Academia Sinica
<120>a kind of method for producing glucuronic acid and its dedicated engineering bacteria
<130> GNCYX171493
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<170> PatentIn version 3.5
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gacgccgttg caatggttaa tatcctgcgt ggcatggcta ccagcgagac aacggcgttc 120
gatgctgagt cgcgtttcga ctcagacaaa gataaatccg cgttccgtaa atatgacgag 180
gcctgcgacc gtgtaaaggg tttctacgcg gagcagcacg ccaaacagac cgtggcgcat 240
aacctggctg cccgccagcg tttttattcg ccgacccgca aacgtccaga gatgaccgta 300
tgggaagcta tcgaaaaact gaacgcatta atcgacaaca gcgatccgga taccgagctg 360
tcccagatcc accacctgct gcaatctgcg gaagcgatcc gtcgtgacgg taaaccgcgc 420
tggatgcagt tagtaggcct gatccacgac ctggggaaac tgatgctgtt ctttgacggc 480
tgtgccgagg gccagtggga agtggtgggt gacactttcc cggtaggctg ccagttcgac 540
gagcgttgca tctacccgga aacgtttaaa gcgaacccgg attcaagcca tgaagtatac 600
gggactcgct acggcatcta tcagccgggt tgcggcatta acaacctgat gatgtgctgg 660
ggccacgatg agtacctgta ccttgtcatt aaagaccagt caaccctgcc gccggaagcc 720
ttggcgatgg tccgcttcca cagcttttac ccgtggcatc gtgagggtgc gtacatggac 780
ttcatggccg aaggggatga agcgcttctg cacgccgttc gtgcgttcaa cccgtacgac 840
ctgtactcta aaagcgacga ggtgccgaaa gttgaagaac ttaaaccgta ctacctggaa 900
ctgattgacg agttcttccc gcagaaagta attaagtggt aa 942
<210> 5
<211> 315
<212> PRT
<213> Cryptococcus neoformans
<400> 5
Met His Ala Pro Glu Val Asn Asp Tyr Ile Lys His Lys Ala Val Lys
1 5 10 15
Leu Asp Gln Val Ser Asp Glu Ile Asp Glu Val Asn Val Leu Lys Leu
20 25 30
Lys Gln Lys Asp Ala Val Glu Lys Thr Gln Ala Glu Ile Asp Tyr Asp
35 40 45
Leu Ala Ser Lys Phe Asp Gln Glu Lys Asp Lys Ala Ala Phe Arg Gln
50 55 60
Tyr Glu Glu Ala Cys Asp Arg Val Lys Asn Phe Tyr Ala Glu Gln His
65 70 75 80
Leu Lys Gln Thr Tyr Glu Tyr Asn Val Lys Ile Arg Gln Glu Phe Arg
85 90 95
Asn Thr Val Arg Ala Arg Met Ser Ile Trp Glu Ala Met Glu Leu Leu
100 105 110
Asp Asn Leu Val Asp Glu Ser Asp Pro Asp Thr Ser Val Gly Gln Ile
115 120 125
Glu His Leu Leu Gln Thr Ala Glu Ala Ile Arg Arg Asp Gly Lys Pro
130 135 140
Glu Trp Met Gln Val Thr Gly Leu Ile His Asp Leu Gly Lys Leu Leu
145 150 155 160
Cys Phe Phe Gly Ala Asp Gly Gln Trp Asp Val Val Gly Asp Thr Phe
165 170 175
Val Val Gly Cys Lys Phe Ser Asp Lys Ile Ile Tyr Pro Asp Thr Phe
180 185 190
Lys Ser Asn Pro Asp Tyr Asn Asn Pro Lys Leu Asn Thr Lys Tyr Gly
195 200 205
Val Tyr Glu Pro Asn Cys Gly Leu Asp Asn Val Leu Leu Ser Trp Gly
210 215 220
His Asp Glu Tyr Met Tyr Glu Ile Cys Lys Asn Gln Ser Thr Leu Pro
225 230 235 240
Gln Glu Ala Leu Ala Met Ile Arg Tyr His Ser Phe Tyr Pro Trp His
