CN106367445A - Method for producing glutaric acid by whole-cell biocatalysis - Google Patents
Method for producing glutaric acid by whole-cell biocatalysis Download PDFInfo
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- CN106367445A CN106367445A CN201610724114.XA CN201610724114A CN106367445A CN 106367445 A CN106367445 A CN 106367445A CN 201610724114 A CN201610724114 A CN 201610724114A CN 106367445 A CN106367445 A CN 106367445A
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- ydt
- 28lgox
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- JFCQEDHGNNZCLN-UHFFFAOYSA-N anhydrous glutaric acid Natural products OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 title claims abstract description 77
- 238000004519 manufacturing process Methods 0.000 title abstract description 6
- RTBFRGCFXZNCOE-UHFFFAOYSA-N 1-methylsulfonylpiperidin-4-one Chemical compound CS(=O)(=O)N1CCC(=O)CC1 RTBFRGCFXZNCOE-UHFFFAOYSA-N 0.000 title abstract description 4
- 238000000034 method Methods 0.000 claims abstract description 46
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims abstract description 31
- 241000894006 Bacteria Species 0.000 claims abstract description 20
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims abstract description 17
- 229960002989 glutamic acid Drugs 0.000 claims abstract description 16
- 239000000758 substrate Substances 0.000 claims abstract description 14
- 108090000790 Enzymes Proteins 0.000 claims abstract description 9
- 102000004190 Enzymes Human genes 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims abstract description 7
- 239000004094 surface-active agent Substances 0.000 claims abstract description 6
- 239000004472 Lysine Substances 0.000 claims abstract description 4
- 238000002156 mixing Methods 0.000 claims abstract 2
- 230000001580 bacterial effect Effects 0.000 claims description 31
- 239000013612 plasmid Substances 0.000 claims description 27
- 230000002018 overexpression Effects 0.000 claims description 23
- 239000012634 fragment Substances 0.000 claims description 12
- 108091008146 restriction endonucleases Proteins 0.000 claims description 12
- 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 7
- 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 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 4
- 241000588722 Escherichia Species 0.000 claims description 3
- 229960005091 chloramphenicol Drugs 0.000 claims description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N lysine Chemical compound NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 3
- 229940097572 chloromycetin Drugs 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
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 2
- 229920001213 Polysorbate 20 Polymers 0.000 claims 1
- 230000008676 import Effects 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
- 238000006555 catalytic reaction Methods 0.000 abstract description 7
- 238000000855 fermentation Methods 0.000 abstract description 4
- 230000014509 gene expression Effects 0.000 abstract description 4
- 230000004151 fermentation Effects 0.000 abstract description 3
- 235000019766 L-Lysine Nutrition 0.000 abstract 2
- 101100001670 Emericella variicolor andE gene Proteins 0.000 abstract 1
- 230000007547 defect Effects 0.000 abstract 1
- 238000005265 energy consumption Methods 0.000 abstract 1
- 238000000605 extraction Methods 0.000 abstract 1
- 239000002207 metabolite Substances 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 6
- 230000006698 induction Effects 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
- 238000001514 detection method 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
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 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
- 230000029087 digestion Effects 0.000 description 2
- 239000000835 fiber Substances 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
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 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
- OFOBLEOULBTSOW-UHFFFAOYSA-L Malonate Chemical compound [O-]C(=O)CC([O-])=O OFOBLEOULBTSOW-UHFFFAOYSA-L 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 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
- 239000003599 detergent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 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
- 239000004220 glutamic acid Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 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
- 238000011068 loading method Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 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
- 239000004014 plasticizer Substances 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000001294 propane Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229940073490 sodium glutamate Drugs 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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)
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
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- 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 whole cell organismA method for catalytically producing glutaric acid. 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 biological technical field and in particular to a kind of Whole Cell Biocatalysis produce 1,3-propanedicarboxylic acid method.
