CN114457133B - Method for producing N-acetylneuraminic acid by using N-acetylglucosamine fermentation broth as substrate through whole cell catalysis - Google Patents
Method for producing N-acetylneuraminic acid by using N-acetylglucosamine fermentation broth as substrate through whole cell catalysis Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 52
- 230000004151 fermentation Effects 0.000 title claims abstract description 52
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 title claims abstract description 49
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 title claims abstract description 38
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 title claims abstract description 38
- 238000006555 catalytic reaction Methods 0.000 title claims abstract description 33
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 title claims abstract description 25
- 229950006780 n-acetylglucosamine Drugs 0.000 title claims abstract description 25
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 19
- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 title claims abstract description 15
- 239000000758 substrate Substances 0.000 title abstract description 16
- 238000006243 chemical reaction Methods 0.000 claims abstract description 51
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 claims abstract description 48
- 229940107700 pyruvic acid Drugs 0.000 claims abstract description 24
- 210000004027 cell Anatomy 0.000 claims description 62
- 239000003054 catalyst Substances 0.000 claims description 18
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
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- 229920004890 Triton X-100 Polymers 0.000 claims description 13
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- 241000588724 Escherichia coli Species 0.000 claims description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 230000001276 controlling effect Effects 0.000 claims description 8
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 230000001105 regulatory effect Effects 0.000 claims description 6
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 claims description 5
- 241001052560 Thallis Species 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- 230000001502 supplementing effect Effects 0.000 claims description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 101150027065 nagE gene Proteins 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
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- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 claims description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 2
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- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
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- 229910000365 copper sulfate Inorganic materials 0.000 claims description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 2
- 230000008878 coupling Effects 0.000 claims description 2
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- 238000005859 coupling reaction Methods 0.000 claims description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 239000011790 ferrous sulphate Substances 0.000 claims description 2
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 2
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- 229940099596 manganese sulfate Drugs 0.000 claims description 2
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 2
- 239000011684 sodium molybdate Substances 0.000 claims description 2
- 235000015393 sodium molybdate Nutrition 0.000 claims description 2
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 2
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- 235000013619 trace mineral Nutrition 0.000 claims description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 2
- 229960001763 zinc sulfate Drugs 0.000 claims description 2
- 241000588722 Escherichia Species 0.000 claims 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims 2
- -1 7H 2 O50mg/L Chemical compound 0.000 claims 1
- 241000579120 Coliiformes Species 0.000 claims 1
- 230000010307 cell transformation Effects 0.000 abstract description 4
- 230000009466 transformation Effects 0.000 abstract description 4
- 230000001580 bacterial effect Effects 0.000 abstract description 3
- SQVRNKJHWKZAKO-LUWBGTNYSA-N N-acetylneuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)CC(O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-LUWBGTNYSA-N 0.000 description 29
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
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- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
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- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 108010043841 Glucosamine 6-Phosphate N-Acetyltransferase Proteins 0.000 description 1
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- DUKURNFHYQXCJG-UHFFFAOYSA-N Lewis A pentasaccharide Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(C(O)C(O)C(CO)O2)O)C(NC(C)=O)C(OC2C(C(OC3C(OC(O)C(O)C3O)CO)OC(CO)C2O)O)OC1CO DUKURNFHYQXCJG-UHFFFAOYSA-N 0.000 description 1
- 102000015728 Mucins Human genes 0.000 description 1
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- 239000012190 activator Substances 0.000 description 1
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- 238000007036 catalytic synthesis reaction Methods 0.000 description 1
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- 229940002612 prodrug Drugs 0.000 description 1
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- 230000002035 prolonged effect Effects 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
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- 239000011780 sodium chloride Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H7/00—Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
- C07H7/02—Acyclic radicals
- C07H7/033—Uronic acids
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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Abstract
The invention discloses a method for producing N-acetylneuraminic acid by using N-acetylglucosamine fermentation broth as a substrate through whole cell catalysis, belonging to the field of bioengineering application. The invention carries out whole-cell catalysis to produce N-acetylneuraminic acid in a conversion system taking N-acetylglucosamine fermentation liquor as a substrate, optimizes the whole-cell conversion condition, and ensures that the temperature is 30 ℃, the initial pH of the conversion liquor is 6.5, the substrate pyruvic acid concentration is 1.38mol/L, tritonX-100, the addition amount is 0.4 percent, and the bacterial OD is provided 600 The whole cell transformation is carried out under the conditions of 30L fermentation tank and the like, so that 180mmol/L N-acetylneuraminic acid can be obtained, and the molar transformation rate reaches 65.45 percent.
