CN106367445B - Method for producing glutaric acid by whole-cell biocatalysis - Google Patents
Method for producing glutaric acid by whole-cell biocatalysis Download PDFInfo
- Publication number
- CN106367445B CN106367445B CN201610724114.XA CN201610724114A CN106367445B CN 106367445 B CN106367445 B CN 106367445B CN 201610724114 A CN201610724114 A CN 201610724114A CN 106367445 B CN106367445 B CN 106367445B
- Authority
- CN
- China
- Prior art keywords
- cell
- glutaric acid
- recombinant
- bacterial strain
- ydt
- 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.)
- Active
Links
- JFCQEDHGNNZCLN-UHFFFAOYSA-N anhydrous glutaric acid Natural products OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 title claims abstract description 38
- RTBFRGCFXZNCOE-UHFFFAOYSA-N 1-methylsulfonylpiperidin-4-one Chemical compound CS(=O)(=O)N1CCC(=O)CC1 RTBFRGCFXZNCOE-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 46
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims abstract description 25
- 241000894006 Bacteria Species 0.000 claims abstract description 19
- 239000000758 substrate Substances 0.000 claims abstract description 14
- 239000004472 Lysine Substances 0.000 claims abstract description 13
- 235000019766 L-Lysine Nutrition 0.000 claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
- 239000004094 surface-active agent Substances 0.000 claims abstract description 6
- 238000002156 mixing Methods 0.000 claims abstract description 3
- 230000001580 bacterial effect Effects 0.000 claims description 31
- 239000013612 plasmid Substances 0.000 claims description 28
- 108091008146 restriction endonucleases Proteins 0.000 claims description 16
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 claims description 13
- 230000003197 catalytic effect Effects 0.000 claims description 8
- XUWPJKDMEZSVTP-LTYMHZPRSA-N kalafungina Chemical compound O=C1C2=C(O)C=CC=C2C(=O)C2=C1[C@@H](C)O[C@H]1[C@@H]2OC(=O)C1 XUWPJKDMEZSVTP-LTYMHZPRSA-N 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 5
- 230000006798 recombination Effects 0.000 claims description 5
- 238000005215 recombination Methods 0.000 claims description 5
- 101100536799 Acinetobacter baylyi (strain ATCC 33305 / BD413 / ADP1) tgnE gene Proteins 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 230000029087 digestion Effects 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 4
- 101150043302 gabD gene Proteins 0.000 claims description 4
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 3
- 229960005091 chloramphenicol Drugs 0.000 claims description 3
- 101150116670 gabT gene Proteins 0.000 claims description 3
- WIIZWVCIJKGZOK-IUCAKERBSA-N 2,2-dichloro-n-[(1s,2s)-1,3-dihydroxy-1-(4-nitrophenyl)propan-2-yl]acetamide Chemical compound ClC(Cl)C(=O)N[C@@H](CO)[C@@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-IUCAKERBSA-N 0.000 claims description 2
- 239000013504 Triton X-100 Substances 0.000 claims description 2
- 229920004890 Triton X-100 Polymers 0.000 claims description 2
- 230000008859 change Effects 0.000 claims description 2
- 238000010276 construction Methods 0.000 claims description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 2
- 229920000053 polysorbate 80 Polymers 0.000 claims description 2
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 claims 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims 1
- 229920001213 Polysorbate 20 Polymers 0.000 claims 1
- 235000018977 lysine Nutrition 0.000 claims 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims 1
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 abstract description 14
- 230000008569 process Effects 0.000 abstract description 9
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 abstract description 5
- 238000006555 catalytic reaction Methods 0.000 abstract description 5
- 108090000790 Enzymes Proteins 0.000 abstract description 4
- 102000004190 Enzymes Human genes 0.000 abstract description 4
- 230000014509 gene expression Effects 0.000 abstract description 4
- 101100001670 Emericella variicolor andE gene Proteins 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 229960002989 glutamic acid Drugs 0.000 abstract 2
- 230000007547 defect Effects 0.000 abstract 1
- 238000005265 energy consumption Methods 0.000 abstract 1
- 238000000605 extraction Methods 0.000 abstract 1
- 238000000855 fermentation Methods 0.000 abstract 1
- 230000004151 fermentation Effects 0.000 abstract 1
- 239000002207 metabolite Substances 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 31