Background technology
1,3-propanedicarboxylic acid, another name: glue acid, a, γ-propane dicarboxylic acids, 1,3- propane dicarboxylic acid, can as important industrial chemicals and
Organic intermediate, has a wide range of applications in all fields.In the industry, 1,3-propanedicarboxylic acid can be used for synthesizing polyvinyl chloride, polyester, gathers
Polyester plasticizer of amide and polrvinyl chloride etc., in addition can be used for synthesizing liquid polyester (the molecule knot of improvement pet fiber
Structure, improves the dyeability of pet fiber, improves dye-uptake).At medical aspect, 1,3-propanedicarboxylic acid can be used for synthesizing various sterilizings to be washed
Liquid and medicine, in addition, in life, 1,3-propanedicarboxylic acid can be used for the flue gases such as the ingredients of detergent, the preparation of binding agent, and sulfur-bearing
Washing etc..
At present in the method for synthesizing glutaric acid, there are chemical synthesiss, such as the multi-step synthetic method with gamma-butyrolacton as raw material,
Choice oxidation process with cyclopentanol-Ketocyclopentane as raw material, aoxidizes hydrolysis with dihydrofuran for raw material, by malonate two
Prepare with formaldehyde condensation in the presence of second ammonium etc..But this method raw material is costly, is not easy to obtain and major part has used strong oxidizer, right
The corrosion of equipment is very big.Reaction is complex, and reactions steps are a lot, and yield is not high, and environmental pollution is more serious, is only limited to test
Room, industrial prospect is little.
si jae parkEt al. using the overexpression restructuring of davab and gabtde. coliWl3110 bacterial strain is containing
There are 20g/l glucose, fermentation culture in the culture medium of 10g/l l- lysine and 10g/l 2-oxoglutaric acid, obtained 1.7g/l
1,3-propanedicarboxylic acid.This method needs external source to add expensive amino acceptor 2-oxoglutaric acid, considerably increases production cost, in addition sends out
Ferment method production 1,3-propanedicarboxylic acid molar yield is relatively low, takes longer, reaction system is more complicated, and product separates more difficult.
jake adkinsEt al. using the overexpression restructuring of davab and davdtescherichia coliBacterial strain is with Portugal
Grape sugar is substrate, creates the 1,3-propanedicarboxylic acid of 0.82g/l, this method reaction system is more complicated after fermentation 48h, 1,3-propanedicarboxylic acid mole
Yield is relatively low.
Through retrieval, a kind of method of double cell coupling production 1,3-propanedicarboxylic acids of economical and efficient, there is not been reported.
Content of the invention
For the deficiencies in the prior art, it is an object of the invention to provide a kind of Whole Cell Biocatalysis produce 1,3-propanedicarboxylic acid
Method, the method is cost-effective, economical and efficient.
For solving prior art problem, the technical scheme that the present invention takes is:
A kind of method that Whole Cell Biocatalysis produce 1,3-propanedicarboxylic acid, comprises the following steps:
A kind of method that Whole Cell Biocatalysis produce 1,3-propanedicarboxylic acid, comprises the following steps:
Step 1, builds the bacterial strain of overexpression davba and gabdte.coliBl-22ab-ydt, then picking restructuring from flat board
Bacterial straine.coliThe single bacterium colony of bl-22ab-ydt is inoculated into and resists containing 100mg/l amicillin resistance and 35mg/l chloromycetin
Property 5ml lb shake in pipe, culture 6-8h after be transferred to containing 100mg/l amicillin resistance and 35mg/l chlorampenicol resistant
100mllb culture medium in, cultivate to od600=0.6-0.8, centrifugation collection bacterium, obtain recombinant bacterial straine.coliBl-22ab-ydt's
Cell；
Step 2, builds the bacterial strain of overexpression lgoxe.coli28lgox, then picking recombinant bacterial strain from flat boarde.coliThe single bacterium colony of 28lgox is inoculated into shaking in pipe containing kalamycin resistance and lb, is transferred to containing card after culture 6-8h
In the shaking flask of that chloramphenicol resistance and lb, cultivate to od600=0.6-0.8, centrifugation collection bacterium, obtain recombinant bacterial straine.coli28lgox's
Cell；
Step 3, by recombinant bacteriume.coliThe cell of 28lgox and recombinant bacteriume.coliThe cell of bl-22ab-ydt proportionally 1:
1-5 mixes, and adds substrate l- lysine (5-100g/l) and l- glutamic acid (5-100g/l), makes l- glutamic acid and l- lysine
(5-100g/l) mol ratio is 1:0.5-4, adds surfactant, and whole-cell catalytic produces 1,3-propanedicarboxylic acid.