245 250 255
Arg Glu Gly Ala Tyr Glu His Leu Met Asn Glu Lys Asp Tyr Ser Gln
260 265 270
Leu Lys Ala Val Lys Ala Phe Asn Pro Tyr Asp Leu Tyr Ser Lys Ser
275 280 285
Asp Asp Pro Pro Lys Lys Glu Glu Leu Lys Pro Tyr Tyr Gln Ser Leu
290 295 300
Ile Ser Lys Phe Phe Pro Glu Glu Val Gln Trp
305 310 315
<210> 6
<211> 948
<212> DNA
<213> Cryptococcus neoformans
<400> 6
atgcatgctc cggaagttaa cgactacata aaacataaag ctgtcaagct ggatcaggtt 60
agcgatgaaa ttgatgaggt gaacgtatta aaactgaagc agaaagacgc agtagaaaag 120
acccaggccg aaatcgacta tgacctggcg agcaaattcg accaggagaa agacaaggcc 180
gccttccgtc aatacgaaga agcgtgcgat cgtgtgaaaa atttttacgc tgaacagcac 240
cttaagcaga cttatgagta taacgtaaaa atccgccagg agttccgcaa cacggtgcgc 300
gctcgtatgt ccatctggga agcaatggag ctgctagata acctggtgga cgaatccgat 360
ccggacacca gtgtaggcca aatcgaacac ttactgcaaa ccgcggaagc tatccgtcgt 420
gatggtaaac ctgagtggat gcaggttact ggtttaattc acgacctggg caaactgctg 480
tgcttcttcg gtgcggatgg tcagtgggat gttgtgggcg ataccttcgt agttggctgt 540
aaattttccg acaaaatcat ttaccctgac acgttcaaat ccaacccgga ctacaacaac 600
ccgaaactga acacgaagta tggtgtgtac gaaccgaact gcggcttaga taacgtactg 660
ctctcttggg gccatgatga gtacatgtat gagatttgta aaaaccagag cacgctgccg 720
caggaagcac tggccatgat ccgttaccat tctttttatc cgtggcatcg tgaaggggcc 780
tacgagcacc tgatgaacga gaaggattat agccagctta aagccgttaa agccttcaac 840
ccgtatgatc tgtacagcaa atccgatgat ccgccgaaga aagaggaact gaaaccgtac 900
tatcaatccc tgatctctaa attcttcccg gaggaggttc agtggtaa 948
<210> 7
<211> 349
<212> PRT
<213> Artificial Sequence
<400> 7
Met Gly Gly Ser His His His His His His Gly Met Ala Ser Met Thr
1 5 10 15
Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp
20 25 30
Pro Ser Ser Ser Met Ala Ala Val Asp Ile Arg Pro Ala Gln Arg Asp
35 40 45
Gly Pro Ala Leu Glu Ala Ile Thr Asp Ala Val Ala Met Val Asn Ile
50 55 60
Leu Arg Gly Met Ala Thr Ser Glu Thr Thr Ala Phe Asp Ala Glu Ser
65 70 75 80
Arg Phe Asp Ser Asp Lys Asp Lys Ser Ala Phe Arg Lys Tyr Asp Glu
85 90 95
Ala Cys Asp Arg Val Lys Gly Phe Tyr Ala Glu Gln His Ala Lys Gln
100 105 110
Thr Val Ala His Asn Leu Ala Ala Arg Gln Arg Phe Tyr Ser Pro Thr
115 120 125
Arg Lys Arg Pro Glu Met Thr Val Trp Glu Ala Ile Glu Lys Leu Asn
130 135 140
Ala Leu Ile Asp Asn Ser Asp Pro Asp Thr Glu Leu Ser Gln Ile His
145 150 155 160
His Leu Leu Gln Ser Ala Glu Ala Ile Arg Arg Asp Gly Lys Pro Arg
165 170 175
Trp Met Gln Leu Val Gly Leu Ile His Asp Leu Gly Lys Leu Met Leu
180 185 190
Phe Phe Asp Gly Cys Ala Glu Gly Gln Trp Glu Val Val Gly Asp Thr
195 200 205
Phe Pro Val Gly Cys Gln Phe Asp Glu Arg Cys Ile Tyr Pro Glu Thr
