CN117683760A - Tryptophan synthase mutant and application thereof in preparation of cysteine and cystine - Google Patents
Tryptophan synthase mutant and application thereof in preparation of cysteine and cystine Download PDFInfo
- Publication number
- CN117683760A CN117683760A CN202410154368.7A CN202410154368A CN117683760A CN 117683760 A CN117683760 A CN 117683760A CN 202410154368 A CN202410154368 A CN 202410154368A CN 117683760 A CN117683760 A CN 117683760A
- Authority
- CN
- China
- Prior art keywords
- cysteine
- cystine
- serine
- mutant
- tryptophan synthase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 title claims abstract description 112
- 229960003067 cystine Drugs 0.000 title claims abstract description 61
- 108010075344 Tryptophan synthase Proteins 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 title abstract description 31
- 235000018417 cysteine Nutrition 0.000 title abstract description 31
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 title abstract 5
- 108090000790 Enzymes Proteins 0.000 claims abstract description 38
- 102000004190 Enzymes Human genes 0.000 claims abstract description 38
- 238000004519 manufacturing process Methods 0.000 claims abstract description 26
- 241000588724 Escherichia coli Species 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 21
- 230000035772 mutation Effects 0.000 claims abstract description 18
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 17
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 101
- 229960001153 serine Drugs 0.000 claims description 73
- 238000006243 chemical reaction Methods 0.000 claims description 60
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 claims description 54
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 50
- 239000004201 L-cysteine Substances 0.000 claims description 42
- 235000013878 L-cysteine Nutrition 0.000 claims description 42
- 238000000855 fermentation Methods 0.000 claims description 41
- 230000004151 fermentation Effects 0.000 claims description 41
- 239000004158 L-cystine Substances 0.000 claims description 37
- 235000019393 L-cystine Nutrition 0.000 claims description 37
- 210000004027 cell Anatomy 0.000 claims description 35
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 14
- HYHCSLBZRBJJCH-UHFFFAOYSA-M sodium hydrosulfide Chemical compound [Na+].[SH-] HYHCSLBZRBJJCH-UHFFFAOYSA-M 0.000 claims description 14
- 150000001413 amino acids Chemical group 0.000 claims description 12
- 244000005700 microbiome Species 0.000 claims description 11
- 239000000758 substrate Substances 0.000 claims description 11
- 108020004707 nucleic acids Proteins 0.000 claims description 9
- 102000039446 nucleic acids Human genes 0.000 claims description 9
- 150000007523 nucleic acids Chemical class 0.000 claims description 9
- 239000012620 biological material Substances 0.000 claims description 7
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 claims description 7
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 claims description 7
- 229960001327 pyridoxal phosphate Drugs 0.000 claims description 7
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 6
- 230000014509 gene expression Effects 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 230000001590 oxidative effect Effects 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 238000010353 genetic engineering Methods 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 239000013612 plasmid Substances 0.000 claims description 3
- 108020004511 Recombinant DNA Proteins 0.000 claims description 2
- 210000000349 chromosome Anatomy 0.000 claims description 2
- 239000013600 plasmid vector Substances 0.000 claims description 2
- 239000013603 viral vector Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 16
- 230000015572 biosynthetic process Effects 0.000 abstract description 9
- 238000003786 synthesis reaction Methods 0.000 abstract description 8
- 238000000338 in vitro Methods 0.000 abstract description 4
- 238000012216 screening Methods 0.000 abstract description 3
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- 239000000047 product Substances 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- 235000010633 broth Nutrition 0.000 description 12
- 239000007788 liquid Substances 0.000 description 12
- 235000001014 amino acid Nutrition 0.000 description 10
- 239000012528 membrane Substances 0.000 description 10
- 229940024606 amino acid Drugs 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 239000002244 precipitate Substances 0.000 description 8
- 238000007254 oxidation reaction Methods 0.000 description 7
- 230000003197 catalytic effect Effects 0.000 description 6
- 239000000919 ceramic Substances 0.000 description 6
- 238000010276 construction Methods 0.000 description 5
- 239000012535 impurity Substances 0.000 description 5
- 230000003647 oxidation Effects 0.000 description 5
- PWKSKIMOESPYIA-UHFFFAOYSA-N 2-acetamido-3-sulfanylpropanoic acid Chemical compound CC(=O)NC(CS)C(O)=O PWKSKIMOESPYIA-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 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 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 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
- 229960000723 ampicillin Drugs 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000010170 biological method Methods 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000000413 hydrolysate Substances 0.000 description 2
- 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
- 230000037353 metabolic pathway Effects 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 238000001728 nano-filtration Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000001223 reverse osmosis Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 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 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 241001302584 Escherichia coli str. K-12 substr. W3110 Species 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102220524118 Pyridine nucleotide-disulfide oxidoreductase domain-containing protein 1_D47R_mutation Human genes 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000009088 enzymatic function Effects 0.000 description 1
- 238000003028 enzyme activity measurement method Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-M hydrosulfide Chemical compound [SH-] RWSOTUBLDIXVET-UHFFFAOYSA-M 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/12—Methionine; Cysteine; Cystine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/01—Hydro-lyases (4.2.1)
- C12Y402/0102—Tryptophan synthase (4.2.1.20)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- General Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to tryptophan synthase mutants and application thereof in preparation of cysteine and cystine. The invention is based on tryptophan synthase Trps from escherichia coli, and mutants with higher enzyme activity are obtained through site-directed mutation screening. The in vitro enzyme catalysis of the mutant is utilized to prepare the cysteine and the cystine, so that the synthesis speed can be improved, the production time can be shortened, the yield of the cysteine and the cystine can be greatly improved, the cost can be effectively reduced, and the method has important significance for the large-scale production of the cysteine and the cystine.