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 230000002194 synthesizing effect Effects 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 229920000728 polyester Polymers 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 241000588722 Escherichia Species 0.000 description 2
- 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
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- 229910052801 chlorine Inorganic materials 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 150000002311 glutaric acids Chemical class 0.000 description 2
- 230000006698 induction Effects 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
- 239000007791 liquid phase Substances 0.000 description 2
- 229920000915 polyvinyl chloride Polymers 0.000 description 2
- 239000004800 polyvinyl chloride Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- JKTCBAGSMQIFNL-UHFFFAOYSA-N 2,3-dihydrofuran Chemical compound C1CC=CO1 JKTCBAGSMQIFNL-UHFFFAOYSA-N 0.000 description 1
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 1
- YEJRWHAVMIAJKC-UHFFFAOYSA-N 4-Butyrolactone Chemical compound O=C1CCCO1 YEJRWHAVMIAJKC-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-L Malonate Chemical compound [O-]C(=O)CC([O-])=O OFOBLEOULBTSOW-UHFFFAOYSA-L 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- LTBFZSXMPHXJJT-UHFFFAOYSA-N cyclopentanol;cyclopentanone Chemical compound OC1CCCC1.O=C1CCCC1 LTBFZSXMPHXJJT-UHFFFAOYSA-N 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000003546 flue gas Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000003317 industrial substance Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 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 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000004014 plasticizer Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000001294 propane Substances 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
-
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
- C12Y104/03011—L-Glutamate oxidase (1.4.3.11)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for producing glutaric acid by whole-cell biocatalysis. The method firstly induces expression and collects recombinant strainsE.coliBL‑22AB‑YDTCells of (2) andE.coli28LGOXthe recombinant strain is culturedE.coli28LGOXCells and recombinant bacteria ofE.coliBL‑22AB‑YDTThe cells of (a) are as follows 1: 1-5, adding substrates L-lysine and L-glutamic acid after mixing, and enabling the molar ratio of the L-glutamic acid to the L-lysine to be 1: 0.5-4, adding a surfactant, and producing glutaric acid by whole-cell catalysis. The method of the invention does not need to add 2-ketoglutaric acid, reduces the production cost, solves the problems of long production period, complex metabolite, low substrate conversion rate, difficult product separation and extraction and high energy consumption of a fermentation method, also solves the defect that the cascade catalysis process in the enzyme catalysis is not easy to realize, improves the catalysis efficiency, and saves the enzyme purification process.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of method of Whole Cell Biocatalysis production glutaric acid.
Background technique
Glutaric acid, alias: glue acid, a, γ-propane dicarboxylic acids, 1,3- propane dicarboxylic acid, can be used as important industrial chemicals and
Organic intermediate has a wide range of applications in all fields.In the industry, glutaric acid can be used for synthesizing polyvinyl chloride, polyester, gather
In addition amide and the polyester plasticizer of polyvinyl chloride etc. can be used for synthesizing liquid polyester (the molecule knot of improvement PET fiber
Structure improves the dyeability of PET fiber, improves dye-uptake).In terms of medicine, glutaric acid can be used for synthesizing various sterilizings and wash
Liquid and drug, in addition, glutaric acid can be used for the ingredients of detergent, the flue gases such as the preparation of adhesive and sulfur-bearing in life
Washing etc..
At present in the method for synthesizing glutaric acid, there is chemical synthesis, such as using gamma-butyrolacton as the multi-step synthetic method of raw material,
Using cyclopentanol-cyclopentanone as the choice oxidation process of raw material, hydrolysis is aoxidized by raw material of dihydrofuran, by malonate two
Prepared in the presence of second ammonium with formaldehyde condensation etc..But this method raw material is costly, is not easy to obtain and major part has used strong oxidizer, it is right
The corrosion of equipment is very big.React complex, there are many reaction step, and yield is not high, and environmental pollution is more serious, are only limited to test
Room, industrial prospect are little.