Description
Technical Field
The invention relates to a method for producing N-acetylneuraminic acid by using N-acetylglucosamine fermentation broth as a substrate through whole cell catalysis, belonging to the field of bioengineering application.
Background
Sialic acid is a generic term for a series of 9-carbon monosaccharides and derivatives thereof, which are named after the initial separation from submaxillary mucins. Neu5Ac (N-acetylneuraminic acid) is one of the main types of sialic acid, and its content is 99% or more of the sialic acid family, and is mainly located at the terminal of non-reducing oligosaccharides such as glycoproteins and glycolipids in the form of α -glycosides. Neu5Ac is involved in a number of physiological processes such as cell recognition, signal transduction, tumorigenesis, etc., and furthermore Neu5Ac can promote the development of the infant brain. Neu5Ac can be used as a potential anti-influenza prodrug for preventing and treating influenza, and the demand of Neu5Ac in the pharmaceutical and biotechnology industries is increasing due to its diverse biological functions.
Neu5Ac is prepared by natural product extraction, chemical synthesis, microbial fermentation, enzymatic synthesis, and whole cell catalysis. The enzymatic method for producing Neu5Ac has the advantages of high conversion rate, simple extraction, high product purity and the like, but has high requirement on raw materials used for synthesis and high price, and the N-acetylglucosamine-2-epimerase needs ATP as an activator, so that the expansion of the production scale is limited. The basic principle of whole cell catalysis is to use whole biological organisms as catalysts for chemical transformations to convert certain precursor molecules into specific products, essentially by the catalytic action of enzymes in biological systems. The whole-cell catalysis method is a better method for producing Neu5Ac because of the advantages of simple and convenient operation, high efficiency, easy separation and purification of products and the like. Compared with the enzyme method, the whole cell catalysis method does not need to additionally add ATP, saves the production cost, and avoids complicated enzyme purification steps; however, in the whole-cell biocatalysis method, expensive N-acetylglucosamine needs to be exogenously added as a substrate, the difficulty of later substrate recovery and product separation is increased due to low substrate conversion rate, and the industrial application of the whole-cell biocatalysis method in Neu5Ac production is limited due to the fact that mass transfer resistance of substrates and products is increased due to the existence of cell membranes and the like. At present, the whole cell catalysis method for producing Neu5Ac takes GlcNAc with higher purity as a substrate, so that the cost is higher, and the production cost and market competitiveness of the Neu5Ac are considered, so that the method is not beneficial to large-scale production.
Disclosure of Invention
The invention provides a method for producing N-acetylneuraminic acid by biocatalysis, which takes fermentation liquor containing N-acetylglucosamine as a raw material and utilizes a cell catalyst to react for at least 24 hours at 30-37 ℃.
In one embodiment, the cell catalyst is recombinant E.coli (Escherichia coli) delta NTE/pET28a- (T7-shnal) - (tac-slr), disclosed in the university of Jiangnan paper, construction of recombinant E.coli for whole cell catalytic production of N-acetylneuraminic acid, published 2017.
In one embodiment, the whole cell catalysis is to add the whole cell catalyst into a reaction system to control the initial OD 600 10-50, initial pH of 6-8, pyruvic acid concentration of 0.83mol/L-2.2mol/L, and surfactant TritonX-100 not more than 1% by volume, and reacting for 24-40 h at 20-50 ℃.
In one embodiment, the whole cell catalyst is obtained by culturing recombinant E.coli in LB medium to OD 600 About 0.6 to 0.8, adding isopropyl-beta-D-thiogalactoside with the final concentration of 1mmol/L, and culturing at 28 ℃ for 8 hours to induce the expression of recombinant protein; after the induction, the cells were collected by centrifugation, washed 2 times with 100mmol/L Tris-HCl buffer, pH7.5, and collected to obtain a whole cell catalyst.
In one embodiment, the recombinant escherichia coli is inoculated in an LB culture medium, cultured for 10 hours at 37 ℃ and 200r/min to obtain seed liquid, and then transferred to the LB culture medium for induction.
In one embodiment, the whole cell catalysis is performed under the following reaction parameters: cell catalyst in reaction system 600 20 to 30, the initial pH of the conversion solution is 6 to 8, and the ratio of GlcNAc to pyruvic acid is 1 (5 to 8); the addition amount of the surfactant TritonX-100 is 0-1%, and the surface is converted for 30-40h at the temperature of 30-37 ℃.