Description
Technical Field
The invention relates to a tryptophan synthase mutant and application thereof in cysteine production, belonging to the technical fields of genetic engineering and enzyme engineering.
Background
L-cysteine and L-cystine are sulfur-containing nonessential amino acids, which are one of the basic units of various proteins constituting living bodies, and are particularly high in human and animal hair and finger keratin. Because the sulfhydryl groups in cysteine are unstable and are very easily oxidized, two cysteine molecules can be linked by disulfide bonds to form a more stable cystine molecule. L-cysteine and L-cystine are widely used for processing and manufacturing medicines, foods, cosmetics, feeds and the like, and particularly can replace methionine with higher price to be used as a feed additive, so that the L-cysteine and L-cystine have wide market demands and prospects.
Currently, the industrial production of L-cystine mainly adopts hair hydrolysate extraction, chemical synthesis and biological methods. The hair hydrolysate extraction method and the chemical synthesis method have lower yield and more operation steps, and a large amount of waste which is difficult to treat is produced in the production process, so that the environment is polluted, and the environmental protection pressure is high. The biological rule has the advantages of strong specificity, simple production process, less side reaction and by-product, less pollution and the like, and has wider development potential.
Biological methods for preparing cysteine include in vivo microbial fermentation and in vitro enzymatic conversion. In recent years, various scientists have studied a great deal for synthesizing L-cysteine by a microbial fermentation method, but the method is limited by the complexity of a microbial intracellular metabolic network and the mutual influence and restriction among different metabolic pathways, and the yield of the L-cysteine produced by the fermentation method is still not ideal, the sulfur conversion rate is low, and the requirement of industrial production cannot be met. The enzyme catalytic conversion method has the advantages of strong specificity, simple reaction steps, mild conditions, environmental friendliness and the like, thereby becoming a new direction of research and attention of people.
The method for producing L-cysteine by using the enzyme catalytic conversion method comprises the steps of expressing a required catalytic enzyme through an engineering strain, then treating the engineering strain, and using the engineering strain as a catalyst to catalyze serine and hydrosulfide to react in vitro to generate cysteine. If the production of L-cystine is needed to be continued, then the reaction solution is subjected to oxidation treatment to oxidize the L-cysteine into L-cystine; and then the L-cystine with extremely low solubility in water and easy precipitation is utilized to obtain the L-cystine product with higher purity by filtering and separating from the reaction liquid.
Tryptophan synthase (Tryptophan synthase, trps) is a key enzyme in the tryptophan synthesis pathway in organisms and consists of two subunits, the β -subunit (TrpB) and the α -subunit (TrpA), which catalyze the reaction of indole with serine to synthesize tryptophan. The research shows that tryptophan synthase (EcTrps) from escherichia coli not only has the function of synthesizing tryptophan, but also can catalyze serine and sodium hydrosulfide to generate cysteine, which opens up a new idea for the enzyme catalysis production of cysteine. However, the study of EcTrps focuses on the tryptophan synthesis and metabolic pathways in which it is involved in the cell, with little attention paid to its ability to synthesize cysteine. In the current process of producing cysteine by using EcTrps enzyme catalysis, the yield and conversion rate of the cysteine are still not high, so that the production cost cannot be further reduced. In recent years, the demand of L-cysteine in various industries is increasing, and the existing yield can not meet the demands of domestic and foreign markets. Therefore, the molecular modification of the tryptophan synthase EcTrps has important significance for improving the yield and conversion rate of cysteine and reducing the cost.
Disclosure of Invention
In order to improve the yield and substrate conversion rate in the process of producing L-cystine by an enzyme catalysis method and reduce the impurity content in the product, the invention carries out mutation and screening on tryptophan synthase EcTrps from escherichia coli to obtain EcTrps mutant with high enzyme activity for catalyzing serine and sodium hydrosulfide to synthesize L-cysteine (the enzyme activity, reaction enzyme activity, enzyme function, catalytic function and the like referred to in the invention are referred to as activity or function for catalyzing serine and sodium hydrosulfide to synthesize L-cysteine or similar expressions with the same meaning, unless specified otherwise).
The invention aims to provide a novel tryptophan synthase mutant and application thereof in cysteine production.
