CN103060301A - N-acetyl-D-neuraminic acid (Neu5Ac) aldolase from pediococcus acidilactici and gene and application thereof - Google Patents
N-acetyl-D-neuraminic acid (Neu5Ac) aldolase from pediococcus acidilactici and gene and application thereof Download PDFInfo
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Abstract
The invention discloses N-acetyl-D-neuraminic acid (Neu5Ac) aldolase from pediococcus acidilactici and a gene and application thereof. The Neu5Ac aldolase provided by the invention is a) a protein formed by amino acid sequences shown in sequence 2 in a sequence table or b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequences shown in the sequence 2 in the sequence table, has Neu5Ac aldolase activity and is derived from a). The Neu5Ac aldolase protein can catalyze N-acetylmannosamine and pyruvic acid to generate Neu5Ac and can be used for producing Neu5Ac.
Description
Technical field
The present invention relates to a kind of N-acetyl-neuraminate zymohexase and encoding gene and application, particularly derive from N-acetyl-neuraminate zymohexase and gene and the application of pediococcus acidilactici.
Background technology
Sialic acid (Sialic acid) is the derivative of a class neuraminic acid, and the natural sialic acid derivative that has been found that at present reaches kind more than 40.N-acetyl-neuraminate (N-acetyl-D-neuraminic acid, Neu5Ac) most importantly wherein.Neu5Ac is usually located at the non-reduced end of sugar chain of cell surface glycoprotein and glycolipid, plays a part very important in many and the sugar physiological process relevant with protein-interacting: such as the transfer of stability, Pathogenicity of Bacteria, virus infection, inflammation and the tumour of the adhesion of cell, signal transmission, glycoprotein etc.The biological function variation of N-acetyl-neuraminate, have important pharmaceutical value at aspects such as treatment influenza, nervous system disease, inflammation and tumours, particularly aspect the prevention of influenza (H1N1 that comprises H 5 N 1 avian influenza and 2009) and the treatment good effect is being arranged.Simultaneously, N-acetyl-neuraminate can promote the growth of nerve synapse, and is extremely important to the growth of infant's brain, therefore also is used to the baby milk powder additive.It no matter is prospect in medicine or as foodstuff additive, N-acetyl-neuraminate all has higher marketable value.
N-acetyl-neuraminate can obtain from following several approach: natural product extraction, chemosynthesis, chemical enzyme process, enzyme process are synthetic etc.N-acetyl-neuraminate can extract from bird's nest, milk or birds, beasts and eggs, but because amount very little, is extracted the purifying difficulty, yield poorly (10-20%), purity is low.
Chemical synthesis is synthesized N-acetyl-neuraminate, because the complicacy of product structure needs various protection and goes to protect step.Use the synthetic N-acetyl-neuraminate severe reaction conditions of chemical method, need some noble metals such as indium as catalyzer, the N-acetyl-neuraminate yield is low.Therefore, chemical method usually is combined with enzyme process.
N-acetyl-neuraminate can be synthetic by enzyme process, and ManNAc (ManNAc) and pyruvic acid are at the lower N-acetyl-neuraminate that generates of N-acetyl-neuraminate zymohexase (Neu5Ac aldolase or Neu5Ac lyase, NanA, EC4.1.3.3) effect.But ManNAc cost for industrial production is higher.The chemistry enzyme process is a kind of method of more economical effective production N-acetyl-neuraminate, namely under alkaline condition, makes N-Acetyl-D-glucosamine (GlcNAc) isomerization generate ManNAc; Then adopt N-acetyl-neuraminate zymohexase catalyzing N-acetylmannosamine and pyruvic acid to generate N-acetyl-neuraminate.Or synthetic with double-enzyme method: namely use GlcNAc 2-isomerase (GlcNAc 2-epimerase, AGE, EC5.1.3.8) catalysis GlcNAc isomery to become ManNAc, then the condensation under the effect of N-acetyl-neuraminate zymohexase of ManNAc and pyruvic acid produces Neu5Ac.
In industrial production, be raw material production Neu5Ac mainly with GlcNAc, no matter be to adopt chemical enzyme process or double-enzyme method, all need the N-acetyl-neuraminate zymohexase.Although the N-acetyl-neuraminate zymohexase has existence in higher animal and some microorganisms, its content is atomic in the organism, is difficult to separate the N-acetyl-neuraminate zymohexase that obtains q.s.Therefore how acquired good N-acetyl-neuraminate zymohexase of fine quality is extremely important to the production of N-acetyl-neuraminate.