210 215 220
Phe Lys Ala Asn Pro Asp Ser Ser His Glu Val Tyr Gly Thr Arg Tyr
225 230 235 240
Gly Ile Tyr Gln Pro Gly Cys Gly Ile Asn Asn Leu Met Met Cys Trp
245 250 255
Gly His Asp Glu Tyr Leu Tyr Leu Val Ile Lys Asp Gln Ser Thr Leu
260 265 270
Pro Pro Glu Ala Leu Ala Met Val Arg Phe His Ser Phe Tyr Pro Trp
275 280 285
His Arg Glu Gly Ala Tyr Met Asp Phe Met Ala Glu Gly Asp Glu Ala
290 295 300
Leu Leu His Ala Val Arg Ala Phe Asn Pro Tyr Asp Leu Tyr Ser Lys
305 310 315 320
Ser Asp Glu Val Pro Lys Val Glu Glu Leu Lys Pro Tyr Tyr Leu Glu
325 330 335
Leu Ile Asp Glu Phe Phe Pro Gln Lys Val Ile Lys Trp
340 345
<210> 8
<211> 1050
<212> DNA
<213> Artificial Sequence
<400> 8
atggggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60
atgggtcggg atctgtacga cgatgacgat aaggatccga gctcgagcat ggcagcggta 120
gacattcgcc ctgcacagcg tgatggtccg gctctggagg cgatcactga cgccgttgca 180
atggttaata tcctgcgtgg catggctacc agcgagacaa cggcgttcga tgctgagtcg 240
cgtttcgact cagacaaaga taaatccgcg ttccgtaaat atgacgaggc ctgcgaccgt 300
gtaaagggtt tctacgcgga gcagcacgcc aaacagaccg tggcgcataa cctggctgcc 360
cgccagcgtt tttattcgcc gacccgcaaa cgtccagaga tgaccgtatg ggaagctatc 420
gaaaaactga acgcattaat cgacaacagc gatccggata ccgagctgtc ccagatccac 480
cacctgctgc aatctgcgga agcgatccgt cgtgacggta aaccgcgctg gatgcagtta 540
gtaggcctga tccacgacct ggggaaactg atgctgttct ttgacggctg tgccgagggc 600
cagtgggaag tggtgggtga cactttcccg gtaggctgcc agttcgacga gcgttgcatc 660
tacccggaaa cgtttaaagc gaacccggat tcaagccatg aagtatacgg gactcgctac 720
ggcatctatc agccgggttg cggcattaac aacctgatga tgtgctgggg ccacgatgag 780
tacctgtacc ttgtcattaa agaccagtca accctgccgc cggaagcctt ggcgatggtc 840
cgcttccaca gcttttaccc gtggcatcgt gagggtgcgt acatggactt catggccgaa 900
ggggatgaag cgcttctgca cgccgttcgt gcgttcaacc cgtacgacct gtactctaaa 960
agcgacgagg tgccgaaagt tgaagaactt aaaccgtact acctggaact gattgacgag 1020
ttcttcccgc agaaagtaat taagtggtaa 1050
Claims (10)
1. a kind of method for producing glucuronic acid includes the following steps: using inositol as raw material, raw under the action of engineering bacteria
Malaga uronic acid；The engineering bacteria is the recombinant bacterium of expressive function albumen, is the functional gene that will encode the functional protein
Import out what bacterium germination obtained；
The functional protein is following (a1) or (a2) or (a3):
(a1) protein shown in the sequence 3 of sequence table；
(a2) fused protein obtained in N-terminal or/and C-terminal the connection label of (a)；
(a3) protein shown in the sequence 7 of sequence table.