The invention provides a tryptophan synthase mutant which is derived from Escherichia coliEscherichia coli) The EcTrps of (E) is subjected to amino acid substitution mutation, wherein the mutant consists of an alpha-subunit and a beta-subunit, and the amino acid sequence of the alpha-subunit is shown as SEQ ID NO:2 is shown in SEQ ID NO:1, and a mutation at a site selected from the group consisting of:
1) Mutation of amino acids 36-40 from QKDPE to AAAPA;
2) Mutation of amino acid 47 from D to W or R;
3) Mutation of amino acid 64 from N to W;
4) Mutation of amino acid 127 from G to R;
5) Mutation of amino acid 143 from S to W or R;
6) Mutation of amino acid 170 from C to A;
7) Mutation of amino acid 246 from N to A;
the present invention provides nucleic acid molecules encoding the tryptophan synthase mutants.
The present invention provides biological materials comprising the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, or engineered bacteria.
The invention provides a recombinant microorganism which is constructed by introducing a nucleic acid molecule encoding the tryptophan synthase mutant into escherichia coli through a plasmid or integrating the nucleic acid molecule into the escherichia coli chromosome through a genetic engineering means.
In one embodiment of the invention, the recombinant microorganism uses E.coli as a host cell in which a tryptophan synthase mutant is expressed.
The present invention provides the use of said tryptophan synthase mutant or said biological material or said recombinant microorganism in cysteine production.
The recombinant microorganism may be a host cell of E.coli in which the tryptophan synthase mutant is expressed.
The E.coli strain may be an engineering strain commonly used in the field of genetic engineering, preferably, for example, BL21 (DE 3), BW25113, W3110 (DE 3), M5 HiPer BLR (DE 3), BL21 (DE 3) DUOs, and particularly preferably E.coli BL21 (DE 3).
The invention provides a preparation method of L-cysteine, which is characterized in that the tryptophan synthase mutant, a nucleic acid molecule encoding the tryptophan synthase mutant or a biological material containing the nucleic acid molecule is used.
The preparation method of the L-cysteine takes serine and sodium hydrosulfide as substrates, the substrates are contacted with the mutant, and the L-cysteine is synthesized by enzyme catalysis.
In one embodiment of the present invention, recombinant E.coli cells expressing the tryptophan synthase mutant are cultured, and the mutant cell disruption solution obtained after disrupting the recombinant cells at high pressure is used as a reaction enzyme solution, and is added to the reaction solution for enzyme-catalyzed reaction.
In one embodiment of the present invention, the enzyme-catalyzed reaction system comprises: l-serine, sodium hydrosulfide, pyridoxal phosphate, disodium hydrogen phosphate, mutant cell disruption solution.
The enzyme catalytic reaction system is as follows: l-serine 0.5-2M, sodium hydrosulfide 0.5-2M, pyridoxal phosphate 0.1-1 mM, disodium hydrogen phosphate 20-100 mM, and mutant cell disruption solution corresponding to 10-50 g/L wet cell.
The mutant cell disruption solution corresponding to 10-50 g/L wet cell is a cell disruption solution obtained by disrupting 10-50g of wet cell expressing the mutant under high pressure in each liter of an enzyme-catalyzed reaction system.
The method comprises the following steps of: 30-45 ℃ and pH 7.5-8.5.
The invention provides a preparation method of L-cystine, which is used for oxidizing the L-cysteine prepared by the method to obtain L-serine.
The invention provides a preparation method of L-cysteine or L-cystine, which specifically comprises the following steps:
(1) Culturing recombinant E.coli cells expressing said tryptophan synthase mutant;
(2) Performing cell disruption treatment on the culture, wherein the obtained cell disruption product is a reaction enzyme solution containing the tryptophan synthase mutant;
(3) The reaction enzyme liquid is used for catalyzing serine and sodium hydrosulfide to react to synthesize L-cysteine;
if L-cystine is to be prepared, the preparation method further comprises the following steps:
(4) Oxidizing the L-cysteine to obtain a crude L-cystine product;
(5) And separating and purifying the crude L-cystine to obtain a finished L-cystine product.
In the preparation method, the L-serine in the step (3) is L-serine pure product or L-serine fermentation liquor obtained by fermenting serine producing bacteria.
In the preparation method, the oxidation in the step (4) is to introduce air into a reaction solution obtained by enzyme catalysis synthesis of L-cysteine, and then add FeCl 2 Oxidation is performed.
The serine producing strain is constructed by the construction method of serine high-producing strain ES-134/pSC-08.
The preparation method of the L-serine fermentation broth comprises the following steps: inoculating the constructed serine producing strain into serine fermentation medium for fermentation.
Serine was measured at the end of the fermentation to be at a concentration of about 36 g/L.