Summary of the invention
An object of the present invention is to provide a kind of N-acetyl-neuraminate zymohexase.
N-acetyl-neuraminate zymohexase provided by the present invention, the name be called PaNanA, derive from pediococcus acidilactici (Pediococcus acidilactici), be following a) or b) protein:
A) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;
B) with the aminoacid sequence shown in the sequence in the sequence table 2 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have the N-acetyl-neuraminate aldolase activity by the protein of a) deriving.
Wherein, sequence 2 is comprised of 305 amino-acid residues in the sequence table.
Albumen in above-mentioned in order to make (a) is convenient to purifying, label as shown in table 1 on N-terminal that can the protein that the aminoacid sequence shown in the sequence 2 forms in by sequence table or C-terminal connect.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6(is generally 5) | RRRRR |
| Poly-His | 2-10(is generally 6) | HHHHHH |
| |
8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
PaNanA in above-mentioned (b) can synthesize first its encoding gene, carries out biological expression again and obtains.The encoding gene of PaNanA in above-mentioned (b) can be by the codon with sequence in the sequence table 1 one or several amino-acid residue of disappearance in the dna sequence dna shown in 5 ' end the 1st to 918 bit base, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The nucleic acid molecule of coding N-acetyl-neuraminate zymohexase PaNanA also belongs to protection scope of the present invention.
Wherein, described nucleic acid molecule can be DNA, such as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule also can be RNA, such as mRNA or hnRNA etc.
Described nucleic acid molecule specifically can be following 1) or 2) or 3) shown in gene:
1) its encoding sequence is the dna molecular of the 1-918 position Nucleotide of sequence 1 in the sequence table;
2) under stringent condition with 1) dna molecule hybridize that limits and the dna molecular of the above-mentioned N-acetyl-neuraminate zymohexase PaNanA that encodes;
3) with 1) or 2) dna molecular that limits has the identity more than 90% and the dna molecular of the above-mentioned N-acetyl-neuraminate zymohexase PaNanA that encodes.
Above-mentioned stringent condition can be with 6 * SSC, and the solution of 0.5% SDS 65 ℃ of lower hybridization, is then used 2 * SSC, 0.1% SDS and 1 * SSC, and 0.1% SDS respectively washes film once.
Wherein, the sequence 1 in the sequence table is comprised of 918 Nucleotide, the protein shown in the sequence 2 in the code sequence tabulation.
Following 1) any biomaterial-4) also belongs to protection scope of the present invention:
1) contains the expression cassette of the nucleic acid molecule of coding N-acetyl-neuraminate zymohexase PaNanA;
2) contain the recombinant expression vector of the nucleic acid molecule of coding N-acetyl-neuraminate zymohexase PaNanA;
3) contain the recombinant microorganism of the nucleic acid molecule of coding N-acetyl-neuraminate zymohexase PaNanA;
4) contain the transgenic cell line of the nucleic acid molecule of coding N-acetyl-neuraminate zymohexase PaNanA.
In the above-mentioned biomaterial, 1) the described expression cassette that contains the nucleic acid molecule of coding N-acetyl-neuraminate zymohexase PaNanA, refer in host cell, to express the DNA of N-acetyl-neuraminate zymohexase PaNanA, this DNA not only can comprise the promotor that starts N-acetyl-neuraminate zymohexase PaNanA genetic transcription, also can comprise stopping the terminator that N-acetyl-neuraminate zymohexase PaNanA transcribes.Further, the described N-acetyl-neuraminate zymohexase PaNanA expression cassette that contains also can comprise enhancer sequence.2) the described recombinant expression vector that contains the nucleic acid molecule of coding N-acetyl-neuraminate zymohexase PaNanA specifically can be in the multiple clone site of carrier 6HisT-pRSET and inserts the recombinant expression vector that N-acetyl-neuraminate zymohexase PaNanA encoding gene obtains.3) described recombinant microorganism specifically can be bacterium, yeast, algae and fungi.Wherein, bacterium can be from Escherichia (Escherichia), such as e. coli bl21 (DE3).4) described transgenic cell line does not comprise the reproductive material of plant.
Another purpose of the present invention provides a kind of method for preparing N-acetyl-neuraminate zymohexase PaNanA.