Si Jae ParkEt al. using being overexpressed the recombination of davAB and gabTDE. coliWL3110 bacterial strain is containing
There is 20g/L glucose, fermented and cultured in the culture medium of 10g/L L-lysine and 10g/L 2-oxoglutaric acid has obtained 1.7g/L
Glutaric acid.The amino acceptor 2-oxoglutaric acid that this method needs external source addition expensive, considerably increases production cost, in addition sends out
Ferment method production glutaric acid molar yield is lower, takes a long time, and reaction system is more complex, and product separation is more difficult.
Jake AdkinsEt al. using being overexpressed the recombination of davAB and davDTEscherichia coliBacterial strain is with Portugal
Grape sugar is substrate, produces the glutaric acid of 0.82g/L after the 48h that ferments, this method reaction system is more complex, mole of glutaric acid
Yield is lower.
Through retrieving, a kind of method of double cells coupling production glutaric acid of economical and efficient, there is not been reported.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of Whole Cell Biocatalysis production glutaric acids
Method, this method save the cost, economical and efficient.
To solve prior art problem, the technical scheme adopted by the invention is as follows:
A kind of method of Whole Cell Biocatalysis production glutaric acid, comprising the following steps:
A kind of method of Whole Cell Biocatalysis production glutaric acid, comprising the following steps:
Step 1, building is overexpressed the bacterial strain of DavBA and gabDTE.coliThen BL-22AB-YDT is picked from the plate
Recombinant bacterial strainE.coliThe single colonie of BL-22AB-YDT is inoculated into mould containing 100mg/L amicillin resistance and 35mg/L chlorine
The 5ml LB of plain resistance shakes in pipe, is transferred to after cultivating 6-8h containing 100mg/L amicillin resistance and 35mg/L chloramphenicol
In the 100MLLB culture medium of resistance, culture to OD600=0.6-0.8, centrifugation collection bacterium, obtains recombinant bacterial strainE.coliBL-22AB-
The cell of YDT；
Step 2, building is overexpressed the bacterial strain of LGOXE.coliThen 28LGOX picks from the plate recombinant bacterial strainE.coliThe single colonie of 28LGOX is inoculated into the shaking in pipe of resistance containing kanamycin and LB, is transferred to after cultivating 6-8h containing card
In the shaking flask of that chloramphenicol resistance and LB, culture to OD600=0.6-0.8, centrifugation collection bacterium, obtains recombinant bacterial strainE.coli28LGOX's
Cell；
Step 3, by recombinant bacteriumE.coliThe cell and recombinant bacterium of 28LGOXE.coliThe cell of BL-22AB-YDT according to than
Example 1:1-5 mixing, is added substrate L-lysine (5-100g/L) and Pidolidone (5-100g/L), and Pidolidone and L- is made to rely ammonia
The molar ratio of sour (5-100g/L) is 1:0.5-4, and surfactant is added, and whole-cell catalytic produces glutaric acid.