The fermentation medium is an M9 medium, and comprises the following specific components: 6.8 g/L Na 2 HPO 4 、3 g/L KH 2 PO 4 、1g/L NaCl、5 g/L (NH 4 ) 2 SO 4 、0.015 g/L CaCl 2 ·2H 2 O、3g/L MgSO 4 ·7H 2 O、0.07 g/L FeSO 4 ·7H 2 O, 0.11. 0.11 g/L sodium citrate, 2 g/L Yeast extract and 8 g/L glucose, and trace elements (7 mg/L CoCl) 2 ·6H 2 O、2.5 mg/L CuSO 4 ·5H 2 O、25 mg/L H 3 BO 3 、16 mg/L MnCl 2 ·4H 2 O、1.5 mg/L Na 2 MoO 4 ·2H 2 O、3 mg/L ZnSO 4 ·7H 2 O)。
The preparation method of the L-serine fermentation liquor further comprises the following steps:
(1) After the fermentation, the pH of the serine fermentation broth is adjusted, and the serine fermentation broth is heated to inactivate and denature the protein, so that the serine fermentation broth becomes insoluble protein.
(2) And (3) passing the serine fermentation liquor after heat treatment through a ceramic membrane, and removing thallus fragments and denatured proteins to obtain ceramic membrane clear liquid.
(3) And concentrating the clear ceramic membrane liquid by using a reverse osmosis membrane or a nanofiltration membrane to obtain L-serine concentrated fermentation liquor.
Each 1L of the reaction system contains: l-serine concentrate fermentation broth, sodium hydrosulfide, pyridoxal phosphate, disodium hydrogen phosphate, recombinant E.coli cell disruption solution of the tryptophan synthase mutant.
The invention obtains mutant Trps with high enzyme activity by mutating and screening wild tryptophan synthase QKDPE36-40AAAPA 、Trps N64W 、Trps G127R 、Trps S143R 、Trps N246A In particular mutant Trps QKDPE36-40AAAPA 、Trps N64W 、Trps G127R The enzyme activities respectively reach 2.87 times, 2 times and 1.8 times of the wild tryptophan synthase, and the mutants all improve the production of cysteine. The invention greatly improves the output of the cysteine and the cystine, reduces the cost and has important significance for the large-scale high-yield production of the cysteine and the cystine.
Drawings
FIG. 1 is a comparison of the relative enzyme activities of E.coli tryptophan synthase and mutants thereof;
FIG. 2 shows the results of cysteine and cystine production using a crude recombinant E.coli enzyme solution expressing tryptophan synthase and mutants thereof.
FIG. 3 shows the results of cysteine production using a recombinant E.coli crude enzyme solution expressing tryptophan synthase and mutants thereof to catalyze serine fermentation broths.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
EXAMPLE 1 construction of Tryptophan synthase EcTrps mutants
(1) Template construction
Two subunits of tryptophan synthase are expressed simultaneously by using an escherichia coli dual expression vector pETDuet-1 with dual promoters, beta-subunit (TrpB) of tryptophan synthase Trps of escherichia coli is cloned to a multiple cloning site 1 of pETDuet-1, alpha-subunit (TrpA) is cloned to a multiple cloning site 2 of pETDuet-1, and a vector pETDuet-1-Trps capable of expressing TrpB and TrpA simultaneously is obtained, and the TrpB and TrpA gene sequences are shown as SEQ ID NO. 3 and SEQ ID NO. 4.
(2) Construction of mutant recombinant strains
The vector pETDuet-1-Trps is used as a template, a mutation primer pair (shown in table 1) is used for carrying out corresponding site mutation on a coding gene of beta-subunit (TrpB) of the Trps, a PCR amplification product is digested with Dpn I for 1 hour and then transferred into an escherichia coli DH5 alpha competent cell, and a monoclonal is selected for sequencing identification. The plasmid containing the mutant gene was transferred into E.coli W3110 (DE 3) to obtain mutant recombinant strain W3110-Trps D47W 、W3110-Trps D47R 、W3110-Trps N64W 、W3110-Trps G127R 、W3110-Trps S143R 、W3110-Trps S143W 、W3110-Trps C170A 、W3110-Trps N246A 、W3110-Trps QKDPE36-40AAAPA . Meanwhile, the unmutated vector pETDuet-1-Trps is transferred into escherichia coli W3110 (DE 3) to obtain recombinant strain W3110-Trps expressing wild-type Trps as a control. (Note: the above mutation sites are numbered 36-40, 47, 64, 127, 143, 170, 246 all refer to beta of Trps of E.coli)-amino acid sequence numbering of subunits (TrpB)
TABLE 1 TrpsB Gene site-directed mutagenesis primer
Example 2 Tryptophan synthase mutant enzyme Activity test
(1) Recombinant protein induced expression
The mutant recombinant strain and the control recombinant strain constructed in example 1 were respectively cultured in LB medium containing ampicillin under shaking at 37℃until the bacterial concentration reached OD 600 When the concentration was 1.0, IPTG was added to a final concentration of 0.4. 0.4mM to induce expression, and cells were collected after culturing at 30℃for 20. 20 h.