In said method preferably, in step 1, the construction step of overexpression davba and gabdt bacterial strain is:
Dava and davb fragment is connected with expression vector pet-22b, obtains recombiant plasmid pet22b-davba, by recombiant plasmid
Pet22b-davba importse.coli bl21Competent cell in, obtain the recombinant bacterium of overexpression davbae.colibl-
This recombinant bacterial strain is simultaneously made competent cell by 22ba, gabd and gabt fragment is connected with expression vector pacyc-duet, obtains
To recombiant plasmid pacyc-gabdt, recombiant plasmid is imported recombinant bacteriume.coliIn the competent cell of bl-22ab, obtained
Have expressed the recombinant bacterial strain of davba and gabdte.colibl-22ab-ydt.
In said method preferably, step 2 builds the bacterial strain of overexpression lgoxe.coliThe method of 28lgox is: by piece
Lgox is with ncoi and bamhi as restriction enzyme site for section, is connected with the expression vector 28a crossing through ncoi and bamhi enzyme action, is recombinated
Plasmid 28a-lgox, recombiant plasmid is importede.coliIn the competent cell of bl21, obtain the recombinant bacterium of overexpression lgoxe.coli28lgox.
Step 3, recombinant bacterial straine.coliThe process of bl-22ab-ydt whole-cell catalytic l- lysine and recombinant bacterium
The method that the process of e.coli28lgox whole-cell catalytic l- glutamic acid is coupled
By recombinant bacteriume.coli28lgox and recombinant bacteriume.coliThe cell of bl-22ab-ydt proportionally mixes, and adds substrate
L- lysine and l- glutamic acid, and add 0.5% x-100, in 37 ° of c, carry out whole-cell catalytic reaction under the conditions of 200rmp, often
Sample at regular intervals, the amount of Liquid Detection 1,3-propanedicarboxylic acid and 2-oxoglutaric acid；
As said method preferably, recombinant bacterium described in step 3e.coli28lgox and recombinant bacteriume.colibl-22ab-
The cell proportion of ydt is 1:4.
As said method preferably, the mol ratio of the substrate l- glutamic acid described in step 3 and substrate l- lysine
1:2.
As said method preferably, the surfactant described in step 3 is sds, triton x-100, tween-
One or more of 20, tween-80.
As surfactant preferably, in step 3, the concentration of l- lysine and l- glutamic acid is 5-100g/l.
As said method preferably, in step 3, catalytic reaction, at 37 DEG C, is carried out under the conditions of speed of agitator 200rpm.
Beneficial effect
A kind of method that Whole Cell Biocatalysis produce 1,3-propanedicarboxylic acid, by overexpression l- dglutamic oxidase (lgox) it is achieved that
Produce the amino acceptor 2-oxoglutaric acid (2-kg) of high value with cheap l- glutamic acid for substrate, solve whole-cell catalytic l-
During lysine is converted into 1,3-propanedicarboxylic acid, external source is needed to add the economic problems of expensive amino acceptor 2-oxoglutaric acid, meanwhile,
By adjusting the mol ratio of substrate l- glutamic acid and substrate l- lysine, realize substrate and more effectively utilize.Additionally by regulation
Recombinant bacteriume.coli28lgox and recombinant bacteriume.coliThe cell od of bl-22ab-ydt600Ratio, makes the molar yield of 1,3-propanedicarboxylic acid
Reach 68.34%.