In the above method preferably, the construction step of DavBA and gabDT bacterial strain is overexpressed in step 1 are as follows:
DavA and davB segment is connected with expression vector pET-22b, recombinant plasmid pET22b-DavBA is obtained, will recombinate
Plasmid pET22b-DavBA is importedE.coli BL21Competent cell in, the recombinant bacterium for expressing davBA is obtainedE.coliThe recombinant bacterial strain is simultaneously made into competent cell by BL-22BA, by gabD and gabT segment and expression vector pACYC-
Duet is connected, and obtains recombinant plasmid pACYC-gabDT, recombinant plasmid is imported recombinant bacteriumE.coliThe competence of BL-22AB is thin
In born of the same parents, the recombinant bacterial strain for expressing DavBA and gabDT is obtainedE.coliBL-22AB-YDT。
In the above method preferably, step 2 building is overexpressed the bacterial strain of LGOXE.coliThe method of 28LGOX is: by piece
LGOX is using NcoI and BamHI as restriction enzyme site for section, is connected with the expression vector 28a through NcoI and BamHI digestion, is recombinated
Plasmid 28a-LGOX, recombinant plasmid is importedE.coliIn the competent cell of BL21, the recombinant bacterium for expressing LGOX is obtainedE.coli28LGOX。
Step 3, recombinant bacterial strainE.coliThe process and recombinant bacterium of BL-22AB-YDT whole-cell catalytic L-lysine
The method that the process of E.coli28LGOX whole-cell catalytic Pidolidone is coupled
By recombinant bacteriumE.coli28LGOX and recombinant bacteriumE.coliThe cell of BL-22AB-YDT proportionally mixes, and is added
Substrate L-lysine and Pidolidone, and 0.5% X-100 is added, it is anti-that whole-cell catalytic is carried out at 37 °C, under the conditions of 200rmp
It answers, per sampling at regular intervals, liquid phase detects the amount of glutaric acid and 2-oxoglutaric acid；
It is preferably as the above method, recombinant bacterium described in step 3E.coli28LGOX and recombinant bacteriumE.coliBL-
The cell proportion of 22AB-YDT is 1:4.
It is preferably as the above method, the molar ratio of substrate Pidolidone and substrate L-lysine described in step 3
1:2.
It is preferably as the above method, surfactant described in step 3 is SDS, Triton X-100, Tween-
One or more of 20, Tween-80.
It is preferably as surfactant, the concentration of L-lysine and Pidolidone is 5-100g/L in step 3.
It is preferably as the above method, catalysis reaction carries out under the conditions of 37 DEG C, speed of agitator 200rpm in step 3.
Beneficial effect
A kind of method of Whole Cell Biocatalysis production glutaric acid, it is real by being overexpressed L-GLOD (LGOX)
The amino acceptor 2-oxoglutaric acid (2-KG) for producing high value using cheap Pidolidone as substrate is showed, has solved full cell and urge
Change L-lysine to be converted into during glutaric acid, the economic problems for the amino acceptor 2-oxoglutaric acid for needing external source addition expensive,
Meanwhile the molar ratio by adjusting substrate Pidolidone and substrate L-lysine, realize that substrate more effectively utilizes.Additionally by
Adjust recombinant bacteriumE.coli28LGOX and recombinant bacteriumE.coliThe cell OD of BL-22AB-YDT600Ratio makes mole of glutaric acid
Yield reaches 68.34%.
Detailed description of the invention
Fig. 1 is the schematic diagram of double cell coupling production glutaric acids；
Fig. 2 is the SDS-PAGE figure that inducing expression LGOX is expressed under different temperatures.
Fig. 3 whole-cell catalytic Pidolidone is converted into the coupling of 2-oxoglutaric acid Yu glutaric acid process.
Specific embodiment
Embodiment 1
It is overexpressed the building of DavBA and gabDT bacterial strain:
(1) it has been overexpressed the recombinant bacterial strain of davBAE.coliBL-22AB is provided by this laboratory, by the recombinant bacterial strainE.coliBL-22AB is made into competent cell.
(2) gabT is synthesized by Jin Weizhi, and restriction enzyme site is NdeI and XhoI and is connected on pACYC carrier, is recombinated
Plasmid pACYC-gabT,
(3) gabD is synthesized by Jin Weiwei intelligence, and restriction enzyme site is NcoI and HindIII, and is connected on pACYC carrier,
Obtain recombinant plasmid pACYC-gabD.
(4) recombinant plasmid pACYC-gabD is obtained into enzyme after restriction enzyme NcoI and HindIII processing recycling
Enzyme site is the segment gabD of NcoI and HindIII, by the processed recombination matter of segment restriction enzyme identical as process
Grain pACYC-gabT is connected, and uses 25 °C of connection 30min of T4DNA ligase.
(5) above-mentioned connection liquid is transferred to Escherichia coliTrans1-T1Competent cell in, be coated on 35mg/L chlorine
The LB plate of chloramphenicol resistance, 37 °C are incubated overnight.
(6) single colonie grown on plate in picking (5), is transferred to the LB culture medium containing 35mg/L chlorampenicol resistant
In, plasmid is then extracted, then carry out digestion verification through restriction enzyme NcoI and HindIII, finally obtained recombinant plasmid
pACYC-gabT-gabD。
(7) recombinant plasmid pACYC-gabT-gabD is transferred to recombinant bacteriumE.coliIn the competent cell of BL-22AB, and
It is coated on the LB plate with 100mg/L amicillin resistance and 35mg/L chlorampenicol resistant, is obtained and expresses
The recombinant bacterial strain of davBA and gabDTE.coliBL-22AB-YDT 。
It is overexpressed the building of LGOX bacterial strain:
LGOX segment is synthesized by Jin Weizhi, and restriction enzyme site is NcoI and BamHI, and is connected on pACYC carrier, obtains weight
Group plasmid pACYC-LGOX.After recombinant plasmid pACYC-LGOX is recycled after restriction enzyme NcoI and BamHI processing, obtain
The segment LGOX for being NcoI and BamHI to restriction enzyme site, by the segment processed plasmid of restriction enzyme identical as process
28a is connected, and uses 25 °C of connection 30min of T4DNA ligase.Above-mentioned connection liquid is transferred to Escherichia coliTrans1-T1Impression
In state cell, it is coated on the LB plate with 50mg/L kalamycin resistance, 37 °C are incubated overnight.Plate in picking previous step
The single colonie of upper growth is transferred in the LB culture medium containing 50mg/L kalamycin resistance, then extracts plasmid, then through limiting
Property restriction endonuclease NcoI and BamHI carry out digestion verification, finally obtained recombinant plasmid 28a-LGOX.By recombinant plasmid 28a-LGOX
It is transferred to recombinant bacteriumE.coliBL21(DE3)Competent cell in, and be coated on 50mg/L kalamycin resistance
On LB plate, the recombinant bacterial strain for expressing LGOX is obtainedE.coli28LGOX 。
Embodiment 2
Induce the optimum temperature screening of LGOX expression
Pick from the plate recombinant bacterial strainE.coliThe single bacterium of 28LGOX falls on the 5ML containing 50mg/L kalamycin resistance
LB shakes in pipe, is transferred in the 100ml LB containing 50mg/L kalamycin resistance after cultivating 6-8h, until OD600=0.6, it is added
The IPTG of 0.5mmol is cultivated in 20 °C, 25 °C, 30 °C, 37 °C of shaking tables respectively, and after 4h, 6000g is centrifuged 5min, and table is obtained
The cell of LGOX is reached.
The obtained cell 100mmolPBS of 5ml is resuspended, broken using cell crushing instrument, broken condition is that super 2s stops
2s, temperature are 4 °C, and power 30%, being crushed the time is 10min, are then centrifuged 20min in 7000g.
The supernatant induced under condition of different temperatures precipitates after measuring protein concentration, is 20ug loading by protein concentration,
It is analyzed through SDS-PAGE, the protein expression situation after obtaining different temperatures induction, as shown in Fig. 2, when 25 °C of inductions, in supernatant
LGOX has apparent overexpression.
Embodiment 3
The process and whole-cell catalytic L-lysine of whole-cell catalytic glutamic acid production 2-oxoglutaric acid are converted into glutaric acid
The method that is coupled of process:
Culture and the recombinant bacterium for being overexpressed LGOX is collected in the way of embodiment 2E.coli28LGOX cell conduct
Catalyst.
Meanwhile picking from the plate recombinant bacterial strainE.coliThe single colonie of BL-22AB-YDT, is inoculated into containing 100mg/L
The 5mlLB of amicillin resistance and 35mg/L chlorampenicol resistant shakes in pipe, after cultivating 6-8h, is transferred to containing 100mg/L ammonia
In parasiticin resistance and the 100ml shaking flask of 35mg/L chlorampenicol resistant, until OD600=0.6, it is added the IPTG of 0.5mmol, 20 °
After C Fiber differentiation 12h, 7000g is centrifuged 5min, obtains recombinant bacterial strainE.coliThe cell of BL-22AB-YDT, and as
Catalyst.
Recombinant bacterium is resuspended respectively using 100mmolPBSE.coli28LGOX andE.coliThe cell of BL-22AB-YDT.
In the reaction system, recombinant bacterium is addedE.coli28LGOX and recombinant bacteriumE.coliThe cell of BL-22AB-YDT, makes
In system,E.coliThe OD of 28LGOX600=5,E.coliThe OD of BL-22AB-YDT600=20, L-sodium: 10g/L, L- rely
Propylhomoserin: 10g/L, X-100:0.5% are sampled at regular intervals, and liquid phase detects the accumulation of glutaric acid and 2-oxoglutaric acid,
Wherein detection method is chromatographic column: Bio-Rad Aminex HPX-87H (300 mm *, 7.8 mm), column temperature: 55 °C, is flowed
Phase: 8 mM H2SO4, flow velocity: 0.6 mL/min, detector: ultraviolet, RID.To 10h, glutaric acid builds up to 3.16g/L, 2- for reaction
KG:6.39g/L, then, 2-oxoglutaric acid are begun to decline, and after reaction to 46h, 2-oxoglutaric acid is depleted, and glutaric acid is built up to
6.14g/L, molar yield 68.34%, concrete condition is as shown in Figure 3.
The method of the present invention withSi Jae ParkEt al. using being overexpressed the recombinant bacterial strain of davAB and gabTDE. coli WL3110Fermented and cultured obtains 1.7g/L glutaric acid, and (molar yield: method 18.92%) is compared, and this method does not need not only
The expensive amino acceptor 2-oxoglutaric acid of external source addition, and the molar yield of glutaric acid improves 49.42%, and side of the present invention
The product that method obtains is easier to isolate and purify.
Claims (6)
1. a kind of method of Whole Cell Biocatalysis production glutaric acid, which comprises the following steps:
Step 1, building is overexpressed the bacterial strain E.coliBL-22AB-YDT of DavBA and gabDT, then picks from the plate recombination
The single colonie of bacterial strain E.coliBL-22AB-YDT is inoculated into anti-containing 100mg/L amicillin resistance and 35mg/L chloramphenicol
Property 5ml LB shake in pipe, cultivate 6-8h after be transferred to containing 100mg/L amicillin resistance and 35mg/L chlorampenicol resistant
100MLLB culture medium in, culture is to OD600=0.6-0.8, centrifugation collection bacterium, obtains recombinant bacterial strain E.coliBL-22AB-YDT
Cell；
Step 2, building is overexpressed the bacterial strain E.coli28LGOX of LGOX, then picks from the plate recombinant bacterial strain
The single colonie of E.coli28LGOX is inoculated into shaking in pipe containing kalamycin resistance and LB, cultivate be transferred to after 6-8h containing card that
In the shaking flask of chloramphenicol resistance and LB, culture to OD600=0.6-0.8, centrifugation collection bacterium, obtains recombinant bacterial strain E.coli28LGOX's
Cell；
Step 3, by the cell of the cell of recombinant bacterium E.coli28LGOX and recombinant bacterium E.coliBL-22AB-YDT proportionally 1:
1-5 mixing, is added substrate L-lysine and Pidolidone, makes the molar ratio 1:0.5-4 of Pidolidone and L-lysine, is added
Surfactant, whole-cell catalytic produce glutaric acid；The cell and recombinant bacterium of recombinant bacterium E.coli28LGOX in step 3
Proportionally 1:5 is mixed the cell of E.coliBL-22AB-YDT；Mole of the substrate Pidolidone and substrate L-lysine
Compare 1:2.
2. the method for Whole Cell Biocatalysis production glutaric acid according to claim 1, it is characterised in that: described in step 1
The construction method for being overexpressed the bacterial strain E.coliBL-22AB-YDT of DavBA and gabDT is: by segment DavB with restriction enzyme site
NdeI and XhoI is connected on carrier 22b, obtains recombinant plasmid 22b-DavB, by segment DavA with restriction enzyme site AgeI and XmaI
It is connected on recombinant plasmid 22b-DavB, obtains recombinant plasmid 22b-DavBA, in addition, by segment gabT with restriction enzyme site NdeI
Be connected on plasmid pACYC with XhoI, obtain recombinant plasmid pACYC-gabT, by segment gabD with restriction enzyme site NcoI and
HindIII is connected on recombinant plasmid pACYC-gabT, obtains recombinant plasmid pACYC-gabT-gabD, by recombinant plasmid 22b-
DavBA and pACYC-gabT-gabD is imported in e. coli bl21 (DE3), and the recombination for expressing DavBA and gabDT is obtained
Bacterial strain E.coliBL-22AB-YDT.
3. the method for Whole Cell Biocatalysis production glutaric acid according to claim 1, it is characterised in that: step 2 building
The method for being overexpressed the bacterial strain E.coli28LGOX of LGOX is: by segment LGOX using NcoI and BamHI as restriction enzyme site, with warp
The expression vector 28a of NcoI with BamHI digestion is connected, and obtains recombinant plasmid 28a-LGOX, and recombinant plasmid is imported E.coli
In the competent cell of BL21, the recombinant bacterium E.coli28LGOX for expressing LGOX is obtained.
4. the method for Whole Cell Biocatalysis production glutaric acid according to claim 1, it is characterised in that: institute in step 3
The surfactant stated is SDS, Triton X-100, Tween-20, one or more of Tween-80.
5. the method for Whole Cell Biocatalysis production glutaric acid according to claim 1, it is characterised in that: L- in step 3
The concentration of lysine and Pidolidone is 5-100g/L.
6. the method for Whole Cell Biocatalysis production glutaric acid according to claim 1, it is characterised in that: urged in step 3
Change reaction to carry out under the conditions of 37 DEG C, speed of agitator 200rpm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610724114.XA CN106367445B (en) | 2016-08-25 | 2016-08-25 | Method for producing glutaric acid by whole-cell biocatalysis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610724114.XA CN106367445B (en) | 2016-08-25 | 2016-08-25 | Method for producing glutaric acid by whole-cell biocatalysis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106367445A CN106367445A (en) | 2017-02-01 |
| CN106367445B true CN106367445B (en) | 2019-08-20 |
Family
ID=57879478
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610724114.XA Active CN106367445B (en) | 2016-08-25 | 2016-08-25 | Method for producing glutaric acid by whole-cell biocatalysis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106367445B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136295B (en) * | 2018-08-17 | 2022-04-15 | 北京化工大学 | Method for biologically synthesizing glutaric acid |
| CN109266664B (en) * | 2018-10-23 | 2022-02-08 | 南京工业大学 | Method for improving stability of glutamate oxidase by using fusion truncated expression strategy |
| CN109295116A (en) * | 2018-11-06 | 2019-02-01 | 南京工业大学 | Method for producing glutaric acid by coupling and catalyzing two cells |
| CN109868297A (en) * | 2019-03-19 | 2019-06-11 | 南京工业大学 | Method for producing glutaric acid by using escherichia coli to express DavA, DavB, GabD, GabT and LGOX |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104109698A (en) * | 2013-04-17 | 2014-10-22 | 上海工业生物技术研发中心 | Enzymic method for producing [alpha]-ketoglutaric acid |
| CN105331642A (en) * | 2015-11-30 | 2016-02-17 | 浙江汇宁生物科技有限公司 | Method for producing alpha-oxoglutarate under catalysis of L-glutamate oxidase |
-
2016