(2) Tryptophan synthase mutant enzyme activity assay
After the collected tryptophan synthase mutant strain is resuspended in water, the culture is subjected to cell disruption by a high-pressure homogenizer (800-1000 bar) to obtain a cell disruption solution as an enzyme solution, and enzyme activity measurement is performed. The enzyme activity reaction system comprises the following components: tris-HCL 100 mM, L-serine 100 mM, sodium hydrosulfide 110 mM, pyridoxal phosphate 0.4mM, a cell disruption solution, wherein the addition amount of the cell disruption solution is an amount equivalent to 1g of wet cell per liter of the reaction system; the reaction conditions were 40℃and pH 8.0. After the reaction was completed, the reaction was terminated with an equal volume of 100. Mu.L of 1M HCL. Samples were tested by HPLC and mutant enzyme activity was determined.
As shown in FIG. 1, the mutant Trps compared to the wild type N64W 、Trps G127R 、Trps QKDPE36-40AAAPA All have higher enzyme activity and respectively reach 1.8-3 times of wild Trps, in particular to mutant Trps obtained by carrying out continuous mutation on 36-40 amino acids of beta subunit QKDPE36-40AAAPA The enzyme activity of (a) is nearly 3 times that of the wild type, and is far less than that of other single-site mutations.
Example 3 catalytic production of L-cysteine Using Tryptophan synthase mutants
Selecting mutant strain W3110-Trps with highest catalytic activity QKDPE36-40AAAPA And the control strain W3110-Trps was inoculated into LB medium containing ampicillin, and cultured with shaking at 37 ℃. When the concentration of the bacterial liquid reaches OD 600 At 1.0, IPTG was added at a final concentration of 0.4mM to induce expression, and the mixture was cultured at 30℃for 20. 20 h to obtain a recombinant cell culture.
And respectively carrying out high-pressure disruption on the recombinant cell culture to obtain a cell disruption solution serving as a reaction enzyme solution, and adding the cell disruption solution into an enzyme catalysis reaction solution for producing L-cysteine. The reaction liquid comprises the following components: l-serine 0.95M, sodium hydrosulfide 1.1M, pyridoxal phosphate 0.4mM, disodium hydrogen phosphate 50 mM, and cell disruption liquid corresponding to 20g/L wet cell culture (namely, cell disruption liquid obtained by disrupting 20g of cell-wet cell culture of mutant strain or control strain under high pressure per liter of reaction system). The reaction conditions are as follows: reaction at 40℃with 2M H 2 SO 4 The pH was adjusted to 8.0. The formation of L-cysteine or L-cystine within 4 hours was examined by HPLC, and the remaining amount of substrate serine: 10 min,60 min,120 min,180 min,240 min reaction mixtures were each assayed and quenched with 1M hydrochloric acid.
Note that: since L-cysteine is unstable, a small amount of L-cysteine as a product is naturally oxidized to L-cystine in the reaction system during the above enzyme-catalyzed reaction, and 1L-cystine molecule is generated from two L-cysteine molecules by the oxidation reaction, the molar concentration of L-cystine to be measured needs to be doubled when the yield is calculated. The method comprises the following steps:
yield = molar concentration of L-cysteine +2 x molar concentration of L-cystine
Serine conversion = (molar concentration of L-cysteine+2×molar concentration of L-cystine)/(initial molar concentration of L-serine×100%
As a result, as shown in FIG. 2, when the wild-type Trps catalytic reaction was used for 4 hours, the yield was 868 mM (cysteine 781mM, cystine 43.5 mM) and the serine conversion rate was 91.4%. While using mutant Trps QKDPE36-40AAAPA When the reaction is carried out for 3 hours, the yield can reach 949.5 mM (878.5 mM of cysteine and 35.5mM of cystine), and the serine is basically completely converted into the cysteine, so that the conversion rate is as high as 99.9%.
The above results demonstrate that the use of mutated tryptophan synthase Trps relative to wild-type Trps QKDPE36-40AAAPA The method has the advantages of catalyzing the synthesis of L-cysteine, shortening the reaction time by 25%, greatly improving the production efficiency, greatly improving the yield and the conversion rate, and particularly basically realizing the complete conversion of serine serving as a substrate and effectively reducing the production cost.
EXAMPLE 4 preparation of L-cysteine Using Tryptophan synthase mutant to catalyze serine fermentation broths
Because of the high price of the pure serine, the fermentation broth of serine producing bacteria is used as a substrate instead of the pure serine to perform the enzyme-catalyzed synthesis of L-cysteine to further reduce the cost.
Preparation of serine fermentation liquor
The purpose of serine fermentation production is to obtain fermentation liquor containing serine with higher concentration through fermentation, which is used for the subsequent preparation of L-cysteine, so as to reduce the high production cost caused by adopting pure serine. Thus, any strain in the art capable of producing serine by fermentation can be used in this step.