Brief description
Fig. 1 is the schematic diagram that double cell couplings produce 1,3-propanedicarboxylic acid；
Fig. 2 is the sds-page figure of abduction delivering lgox expression under different temperatures.
Fig. 3 whole-cell catalytic l- glutamic acid rotating turns to coupling of 2-oxoglutaric acid and 1,3-propanedicarboxylic acid process.
Specific embodiment
Embodiment 1
Overexpression davba and the structure of gabdt bacterial strain:
(1) the overexpression recombinant bacterial strain of davbae.coliBl-22ab is provided by this laboratory, by this recombinant bacterial straine.coliBl-22ab makes 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 the big only intelligence of gold, and restriction enzyme site is ncoi and hindiii, and is connected on pacyc carrier, obtains
Recombiant plasmid pacyc-gabd.
(4) recombiant plasmid pacyc-gabd is processed after recovery through restricted enzyme ncoi and hindiii, obtain enzyme
Enzyme site is fragment gabd of ncoi and hindiii, by this fragment and the restructuring matter crossed through identical restriction enzyme ferment treatment
Grain pacyc-gabt is connected, and connects 30min using 25 ° of c of t4dna ligase.
(5) above-mentioned connection liquid is proceeded to escherichia colitrans1-t1Competent cell in, be coated on 35mg/l chlorine
The lb flat board of chloramphenicol resistance, 37 ° of c incubated overnight.
(6) single bacterium colony growing on picking (5) middle plateform, is transferred to the lb culture medium containing 35mg/l chlorampenicol resistant
In, then extract plasmid, then carry out digestion verification through restricted enzyme ncoi and hindiii, finally obtained recombiant plasmid
pacyc-gabt-gabd.
(7) recombiant plasmid pacyc-gabt-gabd is proceeded to recombinant bacteriume.coliIn the competent cell of bl-22ab, and
It is coated on the lb flat board with 100mg/l amicillin resistance and 35mg/l chlorampenicol resistant, obtained overexpression
The recombinant bacterial strain of davba and gabdte.colibl-22ab-ydt .
The structure of overexpression lgox bacterial strain:
Lgox fragment is synthesized by Jin Weizhi, and restriction enzyme site is ncoi and bamhi, and is connected on pacyc carrier, obtains matter of recombinating
Grain pacyc-lgox.After recombiant plasmid pacyc-lgox is reclaimed after restricted enzyme ncoi and bamhi process, obtain enzyme
Enzyme site is fragment lgox of ncoi and bamhi, by this fragment and the plasmid 28a phase crossed through identical restriction enzyme ferment treatment
Even, connect 30min using 25 ° of c of t4dna ligase.Above-mentioned connection liquid is proceeded to escherichia colitrans1-t1Competent cell
In, it is coated on the lb flat board with 50mg/l kalamycin resistance, 37 ° of c incubated overnight.Grow on picking previous step middle plateform
Single bacterium colony, be transferred in the lb culture medium containing 50mg/l kalamycin resistance, then extract plasmid, then through restriction enzyme
Enzyme ncoi and bamhi carries out digestion verification, finally obtained recombiant plasmid 28a-lgox.Recombiant plasmid 28a-lgox is proceeded to weight
Group bacteriumE.colibl21(de3)Competent cell in, and be coated on the lb flat board with 50mg/l kalamycin resistance
On, obtain the recombinant bacterial strain of overexpression lgoxe.coli28lgox .
Embodiment 2
The optimum temperature screening of induction lgox expression
Picking recombinant bacterial strain from flat boarde.coliThe single bacterium colony of 28lgox is to the 5ml lb containing 50mg/l kalamycin resistance
Shake in pipe, be transferred in the 100ml lb containing 50mg/l kalamycin resistance, to od after culture 6-8h600=0.6, add
The iptg of 0.5mmol, respectively in 20 ° of c, 25 ° of c, 30 ° of c, cultivate in 37 ° of c shaking tables, after 4h, 6000g is centrifuged 5min, obtains table
Reach the cell of lgox.
The cell obtaining is resuspended with the 100mmolpbs of 5ml, crushed using cell crushing instrument, broken condition stops for super 2s
2s, temperature is 4 ° of c, and power is 30%, and the broken time is 10min, is then centrifuged 20min in 7000g.
The supernatant precipitation of induction under condition of different temperatures, after measurement protein concentration, is 20ug loading by protein concentration,
Through sds-page analysis, obtain the protein expression situation after different temperatures induction, as shown in Fig. 2 during 25 ° of c inductions, in supernatant
Lgox has obvious overexpression.
Embodiment 3
Whole-cell catalytic glutamic acid produces the process of 2-oxoglutaric acid and whole-cell catalytic l- lysine is converted into the mistake of 1,3-propanedicarboxylic acid
The method that journey is coupled:
Mode according to embodiment 2 is cultivated and is collected the recombinant bacterium of overexpression lgoxe.coli28lgox cell is as catalysis
Agent.
Meanwhile, picking recombinant bacterial strain from flat boarde.coliThe single bacterium colony of bl-22ab-ydt, is inoculated into containing 100mg/l
The 5mllb of amicillin resistance and 35mg/l chlorampenicol resistant shakes in pipe, after culture 6-8h, is transferred to containing 100mg/l ammonia
In the 100ml shaking flask of benzylpcnicillin resistance and 35mg/l chlorampenicol resistant, to od600=0.6, the iptg of addition 0.5mmol, 20 °
After c inducing culture 12h, 7000g, it is centrifuged 5min, obtain recombinant bacterial straine.coliThe cell of bl-22ab-ydt, and as
Catalyst.
Using 100mmolpbs resuspended recombinant bacterium respectivelye.coli28lgox ande.coliThe cell of bl-22ab-ydt.
In reaction system, add recombinant bacteriume.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 glutamate: 10g/l, l- rely
Propylhomoserin: 10g/l, x-100:0.5%, sample at regular intervals, the accumulation of Liquid Detection 1,3-propanedicarboxylic acid and 2-oxoglutaric acid,
Wherein detection method is chromatographic column: bio-rad aminex hpx-87h (300 mm * 7.8 mm), column temperature: 55 ° of c, flowing
Phase: 8 mm h2so4, flow velocity: 0.6 ml/min, detector: ultraviolet, rid.React to 10h, 1,3-propanedicarboxylic acid builds up to 3.16g/l, 2-
Kg:6.39g/l, subsequently, 2-oxoglutaric acid begins to decline, and reacts to 46h, 2-oxoglutaric acid is depleted, 1,3-propanedicarboxylic acid builds up to
6.14g/l, molar yield is 68.34%, and concrete condition is as shown in Figure 3.
The inventive method withsi jae parkEt al. using the overexpression recombinant bacterial strain of davab and gabtde. coli wl3110Fermentation culture obtains 1.7g/l 1,3-propanedicarboxylic acid, and (molar yield: method 18.92%) is compared, this method does not only need
External source adds expensive amino acceptor 2-oxoglutaric acid, and the molar yield of 1,3-propanedicarboxylic acid improves 49.42%, and side of the present invention
The product that method obtains is more easy to isolate and purify.
Claims (8)
1. a kind of Whole Cell Biocatalysis produce the method for 1,3-propanedicarboxylic acid it is characterised in that comprising the following steps:
Step 1, builds the bacterial strain of overexpression davba and gabdte.coliBl-22ab-ydt, then picking restructuring from flat board
Bacterial straine.coliThe single bacterium colony of bl-22ab-ydt is inoculated into and resists containing 100mg/l amicillin resistance and 35mg/l chloromycetin
Property 5ml lb shake in pipe, culture 6-8h after be transferred to containing 100mg/l amicillin resistance and 35mg/l chlorampenicol resistant
100mllb culture medium in, cultivate to od600=0.6-0.8, centrifugation collection bacterium, obtain recombinant bacterial straine.coliBl-22ab-ydt's
Cell；
Step 2, builds the bacterial strain of overexpression lgoxe.coli28lgox, then picking recombinant bacterial strain from flat boarde.coliThe single bacterium colony of 28lgox is inoculated into shaking in pipe containing kalamycin resistance and lb, be transferred to after culture 6-8h containing card that
In the shaking flask of chloramphenicol resistance and lb, cultivate to od600=0.6-0.8, centrifugation collection bacterium, obtain recombinant bacterial straine.coli28lgox's is thin
Born of the same parents；；
Step 3, by recombinant bacteriume.coliThe cell of 28lgox and recombinant bacteriume.coliThe cell of bl-22ab-ydt proportionally 1:
1-5 mixes, and adds substrate l- lysine and l- glutamic acid, makes l- glutamic acid be 1:0.5-4 with the mol ratio of l- lysine, adds
Surfactant, whole-cell catalytic produces 1,3-propanedicarboxylic acid.
2. Whole Cell Biocatalysis according to claim 1 produce 1,3-propanedicarboxylic acid method it is characterised in that: described in step 1
The bacterial strain of overexpression davba and gabdte.coliThe construction method of bl-22ab-ydt is: by fragment davb with restriction enzyme site
Ndei and xhoi is connected on carrier 22b, obtains recombiant plasmid 22b-davb, by fragment davb with restriction enzyme site agei and xmai
It is connected on recombiant plasmid 22b-davb, obtain recombiant plasmid 22b-davba, in addition, by fragment gabt with restriction enzyme site ndei
Be connected on plasmid pacyc with xhoi, obtain recombiant plasmid pacyc-gabt, by fragment gabd with restriction enzyme site ncoi and
Hindiii is connected on recombiant plasmid pacyc-gabt, obtains recombiant plasmid pacyc-gabt- gabd, by recombiant plasmid 22b-
Davba and pacyc-gabt- gabd imports escherichia coliBl21(de3)In, obtain the restructuring of overexpression davba and gabdt
Bacterial straine.colibl-22ab-ydt.
3. Whole Cell Biocatalysis according to claim 1 produce 1,3-propanedicarboxylic acid method it is characterised in that: step 2 builds
The bacterial strain of overexpression lgoxe.coliThe method of 28lgox is: by fragment lgox with ncoi and bamhi as restriction enzyme site, with warp
The expression vector 28a that ncoi with bamhi enzyme action is crossed is connected, and obtains recombiant plasmid 28a-lgox, recombiant plasmid is importede.coli bl21Competent cell in, obtain the recombinant bacterium of overexpression lgoxe.coli28lgox.
4. Whole Cell Biocatalysis according to claim 1 produce 1,3-propanedicarboxylic acid method it is characterised in that: weight in step 3
Group bacteriume.coliThe cell of 28lgox and recombinant bacteriume.coliThe proportionally 1:5 mixing of the cell of bl-22ab-ydt.
5. Whole Cell Biocatalysis according to claim 1 produce 1,3-propanedicarboxylic acid method it is characterised in that: institute in step 3
The substrate l- glutamic acid stated and mol ratio 1:2 of substrate l- lysine.
6. Whole Cell Biocatalysis according to claim 1 produce 1,3-propanedicarboxylic acid method it is characterised in that: institute in step 3
The surfactant stated is sds, triton x-100, one or more of tween-20, tween-80.
7. Whole Cell Biocatalysis according to claim 1 produce 1,3-propanedicarboxylic acid method it is characterised in that: l- in step 3
The concentration of lysine and l- glutamic acid is 5-100g/l.
8. Whole Cell Biocatalysis according to claim 1 produce 1,3-propanedicarboxylic acid method it is characterised in that: urge in step 3
Change reaction at 37 DEG C, carry out under the conditions of speed of agitator 200rpm.
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| CN109266664A (en) * | 2018-10-23 | 2019-01-25 | 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 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109136295A (en) * | 2018-08-17 | 2019-01-04 | 北京化工大学 | A kind of method of biosynthesis glutaric acid |
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| 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 |
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