- 2016-08-25 CN CN201610724114.XA patent/CN106367445B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104109698A (en) * | 2013-04-17 | 2014-10-22 | 上海工业生物技术研发中心 | Enzymic method for producing [alpha]-ketoglutaric acid |
| CN105331642A (en) * | 2015-11-30 | 2016-02-17 | 浙江汇宁生物科技有限公司 | Method for producing alpha-oxoglutarate under catalysis of L-glutamate oxidase |
Non-Patent Citations (3)
| Title |
|---|
| Enzymatic production of α-ketoglutaric acid from l-glutamic acid via l-glutamate oxidase;Niu P等;《J Biotechnol》;20140321;第179卷;56-62 |
| L-谷氨酸氧化酶高密度发酵及催化合成α-酮戊二酸;樊祥臣等;《过程工程学报》;20160728;第16卷(第2期);292-297 |
| Metabolic engineering of Escherichia coli for the production of 5-aminovalerate and glutarate as C5 platform chemicals;Park SJ等;《Metab Eng》;20121214;第16卷;42-47 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106367445A (en) | 2017-02-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106367445B (en) | Method for producing glutaric acid by whole-cell biocatalysis | |
| CN107502585A (en) | One plant of bacillus licheniformis engineering bacteria for efficiently synthesizing poly- γ glutamic acid | |
| CN104593442A (en) | A method of producing ectoine by high-density culture of recombinant escherichia coli | |
| MX2014000665A (en) | Method for the production of cellulases by a filamentous fungus adapted to a fermenter having a low volumetric oxygen transfer coefficient kla. | |
| CN103667166A (en) | Escherichia coli for producing adipic acid precursor namely cis,cis-muconic acid and application of escherichia coli | |
| CN109880859A (en) | Method for producing pentanediamine by immobilized lysine decarboxylase | |
| CN107881163A (en) | A kind of heat-resisting nitrile hydratase, engineering bacteria and its application in catalysis nitrile compound hydration reaction generation acid amides | |
| CN104178533B (en) | Method for producing R-3-aminobutanol | |
| CN110157653A (en) | Recombinant escherichia coli for high-yield cyclic adenosine monophosphate and application of recombinant escherichia coli in cyclic adenosine monophosphate synthesis | |
| CN109554386A (en) | A kind of utilizing works Escherichia coli are using Corncob hydrolysate as the method for substrate high yield D- xylonic | |
| CN107541532A (en) | A kind of preparation method of the carboxylic acid of 5 methylpyrazine 2 | |
| CN106011191B (en) | A kind of method of Whole Cell Biocatalysis production 5- aminovaleric acid | |
| CN104152500A (en) | New method of biologically synthesizing (R)-3-hydroxylglutarate monoester | |
| CN104762338A (en) | Method of producing nicotinamide by catalysis of rhodococcus | |
| CN103642745B (en) | A kind of method producing mannitol for raw materials through biotransformation with sucrose | |
| CN106399343B (en) | Glutaric acid biology improves synthetic method | |
| CN103937842B (en) | Method for increasing yield of alpha-oxoglutarate produced through whole-cell transformation | |
| CN108060203B (en) | Method for producing 1, 3-propylene glycol by whole-cell mixed transformation of glycerol | |
| CN101469318B (en) | Synthesis of (R)-styrene glycol by coupling acceleration of (R)-carbonyl reduction enzyme and formic dehydrogenase | |
| CN104830744A (en) | Method for preparing (R)-phenylglycol from SD-AS sequence coupled (R)-carbonyl reductase and glucose dehydrogenase | |
| CN109868297A (en) | Method for producing glutaric acid by using escherichia coli to express DavA, DavB, GabD, GabT and LGOX | |
| CN102936590A (en) | Nitrilase, gene sequence and application method thereof | |
| CN106086082A (en) | A kind of method improveing recombination bacillus coli production 9 decenols | |
| CN104560849A (en) | Constructing method and application of gamma-glutamyl transpeptidase and chaperonin coexpression recombinant plasmid | |
| CN109295116A (en) | Method for producing glutaric acid by coupling and catalyzing two cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | 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 |