For example, it can be described in Wang C, wu J, shi B, shi J, zhao Z.Immung L-serine formation byEscherichia coliby reduced uptake of produced L-serine, microb Cell face 2020 Mar 14;19 (1): 66. Doi: 10.1186/s12934-020-01323-2. A serine producing strain was constructed by the disclosed construction method of serine high producing strain ES-134/pSC-08, and serine fermentation broth was prepared by fermentation. The specific method comprises the following steps: the constructed serine producing strain was inoculated into a serine fermentation medium and fermented at 37℃for 51 hours, and the serine concentration was measured to be about 36 g/L at the end of the fermentation. The fermentation medium is consistent with the above documents, is an M9 medium, and comprises the following specific components: 6.8 g/L Na 2 HPO 4 、3 g/L KH 2 PO 4 、1g/L NaCl、5 g/L (NH 4 ) 2 SO 4 、0.015 g/L CaCl 2 ·2H 2 O、3g/L MgSO 4 ·7H 2 O, 0.07 g/L FeSO4.7H2O, 0.11 g/L sodium citrate, 2 g/L Yeast extract and 8 g/L glucose, and trace elements (7 mg/L CoCl) 2 ·6H 2 O、2.5 mg/L CuSO 4 ·5H 2 O、25 mg/L H 3 BO 3 、16 mg/L MnCl 2 ·4H 2 O、1.5 mg/L Na 2 MoO 4 ·2H 2 O、3 mg/L ZnSO 4 ·7H 2 O)。
(II) fermentation liquor treatment
(1) After the fermentation is finished, the pH value of the serine fermentation liquid is adjusted to 5, and the serine fermentation liquid is heated to 70-90 ℃ to inactivate and denature the protein, so that the protein is changed from soluble protein to insoluble protein.
(2) And (3) passing the serine fermentation liquor after heat treatment through a ceramic membrane with the pore size of 50-nM, and removing thallus fragments and denatured proteins to obtain ceramic membrane clear liquid.
(3) And concentrating the clear ceramic membrane liquid by using a reverse osmosis membrane or a nanofiltration membrane with the molecular weight of less than 150 so that the content of L-serine reaches 120g/L, thereby obtaining L-serine concentrated fermentation liquor.
(III) production of L-cysteine
(1) Recombinant mutant strain W3110-Trps QKDPE36-40AAAPA And respectively taking 20g of wet thalli from cultures of the control strain W3110-Trps for high-pressure disruption, and obtaining cell disruption liquid which is the reaction enzyme liquid.
(2) In vitro enzyme catalytic reaction to prepare L-cysteine.
Each 1L of the reaction system contains: l-serine concentrate fermentation broth 833 mL (L-serine concentration 0.95M), sodium hydrosulfide 1.1 mol, pyridoxal phosphate 0.4 mmol, disodium hydrogen phosphate 50 mmol, and cell disruption solution obtained by high-pressure disruption of 20g wet cell in step (1).
The reaction conditions are as follows: reaction at 40℃with 2M H 2 SO 4 The pH was adjusted to 8.0.
(3) The formation of L-cysteine or L-cystine and the residual amount of serine as substrate were measured by HPLC for 4 hours, and 10 min,60 min,120 min,180 min,240 min reaction mixtures were taken and quenched with 1M hydrochloric acid.
Note that: since L-cysteine is unstable, a small amount of L-cysteine as a product is naturally oxidized to L-cystine in the reaction system during the above enzyme-catalyzed reaction, and 1L-cystine molecule is generated from two L-cysteine molecules by the oxidation reaction, the molar concentration of L-cystine measured needs to be doubled when the yield and conversion rate are calculated. The method comprises the following steps:
yield = molar concentration of L-cysteine +2 x molar concentration of L-cystine
Serine conversion = (molar concentration of L-cysteine+2×molar concentration of L-cystine)/(initial molar concentration of L-serine×100%
As a result, as shown in FIG. 3, after 4 hours of the wild-type Trps reaction, the yield was 731 mM (651 mM cysteine, 40mM cystine), the serine conversion was only 76.9%, and about one quarter of serine was not converted to L-cysteine. While using mutant Trps QKDPE36-40AAAPA The reaction is carried out for 4 hours, the yield can reach 897 mM (807 mM of cysteine and 45mM of cystine), the yield is 23% higher than that of the wild type Trps, and the serine conversion rate reaches 94.4% and is far higher than that of the wild type Trps.
In addition, when the reaction was carried out using L-serine as a substrate in comparison with example 3, the conversion rate of the wild-type Trps reaction for 4 hours was reduced from 91.4% to 76.9% when the serine concentrate broth was used as a substrate, which suggests that the impurity components in the L-serine concentrate broth have a large interference and influence on the enzyme activity of the wild-type Trps. While using mutant Trps QKDPE36-40AAAPA In the reaction, the conversion rate is only slightly reduced, and the substantially complete conversion can be still achieved after 4 hours of reaction, which indicates that the mutant Trps QKDPE36-40AAAPA The enzyme activity of (2) is less influenced by impurities in serine concentrated fermentation liquor, and is more suitable for industrial production.