In one embodiment, the concentration of pyruvic acid in the reaction system is 0.83mol/L to 2.2mol/L.
In one embodiment, the whole cell catalysis is performed under the following reaction parameters: the reaction temperature was 30℃and the initial pH was 6.5, the GlcNAc concentration was 0.28mol/L, the pyruvic acid concentration was 1.38mol/L, the Triton X-100 addition was 0.4%, and the cell OD 600 30, converting for 70h, and detecting the content of each component in the conversion liquid by a liquid phase.
In one embodiment, the N-acetylglucosamine-containing fermentation broth is prepared by fermentation under the following conditions:
controlling the initial fermentation temperature to 34 ℃, and when the sugar concentration in the fermentation process is lower than 2g/L, supplementing glucose mother liquor to control the residual sugar in the fermentation system to be 2-5 g/L; when OD is 600 Reaching 55, heating to 37 ℃ and controlling the residual sugar to be 0.2-0.5 g/L; in the fermentation process, ammonia water is automatically supplemented to adjust the pH value to 6.9, the related rotating speed is 200-850 r/min, and dissolved oxygen is adjusted to 10-30%.
In one embodiment, the N-acetylglucosamine broth is prepared by fed-batch fermentation using recombinant E.coli-glmS-gna1- ΔnagE (disclosed in the patent publication No. CN 102268399B), E.coli7107-18 (disclosed in paper Engineering a new pathway for N-acetylglucosamine production: coupling a catabolic enzyme, glucosamine-6-phosphate deaminase, with a biosynthetic enzyme, glucosamine-6-phosphate N-acetyltransferase), or MG: ec Glm S-GNAl (disclosed in paper E.coli acetylglucosamine metabolic engineering research) as a starting strain to obtain a broth having a GlcNAc of 60g/L or more.
In one embodiment, the concentration of the glucose mother liquor is 700g/L.
In one embodiment, the fermentation broth is further centrifuged to remove somatic cells.
The invention also provides the use of said method in the preparation of sialic acid containing chemicals.
The beneficial effects are that: the invention establishes a method for producing N-acetylneuraminic acid by whole cell catalysis, which couples N-acetylglucosamine with the whole cell catalysis method to contain N-acetylglucosamineThe fermentation liquid of sugar amine is used as a substrate to directly carry out the process flow of producing N-acetylneuraminic acid by whole cell catalysis. In one embodiment of the invention, the temperature is controlled to be 30 ℃, the initial pH of the conversion solution is 6.5, the concentration of substrate pyruvic acid is 1.38mol/L, tritonX-100, the addition amount is 0.4%, and the bacterial OD is obtained 600 Whole cell transformation was performed at 30, 180mmol/L of N-acetylneuraminic acid was obtained, and the molar conversion was 65.45%. The method can reduce the cost of industrially producing the Neu5Ac and provide a certain reference basis for the industrial biocatalysis production of the Neu5 Ac.
Drawings
FIG. 1 fermentation process curve for producing GlcNAc by recombinant E.coli ATLWX.
FIG. 2 concentration variation of recombinant E.coli whole cells catalyzing the synthesis of Neu5Ac from GlcNAc fermentation broth.
FIG. 3 is a graph of the results of the condition optimization of whole cell catalysis using fermentation broth as a substrate; a, influence of thallus concentration on synthesis of Neu5 Ac; b, influence of pyruvic acid concentration on synthesis of Neu5 Ac; c, influence of initial pH of the conversion solution on synthesis of Neu5 Ac; d, influence of the addition amount of the surfactant on synthesis of Neu5 Ac; e, influence of temperature on synthesis of Neu5 Ac.
FIG. 4 shows the catalytic synthesis of Neu5Ac by recombinant E.coli whole cells in a 5L fermenter.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
Example 1: preparation of N-acetylglucosamine fermentation broth
Recombinant E.coli strains (E.coli-glmS-gna1-. DELTA.nagE, E.coli7107-18, or MG: ec Glm S-GNAl, in this example E.coli-glmS-gna1-. DELTA.nagE) producing N-acetylglucosamine were picked from the plates and inoculated into a flask containing LB liquid medium containing 100. Mu.g/mL ampicillin resistance, placed in a shaking table at 32℃and cultured at 200rpm for 12 hours to obtain a primary seed liquid. The cultured primary seeds were transferred to a medium containing 100. Mu.g/mL ampicillin resistance at an inoculum size of 1%Culturing in LB liquid medium at 34 deg.C and 200rpm for 6 hr to OD 600 The secondary seed liquid is obtained by the method of (1) =2.5-3.3. The secondary seed liquid is inoculated into a 5L fermentation tank with the liquid loading amount of 2.5L according to the inoculation amount of 16 percent, and the fermentation medium is as follows: 10g/L of glucose, 6.17g/L of ammonium sulfate, 7g/L of monopotassium phosphate, 7g/L of disodium hydrogen phosphate, 2g/L of citric acid and 3g/L of magnesium sulfate heptahydrate; trace elements: calcium chloride 2H 2 O60 mg/L, manganese sulfate H 2 O10 mg/L, zinc sulfate.7H 2 O3 mg/L, ferrous sulfate.7H 2 O50mg/L, aluminum trichloride 6H 2 O5 mg/L, sodium molybdate 2H 2 O3 mg/L, cobalt chloride 6H 2 O0.1mg/L, copper sulfate.5H 2 0.1mg/L of O and 0.5mg/L of boric acid; LB medium: 5g/L yeast powder, 10g/L peptone and 10g/L sodium chloride. The initial fermentation temperature is 34 ℃, the adding amount of glucose is 10g/L, when the initial sugar concentration is lower than 2g/L in the fermentation process, the residual glucose with the concentration of 700g/L is controlled to be 2-5 g/L by manually supplementing glucose, and when the OD is the OD 600 After 55, the temperature is raised to 37 ℃, the residual sugar is controlled to be 0.2-0.5 g/L, the pH value is regulated to be 6.9 by automatically supplementing ammonia water in the fermentation process, the related rotating speed is 200-850 rpm, the dissolved oxygen is regulated to be 10-30 percent, and the thallus concentration OD is regulated when the fermentation is carried out for 45h 600 Up to 135, and the GlcNAc yield up to 61g/L (FIG. 1).
Example 2: whole cell catalysis using GlcNAc fermentation broth as substrate
E.coli delta NTE/pET28a- (T7-shnal) - (tac-slr) preserved in glycerol tubes at-80 ℃ are streaked on a kanamycin (50 mug/mL) resistant LB solid plate, single colonies are selected and inoculated into 20mL of liquid LB medium containing 50 mug/mL kanamycin, and the culture is carried out at 37 ℃ for 10 hours at 200r/min to obtain seed liquid. Transferring the cultured seed solution into LB culture medium at 1% by volume, and culturing in shaking table at 37deg.C until OD 600 When about 0.6-0.8, 1mmol/L isopropyl-beta-D-thiogalactoside is added for culturing at 28 ℃ for 8 hours to induce recombinant protein expression, after the induction is finished, the thalli are collected by centrifugation, washed for 2 times by a pH7.5 and 100mmol/L Tris-HCL buffer solution, and the thalli are collected, thus obtaining the whole cell catalyst.
Centrifuging the fermentation broth obtained in example 1 to remove thallus to obtain N-acetylglucosamineFermenting the supernatant. Controlling the final concentration of N-acetylglucosamine fermentation supernatant to be 0.28mol/L, adding sodium pyruvate with the final concentration of 0.74mol/L and TritonX-100 with the concentration of 0.2 percent by volumeE.coli△NTE/pET28a-(T7-shnal)-(tac-slr)Obtaining the cell, obtaining the final concentration OD of the whole cell catalyst 600 =30, whole cell transformation was performed at 30 ℃, transformation time was 30-40h, and pH of the reaction system was adjusted to 7.5 with NaOH during transformation. As a result, as shown in FIG. 2, when the reaction was carried out for 36 hours, the Neu5Ac yield reached 44.5mmol/L, the conversion was 16.18%, and after the reaction time was prolonged to 50 hours, the product was degraded.
Example 3: whole cell catalysis under different conditions using GlcNAc fermentation broth as substrate
A fermentation supernatant containing N-acetylglucosamine was prepared as in example 2, and whole cell catalysis was performed in a conversion solution of different pH containing a certain concentration of GlcNAc, pyruvic acid, triton X-100, and the reaction was performed for 36 hours.
(1) At different cell concentrations OD 600 Whole cell catalysis is carried out
Adding whole cell catalyst into a reaction system containing 0.28mol/LGlcNAc fermentation broth, 0.74mol/L pyruvic acid and 0.2% Triton X-100, and controlling cell concentration OD of the cell catalyst in the reaction system 600 The initial pH of the conversion solution was 7.5 at (10, 20, 30, 40, 50), and the results showed that the cell concentration OD was 600 When the yield of Neu5Ac is 10-50, the yield of Neu5Ac is 30-43 mmol/L; concentration of thallus OD 600 At 30, neu5Ac may reach 42.8mmol/L with a conversion of 15.57%.
(2) Whole cell catalysis at different pyruvate concentrations
The cell concentration OD was added to a reaction system containing 0.28mol/LGlcNAc fermentation broth and 0.2% Triton X-100 600 30, the concentration of pyruvic acid is controlled to be 0.83mol/L, 1.10mol/L, 1.38mol/L, 1.65mol/L, 1.93mol/L and 2.20mol/L respectively, the ratio of GlcNAc to pyruvic acid is 1:3,1:4,1:5,1:6,1:7 and 1:8 respectively, the initial pH value of the conversion solution is 7.5, the influence of different pyruvic acid concentrations on Neu5Ac synthesis is examined, and the result shows that the pyruvic acid concentration is 0.83-2.20 molWhen the concentration of the pyruvic acid is 1.65mol/L, the yield is 75.9mmol/L, and the conversion rate is improved to 27.6%;
(3) Whole cell catalysis at different initial pH
To a reaction system containing 0.28mol/LGlcNAc fermentation broth, 1.65mol/L pyruvic acid and 0.2% Triton X-100 was added cell concentration OD 600 The whole cell catalyst is 30, the initial pH of the conversion solution is respectively regulated to be 6.0, 6.5, 7.0, 7.5 and 8.0 for whole cell catalysis, and the influence of the initial pH of the conversion solution on the synthesis of Neu5Ac is examined, so that the result shows that the Neu5Ac yield is 87-98 mmol/L at the pH of 6-8, the yield is 97.16mmol/L at the pH of 6.5, and the conversion rate is 35.33%.
(4) Whole cell catalysis at different Triton X-100 addition levels
Adding the bacterial cell concentration OD to a reaction system containing 0.28mol/LGlcNAc fermentation broth and 1.65mol/L pyruvic acid 600 The initial pH of the conversion solution is 7.5, the addition amount of Triton X-100 is controlled to be 0, 0.2%, 0.4%, 0.6%, 0.8% and 1% respectively, the whole cell conversion is carried out under the conditions of examining the influence of different Triton X-100 on the synthesis of Neu5Ac, and the result shows that when the addition amount of Triton X-100 is less than or equal to 0.8%, the Neu5Ac yield is 96-103 mmol/L, and when the addition amount of Triton X-100 is 0.4%, the Neu5Ac yield can reach 102.5mmol/L, and the conversion rate is 37.3%.
(5) Whole cell catalysis at different temperatures
To a reaction system containing 0.28mol/LGlcNAc fermentation broth, 1.65mol/L pyruvic acid and 0.2% Triton X-100 was added cell concentration OD 600 The whole cell catalyst is 30, the initial pH value of the conversion solution is 7.5, the whole cell catalytic reaction is carried out at 20 ℃, 30 ℃,37 ℃, 40 ℃ and 50 ℃ respectively, the influence of the temperature on the synthesis of Neu5Ac is examined, and the result shows that the Neu5Ac yield is 86-101 mmol/L at the temperature of 30-37 ℃; the highest yield at 30℃was 103.4mmol/L and the conversion was 37.6%.
Example 4: orthogonal experiment optimizes conditions for producing Neu5Ac by whole cell catalysis
Three factors including thallus concentration, pyruvic acid concentration and surfactant adding amount are selected for three-factor three-level orthogonal test, and whole cell catalysis test is performed under the condition, and the level arrangement of each factor is shown in table 1
TABLE 1 factor level Table
The Neu5Ac content in the conversion solution was used as an index, L9 (3 4 ) Orthogonal table the test was performed and the results of the orthogonal test are shown in table 2.
TABLE 2 results of orthogonal test table
The influence of the concentration of the thalli on the Neu5Ac yield is the greatest, the addition amount of the surfactant is the least, the influence of the concentration of the pyruvic acid on the yield is the least, and the optimal combination of the three factors is A 2 B 1 C 3 Namely, cell concentration OD 600 30% of pyruvic acid with the concentration of 1.38mol/L and 0.4% of surfactant.
Controlling the concentration OD of the cells 600 30, 0.28mol/L GlcNAc, 1.38mol/L pyruvic acid, 0.4% surfactant, 48h after reaction, neu5Ac yield (141.+ -. 3) mmol/L, conversion (51.27.+ -. 1.09)%. The process has good feasibility and provides a certain basis for the subsequent production process research.
In a 5L fermenter, the catalytic conditions are: the reaction temperature was 30℃and the initial pH was 6.5, the GlcNAc concentration was 0.28mol/L, the pyruvic acid concentration was 1.38mol/L, the Triton X-100 addition was 0.4%, and the cell OD 600 For 30, whole cell transformation is carried out, and the content of each component in the transformation liquid is detected by liquid phase. As shown in FIG. 4, the Neu5Ac yield can reach 180mmol/L at 70h of reaction, the conversion rate is 65.45% and the ratio is not optimized(example 2) was increased by 49.27%.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (4)
1. A method for producing N-acetylneuraminic acid by biological catalysis is characterized in that fermentation liquor containing N-acetylglucosamine is used as a raw material, a cell catalyst is used for preparing the fermentation liquor at 30 ℃, the initial pH value is 6.5, the concentration of the N-acetylglucosamine is 0.28mol/L, the concentration of pyruvic acid is 1.38mol/L, the adding amount of Triton X-100 is 0.4%, and the OD of thalli is 0.4% 600 Reaction 70h at 30; the cell catalyst is recombinant escherichia coliEscherichia coli) Delta NTE/pET28a- (T7-shnal) - (tac-slr); the fermentation liquid containing N-acetylglucosamine is prepared by recombinant escherichia coliE. coliThe glmS-gna1-deltanagE is used as an initial strain to be fermented in a fed-batch manner to obtain fermentation liquor with GlcNAc more than or equal to 60 g/L.
2. A method for producing N-acetylneuraminic acid by coupling N-acetylglucosamine fermentation with whole cell catalysis is characterized in that escherichia coli with N-acetylglucosamine production capacityE. coliCarrying out fed-batch fermentation on glmS-gna1-delta nagE to obtain fermentation liquor containing N-acetylglucosamine, and converting the N-acetylglucosamine in the fermentation liquor by using a cell catalyst to produce N-acetylneuraminic acid;
the cell catalyst is recombinant escherichia coliEscherichia coli)△NTE/pET28a-(T7-shnal)-(tac-slr);
The fed-batch fermentation is performed under the following conditions: taking a culture medium containing glucose and ammonium sulfate as a fermentation culture medium, controlling the initial fermentation temperature to be 33-35 ℃ and taking the initial fermentation temperature as OD 600 Less than 55, controlling the residual sugar in the fermentation system to be 2-5 g/L; when OD is 600 More than or equal to 55, heating to 37 ℃, and controlling the residual sugar to be 0.2-0.5 g/L; in the fermentation process, the pH value is regulated to 6.8-7.0 by automatically supplementing ammonia water, and the related rotating speed is from 200 to 850r/min, and regulating dissolved oxygen by 10-30%; the culture medium containing glucose and ammonium sulfate contains: 10g/L of glucose, 6.17g/L of ammonium sulfate, 7g/L of monopotassium phosphate, 7g/L of disodium hydrogen phosphate, 2g/L of citric acid and 3g/L of magnesium sulfate heptahydrate; trace elements: calcium chloride 2H 2 O60 mg/L, manganese sulfate, H 2 O10 mg/L, zinc sulfate.7H 2 O3 mg/L, ferrous sulfate, 7H 2 O50mg/L, aluminum trichloride 6. 6H 2 O5 mg/L, sodium molybdate 2. 2H 2 O3 mg/L, cobalt chloride.6 6H 2 O0.1mg/L, copper sulfate 5H 2 O0.1mg/L, boric acid 0.5mg/L; the whole cell catalysis is performed under the following reaction parameters: cell catalyst in reaction system 600 The initial pH of the conversion solution is 30, the concentration of N-acetylglucosamine is 0.28mol/L, the concentration of pyruvic acid is 1.38mol/L, the addition amount of TritonX-100 is 0.4%, and the conversion is 70h under the condition of 30 ℃.
3. The method according to claim 2, wherein the fermentation broth containing N-acetylglucosamine is centrifuged to remove somatic cells for whole cell catalysis.
4. Use of the method of any one of claims 1 to 3 for the preparation of a sialic acid containing chemical.
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