The method for preparing N-acetyl-neuraminate zymohexase PaNanA provided by the present invention comprises N-acetyl-neuraminate zymohexase PaNanA encoding gene is expressed the step that obtains the N-acetyl-neuraminate zymohexase in biomass cells; Described biomass cells is microorganism cells, vegetable cell or non-human animal's cell.
A further object of the present invention provides a kind of method for preparing the recombinant microorganism of expressing N-acetyl-neuraminate zymohexase PaNanA.
The method of the recombinant microorganism of N-acetyl-neuraminate zymohexase PaNanA is expressed in preparation provided by the present invention, comprise the encoding gene of N-acetyl-neuraminate zymohexase PaNanA is imported the host microorganism cell, obtain expressing the step of the recombinant microorganism of N-acetyl-neuraminate zymohexase PaNanA.
Described host microorganism specifically can be bacterium, yeast, algae and fungi.Wherein, bacterium can be from Escherichia (Escherichia), such as e. coli bl21 (DE3).
The nucleic acid molecule of N-acetyl-neuraminate zymohexase PaNanA, coding N-acetyl-neuraminate zymohexase PaNanA or contain the application of biomaterial in the preparation N-acetyl-neuraminate of the nucleic acid molecule of coding N-acetyl-neuraminate zymohexase PaNanA also belongs to protection scope of the present invention.
The nucleic acid molecule total length of amplification coding N-acetyl-neuraminate zymohexase PaNanA or the primer of its arbitrary fragment are to also belonging to protection scope of the present invention.Described primer centering, a primer sequence specifically can be shown in sequence in the sequence table 3, and another primer sequence specifically can be shown in sequence in the sequence table 4.
Experiment showed, that but N-acetyl-neuraminate zymohexase PaNanA catalyzing N-acetylmannosamine of the present invention and pyruvic acid generate N-acetyl-neuraminate, can be used for producing N-acetyl-neuraminate.
Description of drawings
Fig. 1 is the pcr amplification result of paNanA.
M is dna molecular amount standard, and Pa is pcr amplification product.
Fig. 2 is that the enzyme of recombinant plasmid pPaNanA is cut qualification result.
M is dna molecular amount standard, and Pa is that the enzyme of pPaNanA is cut product.
Fig. 3 is that the SDS-PAGE of genetic engineering bacterium pPaNanA/BL21 expression product analyzes.
5 swimming lanes from left to right are followed successively by the cytoclasis supernatant liquor of pRSET/BL21, the cytoclasis supernatant liquor of pPaNanA/BL21, the cytoclasis precipitation of pPaNanA/BL21, the full cell of pPaNanA/BL21 and protein molecular weight standard (unit is kDa).
Fig. 4 is that the SDS-PAGE behind the PaNanA purifying analyzes.
M is protein molecular weight standard (unit is kDa), and 1 is the cytoclasis supernatant liquor, and 2 is the albumen behind the purifying.
Fig. 5 is the HPLC collection of illustrative plates of converted product.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The preparation of embodiment 1, N-acetyl-neuraminate zymohexase and functional verification
One, the pcr amplification of the extraction of pediococcus acidilactici genomic dna and paNanA gene
Employing bacterial genomes extraction test kit (day root company) extraction pediococcus acidilactici (Pediococcus acidilactici) CGMCC1.4(China Committee for Culture Collection of Microorganisms common micro-organisms center) genomic dna.With the pediococcus acidilactici genome DNA extracted as template, take Pa-F and Pa-R as primer, with high-fidelity pyrobest enzyme (Takara company) pcr amplification PaNanA gene.Primer sequence is as follows:
Pa-F:5 ' GCCGGCA
CATATGACAAAAAATCTTGATAAAT 3 ' (sequence 3 in the sequence table)
Pa-R:5 ' TCA
GGATCCCTAGCCAACGTATTTCTCAATTG 3 ' (sequence 4 in the sequence table)
The PCR program is: first 95 ℃ 5 minutes; Then carry out under the following conditions 30 circulations: 95 ℃ of 30s, 58 ℃ of 30s, 72 ℃ 1 minute 20 seconds; Last 72 ℃ 10 minutes.
Pcr amplification goes out the fragment (Fig. 1, arrow shows) about 900bp, after the recovery through being connected on the cloning vector behind the NdeI/BamHI double digestion, Screening and Identification positive colony, and carry out sequencing; Sequencing result shows that the dna sequence dna that the clone obtains is shown in sequence in the sequence table 1, and the sequence 1 in the sequence table is comprised of 918 Nucleotide, the protein shown in the sequence 2 in the code sequence tabulation.Sequence 2 is comprised of 305 amino-acid residues in the sequence table.Be paNanA with this unnamed gene, with the albumen called after PaNanA of its coding.
Two, the structure that contains the recombinant expression vector of paNanA
The pcr amplified fragment that above-mentioned steps one obtains is cut with NdeI and BamHI enzyme, reclaimed the purpose fragment; Cutting the carrier 6HisT-pRSET(public with NdeI and BamHI enzyme simultaneously can obtain from Microbe Inst., Chinese Academy of Sciences, and the non-patent literature of putting down in writing this material is: TAO, and Y., Guermah, M., Martinez, E.,
T., Hasegawa, S., Takada, R., Yamamoto, T., Horikoshi, M.and Roeder, R.G. (1997) Specific Interactions and Potential Functions of Human TAFII100.Journal of Biological Chemistry272:6714-6721.), reclaim the carrier large fragment; 16 ℃ of the carrier large fragments of the purpose fragment that reclaims and recovery are connected, obtain the purpose plasmid.With purpose plasmid CaCl
2Method transforms bacillus coli DH 5 alpha competent cell (available from Takara, catalog number is D9057A).It is evenly coated on the LB flat board that contains penbritin, cultivated 16 hours for 37 ℃.Single bacterium colony shaking culture is spent the night, extract plasmid and carry out double digestion with NdeI and BamHI and identify, qualification result as shown in Figure 2, as seen from the figure, obtained about 2900bp with 900bp about endonuclease bamhi (arrow shows among Fig. 2).Enzyme is cut the correct plasmid of checking to check order, sequencing result shows, between the NdeI of carrier 6HisT-pRSET and BamHI restriction enzyme site, inserted the paNanA gene fragment shown in the sequence 1 in the sequence table, proved that plasmid construction is correct, with recombinant vectors called after pPaNanA.
Three, the structure of the genetic engineering bacterium of recombinant expressed PaNanA albumen and PaNanA protein expression
Recombinant plasmid pPaNanA is transformed e. coli bl21 (DE3) (available from sky root company with Calcium Chloride Method, catalog number (Cat.No.) CB105-02), the penbritin screening and culturing, the picking mono-clonal, identify as primer carries out PCR take Pa-F and Pa-R, PCR is identified the positive colony that obtains 900bp left and right sides PCR product as genetic engineering bacterium, called after pPaNanA/BL21.37 ℃ of shaking culture of the single bacterium colony of picking pPaNanA/BL21 spend the night to obtain saturated cultivation.Volume ratio according to 1% is inoculated in the LB substratum that contains penbritin (50 μ g/ml), when 37 ℃ of continuation cultivations are 0.6-0.8 to OD600, the adding final concentration is that centrifugal collection thalline carried out the ultrasonic disruption cell behind the resuspended thalline of PBS after the IPTG of 0.8mmol/L induced 5 hours, carry out the SDS-PAGE electrophoresis after the sample preparation, the result as shown in Figure 3, as seen from the figure, PaNanA is efficiently expressed in intestinal bacteria, and albumen is with soluble formal representation, the about 34kDa of size.
Change 6HisT-pRSET over to e. coli bl21 (DE3) according to identical method, with the recombination bacillus coli that obtains pRSET/BL21 by name.PRSET/BL21 is carried out abduction delivering according to the method described above as empty carrier contrast bacterium.Do not obtain size in the as a result empty carrier contrast bacterium and be about the 34kDa target protein.
Four, the purifying of PaNanA albumen
Genetic engineering bacterium pPaNanA/BL21 is seeded among the self-induction substratum ZYM that 3 bottles of 100ml contain ammonia benzyl resistance with 1% inoculum size (volume ratio), 30 ℃, induce (adding final concentration is the IPTG of 0.8mmol/L) under the 250rpm condition, behind the 20h 4 ℃, 12000rpm is centrifugal, collects thalline.Thalline is suspended in the 30ml damping fluid, and (solute and concentration thereof are 20mM Tris; 300mM NaCl; 20% glycerine; Solvent is water) in, ice bath, ultrasonic disruption cell (ultrasonic power 400W, broken 5s, 10s intermittently, total duration 60 minutes).In 4 ℃, centrifugal 20 minutes of 12000rpm gets supernatant liquor with the bacterium liquid after the fragmentation, behind 0.22 μ m membrane filtration, carries out the PaNanA albumen (Fig. 4, swimming lane 1 is the cytoclasis supernatant liquor, swimming lane 2 is the albumen behind the purifying) that purifying obtains single band with the nickel post.Albumen behind the purifying is removed imidazoles with dialysis tubing, obtain the PaNanA albumen of purifying.
The self-induction substratum ZYM prescription that contains ammonia benzyl resistance is as follows: the following percentage concentration of 100mL A+2mL B+2mL C+200 μ LD+100 μ L E(is mass percent concentration);
A.ZY: solute and concentration thereof are 1% Tryptones, 0.5% yeast powder; Solvent is water.
B.50 * and M: solute and concentration thereof are 1.25M Na
2HPO
4, 1.25M KH
2PO
4, 2.5M NH
4Cl and 0.25M Na
2SO
4Solvent is water.
C.50 * 5052: solute and concentration thereof are 25% glycerine, 2.5% glucose, 10% lactose; Solvent is water.
D. solute and concentration thereof are 1M MgSO
4Solvent is water.
E.1000 * and trace element: solute and concentration thereof are 50Mm FeCl
3, 20mM CaCl
2, 10mM MnCl
2, 10mM ZnSO
4, CoCl
2, NiCl
2, Na
2Mo
4, Na
2SeO
3And H
3BO
3Each 2mM; Solvent is water.
Five, the enzyme assay of PaNanA albumen
PaNanA albumen is carried out enzyme activity determination, reaction system is: 50mM Tris-HCl pH7.0,10mM ManNAc (ManNAc), 8.75mM pyruvic acid (pyruvic acid, pH7.0), in the step 4 from genetic engineering bacterium pPaNanA/BL21 abduction delivering product the PaNanA albumen 1mg of purifying, reaction system is 100 μ l.37 ℃ of reactions were boiled 2 minutes termination reactions after 30 minutes.Three repetitions, results averaged are established in experiment.Enzyme live unit definition be under 37 ℃ of conditions per minute to produce the needed enzyme amount of 1 μ mol N-acetyl-neuraminate be a unit (U).Detect the N-acetyl-neuraminate of generation with HPLC, chromatographic condition is: chromatographic column: Aminex HPX-87H (BioRad, CA, USA); Moving phase: 6mM H
2SO
4The aqueous solution; Flow rate of mobile phase: 0.55mL/min; Detector: UV 210nm; Sample size: 10 μ l; Temperature: 65 ℃.HPLC result as shown in Figure 5, A is the HPLC collection of illustrative plates of N-acetyl-neuraminate standard substance (available from sigma company) among Fig. 5, as seen from the figure, the retention time of N-acetyl-neuraminate standard substance is 8.565 minutes; Among Fig. 5 B be in the step 4 from genetic engineering bacterium pPaNanA/BL21 abduction delivering product the HPLC collection of illustrative plates of the PaNanA albumen of purifying, as seen from the figure, retention time is that the 8.6min place is the peak (1) of N-acetyl-neuraminate (Neu5Ac); 9.9min locate the peak (2) into pyruvic acid; 11.9min locate the peak (3) into ManNAc.This presentation of results PaNanA albumen has the enzymic activity of catalyzing N-acetylmannosamine and pyruvic acid generation N-acetyl-neuraminate.With the concentration of external standard standard measure mensuration N-acetyl-neuraminate, under this chromatographic condition, the typical curve of N-acetyl-neuraminate standard substance is C=0.000601*A (A: peak area, C: concentration mM).Calculate enzyme activity (U) according to following formula: U=C* extension rate * 1000/30min/ protein content (mg).Three repetitions are established in experiment, and the result shows that the enzyme work of the PaNanA albumen of purifying from genetic engineering bacterium pPaNanA/BL21 abduction delivering product is 1.29 ± 0.09U/mg.
Claims (9)
1. albumen, be following a) or b) protein:
A) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;
B) with the aminoacid sequence shown in the sequence in the sequence table 2 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have the N-acetyl-neuraminate aldolase activity by the protein of a) deriving.
2. the nucleic acid molecule of coding claim 1 described albumen.
3. nucleic acid molecule according to claim 2, it is characterized in that: described nucleic acid molecule is following 1) or 2) or 3) shown in gene:
1) its encoding sequence is the dna molecular of the 1-918 position Nucleotide of sequence 1 in the sequence table;
2) under stringent condition with 1) dna molecule hybridize that limits and the dna molecular of the described albumen of coding claim 1;
3) with 1) or 2) dna molecular that limits has the dna molecular of the homology 90% or more and the described albumen of claim 1 of encoding.
4. any biomaterial following 1)-4):
1) contains the expression cassette of the described nucleic acid molecule of claim 3;
2) contain the recombinant expression vector of the described nucleic acid molecule of claim 3;
3) contain the recombinant microorganism of claim 2 or 3 described nucleic acid molecule;
4) contain the transgenic cell line of claim 2 or 3 described nucleic acid molecule.
5. the described method of protein of preparation claim 1 comprises the encoding gene of protein claimed in claim 1 is expressed the step that obtains the described protein of claim 1 in biomass cells; Described biomass cells is microorganism cells, vegetable cell or non-human animal's cell.
6. the method for the recombinant microorganism of the described protein of claim 1 is expressed in preparation, comprises that the encoding gene with protein claimed in claim 1 imports the host microorganism cell, obtains expressing the step of the recombinant microorganism of the described protein of claim 1.
7. protein claimed in claim 1 is as the application in the N-acetyl-neuraminate zymohexase.
8. protein claimed in claim 1, claim 2 or 3 described nucleic acid molecule or biomaterial claimed in claim 4 application in the preparation N-acetyl-neuraminate.
9. the primer of amplification claim 2 or 3 described nucleic acid molecule total lengths or its arbitrary fragment pair.
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| CN114507658A (en) * | 2022-04-02 | 2022-05-17 | 深圳瑞德林生物技术有限公司 | Enzyme coexpression system and application thereof in sialic acid synthesis |
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| CN103305450B (en) * | 2013-06-04 | 2015-05-20 | 南京师范大学 | Escherichia coli expression strain and method thereof for producing N-acetyl-D-neuraminic acid |
| CN104878035A (en) * | 2015-04-20 | 2015-09-02 | 江南大学 | Construction method for producing N-acetylneuraminic acid recombinant microorganisms and application of N-acetylneuraminic acid recombinant microorganisms |
| CN107267575B (en) | 2016-04-05 | 2020-11-06 | 孙镧 | Method for producing N-acetyl-D-glucosamine and/or D-glucosamine salt by microbial fermentation |
| WO2017174039A1 (en) * | 2016-04-05 | 2017-10-12 | 孙镧 | Method for producing n-acetyl-d-glucosamine and/or d-glucosamine salt by means of microbial fermentation |
| WO2017174037A1 (en) * | 2016-04-05 | 2017-10-12 | 孙镧 | Method for producing n-acetyl-d-glucosamine and/or d-glucosamine salt by microbial fermentation |
| CN108330095B (en) * | 2018-03-01 | 2020-12-29 | 江南大学 | Recombinant corynebacterium glutamicum for accumulating N-acetylneuraminic acid and application thereof |
| WO2022057854A1 (en) * | 2020-09-16 | 2022-03-24 | 南京迈西可生物科技有限公司 | Pathogen specific nucleic acid fragment and application thereof |
| CN114874967A (en) * | 2022-06-17 | 2022-08-09 | 江南大学 | Recombinant escherichia coli for producing N-acetylneuraminic acid and construction method thereof |
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| CN101603023A (en) * | 2009-07-10 | 2009-12-16 | 山东大学 | The recombination bacillus coli of one temperature-control coexpression exogenous gene and application thereof |
| CN101979644A (en) * | 2010-09-10 | 2011-02-23 | 扬州博生源生物科技有限公司 | Method for preparing N-acetylneuraminic acid by one-step catalysis of fusion protein |
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| CN101165190B (en) * | 2006-10-17 | 2011-08-24 | 中国科学院上海生命科学研究院 | Preparation for N-acetylneuraminic acid by immobilization double-enzyme method |
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| WO2011086834A1 (en) * | 2010-01-15 | 2011-07-21 | 味の素株式会社 | Process for production of n-acetyl-d-neuraminic acid |
| CN101906449B (en) * | 2010-06-24 | 2012-07-04 | 山东大学 | Method for producing N-acetylneuraminic acid by spore surface display system |
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| TW200904458A (en) * | 2007-07-25 | 2009-02-01 | Univ Nat Chunghsing | Method of producing N-acetyl-d-neuraminic acid and application thereof |
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| CN103060358A (en) | 2013-04-24 |
| CN103060358B (en) | 2014-07-16 |
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