EXAMPLE 5 preparation of L-cystine from L-cysteine
(1) Oxidation
Trps Using Tryptophan synthase mutant in example 4 QKDPE36-40AAAPA After the reaction is finished, the pH of the reaction system is adjusted to 7.0, air is introduced, the ventilation amount is 1.67 vvm, and 200mg/L FeCl is added after 30min of ventilation 2 Oxidizing for 6h, and oxidizing the L-cysteine in the reaction system into L-cystine.
(2) Separation
The oxidation product obtained in the step (1) is filtered and separated by a buchner funnel to obtain reaction solution and cystine precipitate, the reaction solution and cystine precipitate are leached for 2 times by a dilute sulfuric acid solution (pH is approximately equal to 5) with the concentration of 5 mu M, and the precipitate is collected.
(3) Purification
Adding 6M sulfuric acid solution into the precipitate obtained in the step (2) until the cystine precipitate is completely dissolved, adding 0.5% active carbon for decoloring treatment for 30min, and filtering and separating supernatant and precipitate by a Buchner funnel. Regulating pH of the supernatant to 5.6 with 10M NaOH, filtering with a Buchner funnel, separating supernatant and cystine precipitate, eluting with pure water for 2 times, drying and crystallizing the cystine precipitate to obtain white powder crystal, namely L-cystine product.
The optical characteristics and purity of the obtained L-cystine product are detected, and the light transmittance is 98%, the specific rotation is-215% and the purity is 99%.
The above results demonstrate that tryptophan synthase mutant Trps was used QKDPE36-40AAAPA The preparation of cystine can obtain high-purity and high-quality L-cystine product with excellent optical properties even though the substrate uses low-cost serine concentrated fermentation liquor which contains more impurities and is not purified.
In conclusion, the tryptophan synthase mutant with high enzymatic activity is obtained by mutating the escherichia coli Trps, so that the yield and the conversion rate of cysteine or cystine are greatly improved, the activity is not easily affected by impurities, the production efficiency is effectively improved, the production cost is reduced, and the method has important significance for large-scale high-yield production of cysteine and cystine.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. A tryptophan synthase mutant comprising an α -subunit and a β -subunit, wherein the amino acid sequence of the α -subunit is set forth in SEQ ID NO:2 is shown in SEQ ID NO:1, and performing mutation at a site selected from the group consisting of: the mutation of N at position 64 to W, G at position 127 to R or QKDPE at positions 36-40 to AAAPA.
2. A nucleic acid molecule encoding the mutant of claim 1.
3. A biological material comprising the nucleic acid molecule of claim 2, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, or an engineering bacterium.
4. A recombinant microorganism, wherein the recombinant microorganism is constructed by introducing the nucleic acid molecule of claim 2 into E.coli by means of a plasmid or integrating it into E.coli chromosome by means of genetic engineering.
5. Use of the mutant according to claim 1 or the biological material according to claim 3 or the recombinant microorganism according to claim 4 for the production of L-cysteine or L-cystine.
6. A process for the preparation of L-cysteine or L-cystine, characterized in that the mutant according to claim 1 or the biological material according to claim 3 or the recombinant microorganism according to claim 4 is used.
7. The method for producing L-cysteine or L-cystine according to claim 6, wherein L-serine and sodium hydrosulfide are used as substrates.
8. A method of preparing as claimed in claim 7, comprising the steps of:
(1) Culturing a recombinant microorganism expressing the recombinant microorganism of claim 4;
(2) Performing cell disruption treatment on the culture, wherein the obtained cell disruption product is a reaction enzyme solution containing the tryptophan synthase mutant;
(3) Adding the reaction enzyme solution into a catalytic reaction system to catalyze L-serine and sodium hydrosulfide to react and synthesize L-cysteine;
in preparing L-cystine, the preparation method further comprises the following steps:
(4) Oxidizing the L-cysteine to obtain a crude L-cystine product;
(5) And separating and purifying the crude L-cystine to obtain a finished L-cystine product.
9. The process according to claim 8, wherein the catalytic reaction system of step (3) comprises L-serine, sodium hydrosulfide, pyridoxal phosphate, disodium hydrogen phosphate, and a cell disruption solution of the recombinant microorganism according to claim 4.