2. the method as described in claim 1, it is characterised in that: in the method, reaction temperature is 30 DEG C -40 DEG C, reacts pH
For 8.0-9.0.
3. method according to claim 1 or 2, it is characterised in that: the engineering bacteria in the method is after carrying out Fiber differentiation
Engineering bacteria；The Fiber differentiation is to be induced and cultivated using L-arabinose.
4. such as method any one of claims 1 to 3, it is characterised in that: the bacterium germination out is Escherichia coli.
5. the method as described in any in Claims 1-4, it is characterised in that: the functional gene is imported by recombinant plasmid
Bacterium germination out；The recombinant plasmid is that functional gene insertion is set out what carrier obtained.
6. method as claimed in claim 5, it is characterised in that: the carrier that sets out is pBAD/HisB carrier.
7. a kind of method for producing glucuronic acid includes the following steps: using inositol as raw material, under the action of functional protein,
Produce glucuronic acid；The functional protein is following (a1) or (a2) or (a3):
(a1) protein shown in the sequence 3 of sequence table；
(a2) fused protein obtained in N-terminal or/and C-terminal the connection label of (a)；
(a3) protein shown in the sequence 7 of sequence table.
8. a kind of recombinant bacterium is that recombinant plasmid is imported out to what bacterium germination obtained；The recombinant plasmid is by encoding function albumen
Functional gene importing sets out what carrier obtained；The carrier that sets out is pBAD/HisB carrier；The bacterium germination out is Escherichia coli
BW25113；The functional protein is following (a1) or (a2) or (a3):
(a1) protein shown in the sequence 3 of sequence table；
(a2) fused protein obtained in N-terminal or/and C-terminal the connection label of (a)；
(a3) protein shown in the sequence 7 of sequence table.
9. recombinant bacterium described in claim 8 is preparing the application in glucuronic acid.
10. functional protein is preparing the application in glucuronic acid；The functional protein is following (a1) or (a2) or (a3):
(a1) protein shown in the sequence 3 of sequence table；
(a2) fused protein obtained in N-terminal or/and C-terminal the connection label of (a)；
(a3) protein shown in the sequence 7 of sequence table.
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| CN112592911A (en) * | 2020-12-30 | 2021-04-02 | 南京师范大学 | Application of heat-resistant alpha-glucuronidase polypeptide fusion in preparation of glucuronic acid |
| CN112608960A (en) * | 2020-12-30 | 2021-04-06 | 江南大学 | Preparation method of uridine diphosphate glucuronic acid |
| CN113249283A (en) * | 2021-04-28 | 2021-08-13 | 济南大学 | Engineering strain for efficiently biologically synthesizing glucuronic acid and application thereof |
| WO2024179615A1 (en) * | 2023-02-28 | 2024-09-06 | 诸城市浩天药业有限公司 | Method for preparing d-glucuronic acid and process for preparing glucurolactone |
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| CN112608960A (en) * | 2020-12-30 | 2021-04-06 | 江南大学 | Preparation method of uridine diphosphate glucuronic acid |
| CN112592911B (en) * | 2020-12-30 | 2022-09-23 | 南京师范大学 | Application of heat-resistant alpha-glucuronidase polypeptide fusion in preparation of glucuronic acid |
| CN113249283A (en) * | 2021-04-28 | 2021-08-13 | 济南大学 | Engineering strain for efficiently biologically synthesizing glucuronic acid and application thereof |
| WO2024179615A1 (en) * | 2023-02-28 | 2024-09-06 | 诸城市浩天药业有限公司 | Method for preparing d-glucuronic acid and process for preparing glucurolactone |
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| CN109423469B (en) | 2020-05-12 |
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