10. The process according to any one of claims 7 to 9, wherein the L-serine is pure L-serine or an L-serine fermentation broth obtained by fermentation of serine producing bacteria.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202410154368.7A CN117683760B (en) | 2024-02-04 | 2024-02-04 | Tryptophan synthase mutant and application thereof in preparation of cysteine and cystine |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202410154368.7A CN117683760B (en) | 2024-02-04 | 2024-02-04 | Tryptophan synthase mutant and application thereof in preparation of cysteine and cystine |
Publications (2)
Publication Number | Publication Date |
---|---|
CN117683760A true CN117683760A (en) | 2024-03-12 |
CN117683760B CN117683760B (en) | 2024-04-19 |
Family
ID=90130408
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202410154368.7A Active CN117683760B (en) | 2024-02-04 | 2024-02-04 | Tryptophan synthase mutant and application thereof in preparation of cysteine and cystine |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117683760B (en) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4769491A (en) * | 1985-11-20 | 1988-09-06 | Mitsui Toatsu Chemicals, Inc. | Method for production of cystine from cysteine |
CN87103767A (en) * | 1986-11-19 | 1988-12-07 | 三井东圧化学株式会社 | Produce the method for L-Gelucystine |
WO2018083692A1 (en) * | 2016-11-01 | 2018-05-11 | Tamar Levin | Novel methods for modulating protein expression in microorganisms |
CN115261365A (en) * | 2022-06-22 | 2022-11-01 | 南通紫琅生物医药科技有限公司 | Tryptophan synthetase mutant and application thereof |
CN117230050A (en) * | 2023-11-10 | 2023-12-15 | 北京量维生物科技研究院有限公司 | Tryptophan synthase and application of mutant thereof in production of cysteine and cystine |
-
2024
- 2024-02-04 CN CN202410154368.7A patent/CN117683760B/en active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4769491A (en) * | 1985-11-20 | 1988-09-06 | Mitsui Toatsu Chemicals, Inc. | Method for production of cystine from cysteine |
CN87103767A (en) * | 1986-11-19 | 1988-12-07 | 三井东圧化学株式会社 | Produce the method for L-Gelucystine |
WO2018083692A1 (en) * | 2016-11-01 | 2018-05-11 | Tamar Levin | Novel methods for modulating protein expression in microorganisms |
CN115261365A (en) * | 2022-06-22 | 2022-11-01 | 南通紫琅生物医药科技有限公司 | Tryptophan synthetase mutant and application thereof |
CN117230050A (en) * | 2023-11-10 | 2023-12-15 | 北京量维生物科技研究院有限公司 | Tryptophan synthase and application of mutant thereof in production of cysteine and cystine |
Non-Patent Citations (1)
Title |
---|
KEN-ICHI ISHIWATA等: "Enzymatic production of l-cysteine with tryptophan synthase of Escherichia coli", JOURNAL OF FERMENTATION AND BIOENGINEERING, vol. 67, no. 3, 31 December 1989 (1989-12-31), pages 169 - 172 * |
Also Published As
Publication number | Publication date |
---|---|
CN117683760B (en) | 2024-04-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN104531629B (en) | A kind of yclodextrin glycosyltransferase mutant of raising AA 2G conversion ratios | |
CN117230050B (en) | Tryptophan synthase and application of mutant thereof in production of cysteine and cystine | |
CN104726478A (en) | Recombinant Escherichia coli for expressing arginine deiminase gene and application of recombinant Escherichia coli | |
CN114525268A (en) | Glutamic acid decarboxylase mutant with improved pH tolerance and application thereof in synthesis of gamma-aminobutyric acid | |
CN113122527B (en) | Aspartase mutant with improved enzyme activity and changed optimal pH | |
CN112251428B (en) | Glutamic acid decarboxylase mutant and application thereof in production of gamma-aminobutyric acid | |
CN113832120B (en) | Formaldehyde conversion mutant protein and application thereof | |
CN113549633A (en) | L-cysteine transporter mutant and application thereof in production of L-cysteine | |
CN117683760B (en) | Tryptophan synthase mutant and application thereof in preparation of cysteine and cystine | |
CN110358751B (en) | Recombinant lipase mutant, encoding gene, recombinant engineering bacterium and application | |
CN109251923B (en) | Serine racemase mutants | |
CN112011495B (en) | Recombinant escherichia coli for expressing thermolysin mutant and application thereof | |
CN114181288A (en) | Process for producing L-valine, gene used therefor and protein encoded by the gene | |
CN110791536B (en) | Biosynthesis method of levodopa | |
CN110872595B (en) | Acid-resistant expression cassette and application thereof in fermentation production of organic acid | |
CN113151378A (en) | Method for preparing nucleoside, nicotinic acid adenine dinucleotide and nicotinic acid mononucleotide of nicotinic acid or derivative thereof, enzyme composition and application | |
CN118207172A (en) | Bifunctional glutathione synthase mutant and application thereof | |
CN115716868B (en) | Transcription factor MrPigB mutant and application thereof | |
CN116355875B (en) | Methionine adenosyltransferase mutant and application thereof in production of S-adenosylmethionine | |
CN115011569B (en) | Bose NemR-PS mutant and application thereof in preparation of (S) -citronellol | |
CN107201355B (en) | High-stereoselectivity phenylalanine deaminase mutant and application thereof | |
CN111575258B (en) | Carbonyl reductase EbSDR8 mutant and construction method and application thereof | |
CN116286695A (en) | L-lactate dehydrogenase and encoding gene and application thereof | |
CN117247914A (en) | Acetohydroxyacid synthase mutant and application thereof | |
CN118006528A (en) | Genetically engineered bacterium for producing tetrahydropyrimidine, preparation method and application |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |