CN109280677A - Method for co-production of 5-keto-D-gluconic acid and 4-chloro-3-hydroxy ethyl butyrate - Google Patents
Method for co-production of 5-keto-D-gluconic acid and 4-chloro-3-hydroxy ethyl butyrate Download PDFInfo
- Publication number
- CN109280677A CN109280677A CN201811247818.8A CN201811247818A CN109280677A CN 109280677 A CN109280677 A CN 109280677A CN 201811247818 A CN201811247818 A CN 201811247818A CN 109280677 A CN109280677 A CN 109280677A
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- CN
- China
- Prior art keywords
- gluconic acid
- chloro
- keto
- coproduction
- ethyl butyrate
- 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.)
- Pending
Links
- IZSRJDGCGRAUAR-MROZADKFSA-N 5-dehydro-D-gluconic acid Chemical compound OCC(=O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O IZSRJDGCGRAUAR-MROZADKFSA-N 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 28
- OBNCKNCVKJNDBV-UHFFFAOYSA-N butanoic acid ethyl ester Natural products CCCC(=O)OCC OBNCKNCVKJNDBV-UHFFFAOYSA-N 0.000 title claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 title description 2
- 101710088194 Dehydrogenase Proteins 0.000 claims abstract description 24
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 claims abstract description 16
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 claims abstract description 16
- 239000000174 gluconic acid Substances 0.000 claims abstract description 16
- 229950006191 gluconic acid Drugs 0.000 claims abstract description 16
- 235000012208 gluconic acid Nutrition 0.000 claims abstract description 16
- 239000002994 raw material Substances 0.000 claims abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims description 44
- 108090000854 Oxidoreductases Proteins 0.000 claims description 28
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 claims description 26
- 102000004316 Oxidoreductases Human genes 0.000 claims description 24
- RGHNJXZEOKUKBD-SQOUGZDYSA-M D-gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O RGHNJXZEOKUKBD-SQOUGZDYSA-M 0.000 claims description 22
- 102000004190 Enzymes Human genes 0.000 claims description 22
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- 239000012071 phase Substances 0.000 claims description 13
- AEQDJSLRWYMAQI-UHFFFAOYSA-N 2,3,9,10-tetramethoxy-6,8,13,13a-tetrahydro-5H-isoquinolino[2,1-b]isoquinoline Chemical compound C1CN2CC(C(=C(OC)C=C3)OC)=C3CC2C2=C1C=C(OC)C(OC)=C2 AEQDJSLRWYMAQI-UHFFFAOYSA-N 0.000 claims description 10
- 239000000176 sodium gluconate Substances 0.000 claims description 10
- 235000012207 sodium gluconate Nutrition 0.000 claims description 10
- 229940005574 sodium gluconate Drugs 0.000 claims description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 9
- 239000012074 organic phase Substances 0.000 claims description 8
- 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 claims description 6
- 241000589220 Acetobacter Species 0.000 claims description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 6
- 238000006555 catalytic reaction Methods 0.000 claims description 6
- 229910052739 hydrogen Inorganic materials 0.000 claims description 6
- 241000589232 Gluconobacter oxydans Species 0.000 claims description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 5
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 5
- -1 ethyl caprilate Chemical compound 0.000 claims description 5
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 claims description 4
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- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 4
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- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 4
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- 150000002148 esters Chemical class 0.000 claims description 3
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- NAOLWIGVYRIGTP-UHFFFAOYSA-N 1,3,5-trihydroxyanthracene-9,10-dione Chemical compound C1=CC(O)=C2C(=O)C3=CC(O)=CC(O)=C3C(=O)C2=C1 NAOLWIGVYRIGTP-UHFFFAOYSA-N 0.000 claims description 2
- 241000589212 Acetobacter pasteurianus Species 0.000 claims description 2
- 241000222173 Candida parapsilosis Species 0.000 claims description 2
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical group CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 claims description 2
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 2
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 claims description 2
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- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical compound CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 claims description 2
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims 3
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims 2
- 241000235649 Kluyveromyces Species 0.000 claims 2
- LDSBAJNSERBHAR-UHFFFAOYSA-N 2-chloroethyl 3-hydroxybutanoate Chemical compound CC(O)CC(=O)OCCCl LDSBAJNSERBHAR-UHFFFAOYSA-N 0.000 claims 1
- 239000007986 glycine-NaOH buffer Substances 0.000 claims 1
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- XNGIFLGASWRNHJ-UHFFFAOYSA-N o-dicarboxybenzene Natural products OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 claims 1
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- 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 10
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- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 4
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
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- AKDAXGMVRMXFOO-UHFFFAOYSA-N 4-chloro-3-hydroxybutanoic acid Chemical compound ClCC(O)CC(O)=O AKDAXGMVRMXFOO-UHFFFAOYSA-N 0.000 description 1
- IZSRJDGCGRAUAR-MROZADKFSA-M 5-dehydro-D-gluconate Chemical compound OCC(=O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O IZSRJDGCGRAUAR-MROZADKFSA-M 0.000 description 1
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- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
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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
- C12P7/58—Aldonic, ketoaldonic or saccharic acids
-
- 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
- C12P7/62—Carboxylic acid esters
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Abstract
The invention discloses a method for coproducing 5-keto-D-gluconic acid and 4-chloro-3-hydroxy ethyl butyrate, which adopts gluconic acid and 4-chloro-3-ethyl acetoacetate as raw materials, and reduces 4-chloro-3-ethyl acetoacetate to 4-chloro-3-hydroxy ethyl butyrate while oxidizing gluconic acid to generate 5-keto-D-gluconic acid through a double-enzyme coupling system consisting of gluconic acid dehydrogenase and carbonyl reductase; the system is also suitable for other alcohol dehydrogenase and carbonyl reductase, and is an economic, convenient and effective biological catalytic system with wide applicability.
Description
Technical field
The present invention relates to biocatalysis technology fields, and in particular to a kind of coproduction 5- keto-D-gluconic acid and the chloro- 3- of 4-
The method of 3-hydroxyethyl butyrate.
Background technique
5- keto-D-gluconic acid (5-keto-D-gluconicacid, 5KGA) is that gluconic acid is further de- at 5-
The Mek-Tol Unit compound that hydrogen is formed, can be with synthesising biological based platform molecule 2,5-furandicarboxylic acid, and food after catalysis oxidation
Product additive, medicine resolving agent L (+)-tartaric acid and glycolic.
Early in nineteen twenty-two, Germany scientist Kiliani etc. proposes nitric acid chemical method and prepares 5KGA for oxidizing glucose,
Yield is only 10 ~ 12%, and the process requirement consumes a large amount of soda acid, and product isolates and purifies process complexity and time-consuming.Compare and
Speech, the bioanalysis property of can choose to the position the 5- hydroxyl dehydrogenation oxidation of gluconic acid, thus highly selective acquisition 5KGA.1880
Year, Harvard University professor Boutroux is just prepared for 5KGA using acetobacter oxidizing glucose for the first time, but yield is lower.In recent years
Come, researchers optimize the metabolic pathway in bacterium and regulate and control fermentation means also by gene recombination technology can be improved 5KGA yield
(woods Jianping etc., CN201510480880.1), also makes some progress, but its process is relative complex, and inefficiency.
Currently, Cao Fei etc. reports one by external enzymatic process, there is high dependency using to coenzyme NAD P+
Gluconate dehydrogenase (Ga5DH), to realize the high efficiency preparation method (CN201711171003.1) of 5KGA.This method efficiency
Height, flexibility ratio are big, strong operability.The coenzyme circulating system for wherein using NAD (P) H oxidizing ferment, can be by the NAD (P) of generation
H is oxidized to NADP+, while producing and converting water for cosubstrate oxygen, realizes the in-situ regeneration of coenzyme to a certain extent.But
During this, there is competition for the oxidation of NAD (P) H in Ga5DH and NAD (P) H oxidizing ferment, wherein NAD (P) H oxidizing ferment and NAD
(P) H joint efficiency is lower, and concentration of the substrate oxygen in water is lower and is difficult to improve, and causes largely to exist in reaction process
The reaction of Ga5DH catalyst body is reverse to be carried out, and 5KGA is reduced into the situation of gluconic acid, as reaction carries out to a certain extent
When, reaction tends to balance, and remaining gluconic acid can not be further converted to 5KGA, is just difficult to realize the complete of gluconic acid at this time
Conversion.Therefore, the external enzymatic preparation of 5KGA realizes the original position of coenzyme there is still a need for more particularly suitable coenzyme circulating system is found
Regeneration and the complete conversion of substrate.
Matched coenzyme circulating system needs needed for 5KGA preparation based on external enzymatic are met the following requirements: 1,
NAD (P) H can be oxidized to NAD (P)+；2, reaction bottom/product is not reacted with main reaction bottom/product, and is easily isolated；3, it reacts
Substrate solubility can be much larger than gluconic acid, in order to the progress of main reaction forward direction.In recent years, using carbonyl acyl reductase biocatalysis
Method asymmetric reduction preparation 4- chloro-3-hydroxyl ethyl butyrate (4-chloro-3-hydroxybutanoate, (S)-CHBE) by
Common concern.It can reach 100% theory using the asyininetric hydrogenation of carbonyl acyl reductase biocatalysis carbonyl ester type compound
Conversion ratio, and prepare in its reduction (S) simultaneous oxidation NADPH during-CHBE, generate a large amount of NADP+, and this reaction can be in having
It carries out in body system, if the NADP+ regenerating system common with gluconate dehydrogenase, can be carried out in diphasic system, realized
Bottom/product and the separation of main reaction bottom/product are assisted, and wherein (S)-CHBE be important drug chirality platform chemicals,
As the critical medication intermediate of manufacture statins, the chiral drug needed for other groups generate can be imported.
Summary of the invention
For present in the chloro- 3- ethyl acetoacetate preparation process of 5- keto-D-gluconic acid and 4- reported at present
Defect, the purpose of the present invention is to provide the chloro- 3- acetoacetates of the coproduction 5- keto-D-gluconic acid and 4- of a kind of simple and effective
The method of ethyl ester, and reduce the resource consumption in preparation process.
To achieve the goals above, the technical solution adopted by the present invention are as follows:
The present invention using external multienzyme catalysis process coproduction 5KGA with (S)-CHBE, enable gluconate dehydrogenase at NAD (P)+It deposits
It is 5KGA that gluconic acid can be catalyzed under, and generates NAD (P) H and H+；It is added after carbonyl acyl reductase in system simultaneously,
Carbonyl acyl reductase is catalyzed COBE in the presence of NAD (P) H and generates CHBE, and synchronous by NAD (P) H dehydrogenation formation NAD (P)+, realize
Coenzyme in-situ regeneration, gluconic acid it is complete conversion and 5KGA with (S)-CHBE effective acquisition, reaction principle is shown below:
。
A kind of method of coproduction 5- keto-D-gluconic acid and 4- chloro-3-hydroxyl ethyl butyrate, this method is with glucose
Acid and the chloro- 3- ethyl acetoacetate of 4- are raw material, with the whole cell containing gluconate dehydrogenase, carbonyl acyl reductase or contain Portugal
Grape saccharic acid dehydrogenase, the immobilized cell of carbonyl acyl reductase or gluconate dehydrogenase, carbonyl acyl reductase resolvase be catalysis
Agent, in the reaction system, by NAD (P)+Or NAD+As hydrogen acceptor, catalysis preparation 5- keto-D-gluconic acid and the chloro- 3- hydroxyl of 4-
Base ethyl butyrate.
Heretofore described gluconate dehydrogenase is as expressed by the recombinant bacterium containing gluconate dehydrogenase gene, institute
The gluconate dehydrogenase gene stated derives from Gluconobacter oxydans (Gluconobacter oxvdans), or
Gluconobacter suboxydans (weak glucose oxidation bacteria) or Acetobacter pasteurianus (Pasteur's vinegar bar
Bacterium) or Acetobacter ascendens (climbing film acetobacter) or Streptococcus suis (Streptococcus suis), or
Klebsiellasp (Klebsiella) or Penicillium chrysogenum (penicillium chrysogenum) etc. other contain glucose
The strain of acidohydrogenase, the gluconate dehydrogenase is by the recombinant bacterium containing the geneE.coliBL21 (DE3) is expressed,
Expression vector is selected but is not limited to pet-28a (+).
Heretofore described carbonyl acyl reductase is as expressed by the recombinant bacterium containing carbonyl acyl reductase gene, the carbonyl acyl
Reductase gene derives from Streptomyces coelicolor(Streptomyces Syancus) or Candida albicans (Candida
Albicans) or Candida magnoliae(candida magnoliae) or Kluyveromyces aestuarii(ooze gram
Tie up yeast in Shandong) Saccharomyces cerevisiae(saccharomyces cerevisiae), the flat nearly sliding false silk ferment of Candida parapsilosis(
It is female), the flat Rhodococcus sp of Rhodococcus erythropolis() etc. other contain the species of carbonyl acyl reductase.The carbonyl acyl is also
Protoenzyme is expressed by the recombinant bacterium E.coliBL21 (DE3) containing the gene, and expression vector is selected but is not limited to pet-28a (+).
Catalystic converter system is water phase or water phase and organic heterogeneous system mixed in the present invention, and wherein water phase can
For water or phosphate-buffered or the systems such as Tris-HCl buffering or Glycine-NaOH buffering；Wherein organic phase can be second
The esters such as acetoacetic ester, butyl acetate, ethyl caprilate, neck dibatyl phithalate, the alkane such as n-hexane, heptane, octane, dichloromethane
The halogenated hydrocarbons such as alkane, chloroform, one or more of ethers such as ether, isopropyl ether；Wherein water phase can be with organic Phase Proportion
10:0-1:9。
Catalystic converter system forms in the present invention are as follows: water phase volume is 1-10mL, pH 6.0-10.0, organic phase volume
For 0-10mL, 1.5-150g/L sodium gluconate, the chloro- 3- ethyl acetoacetate of 4- of 3-300g/L, 50-5000U glucose
The NAD (P)+or NAD of acidohydrogenase, the carbonyl acyl reductase of 70-8000U and 0.05-0.2mmol/L+。
Reaction condition of the present invention are as follows: 25-45 DEG C of reaction temperature, speed of agitator 180-280rpm, reaction time 1-24h.
Compared with prior art, the present invention has the advantage that
1, technical solutions according to the invention generate 5KGA and (S)-CHBE using enzyme law catalysis, and selectivity is high, and reaction temperature is low,
High-efficient, low energy consumption.
2, use in conjunction carbonyl acyl reductase of the present invention is not only able to achieve NAD (P)+Regeneration, co-products isolate and purify mutually not
It influences, and enables gluconic acid conversion ratio up to 99% or more, and obtain (S)-CHBE of high-purity simultaneously.
3, technical solutions according to the invention process is simple, and coenzyme can be recycled during enzymatic, reacts more green
Colour circle is protected.
Detailed description of the invention
Fig. 1, gluconate dehydrogenase SDS-PAGE electrophoresis；
Fig. 2, carbonyl reductase SDS-PAGE electrophoresis；
Fig. 3, embodiment 6 react initial high-efficient liquid phase chromatogram；
Fig. 4, the reaction of embodiment 6 terminate high-efficient liquid phase chromatogram；
Fig. 5, embodiment 6 react initial gas chromatogram；
Fig. 6, the reaction of embodiment 6 terminate gas chromatogram.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, content described in embodiment is merely to illustrate
The present invention, without the present invention described in detail in claims should will not be limited.
Embodiment 1: it derives fromGluconobacteroxydansGluconate dehydrogenase gene (gdh) clone
GluconobacteroxydansIt is purchased from China General Microbiological culture presevation administrative center (CGMCC 1.637).Oxidation
Gluconobacter suboxydans culture uses culture medium prescription (g/L) are as follows: glucose 100, yeast extract 10, and calcium carbonate 20, agar 15,
It is dissolved completely in deionized water, adjusts pH to 6.8, constant volume, high pressure steam sterilization.
Gluconobacter oxvdans are inoculated in LB liquid medium, 37 DEG C of cultures to logarithmic growth phase use
DNAKit (TIANGEN, China) extracts streptococcus thermophilus genome.Primer used in construction of expression vector adds restriction enzyme site,
Primer sequence is as follows:
Upstream primer (gluconate dehydrogenase-sense contains EcoRI) are as follows:
5’-CCGGAATTCATGAGTACCTCTCTTTTTG-3’
Downstream primer (gluconate dehydrogenase-anti contains HindIII) are as follows:
3'-CCCAAGCTTTCAAAGAGAGACCGTAATC-5'
All primers are synthesized by Suzhou Jin Weizhi Biotechnology Co., Ltd.The PCR condition of gene is 94 DEG C of denaturation 5min, is pressed
Following parameter cyclic 30 times: 95 DEG C of denaturation 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 30s.Last 72 DEG C of extensions 6min.Reaction institute
Obtained product uses 0.8% agarose gel electrophoresis to analyze result respectively.Confirmation clip size is being imaged just through gel imaging system
After really, mesh is recycled using the DNA purification and recovery kit (TaKaRaAgaroseGelDNAPurification) of TaKaRa company
Segment be used for recombinant expression carrier pET-28a-gdhBuilding.
Embodiment 2: recombinant expression carrier pET-28a-gdhBuilding
With EcoRI and HindIII difference digestion (being purchased from Novagen Merck China) and expand containing there are two the mesh of restriction enzyme site
Gene, the target fragment and expression vector of glue recycling double digestion respectively, by the expression vector pET-28a and mesh of double digestion
Gene (indexed number be NC_019396.1) in Genbank stayed overnight with T4-DNA ligase (being purchased from TaKaRa company)
Connection, obtains recombinant vector pET-28a-gdh；In the e. coli bl21 competent cell that the connection product of 10 μ L is added,
30min, 42 DEG C of heat shock 90s are placed on ice.2min is placed on ice.The 0.45mLLB culture medium of preheating is added.220rpm, 37 DEG C,
1h.200 μ L bacterium solutions are added on the plate of the kanamycins containing 30 μ g/mL, 37 DEG C are incubated overnight 12-16h, obtain recombinant bacteriumE.coliBL21 (contains pET-28a-gdh)。
Embodiment 3: from ScheffersomycesstipitisCBS6054 carbonyl acyl reductase gene (sou2)
Source and acquisition
Present embodiments providing coding has the active polynucleotide molecule of carbonyl acyl reductase, nucleic acid molecule coding
Indexed number is XP-001387287 in Genbank, and artificial synthesized by Suzhou Jin Weizhi Biotechnology Co., Ltd.
Embodiment 4: recombinant expression carrier: pET-28a-sou2Building
With EcoRI and NcoI difference digestion (being purchased from Novagen Merck China) and containing there are two the target gene of restriction enzyme site (realities
Apply artificial synthesized acquisition in example 3), glue recycles the target fragment and expression vector of double digestion respectively, by the expression of double digestion
Carrier pET-28a and target gene are placed on ice by the e. coli bl21 competent cell of the connection product addition of 10 μ L
30min, 42 DEG C of heat shock 90s.2min is placed on ice.The 0.45mLLB culture medium of preheating is added.220rpm, 37 DEG C, 1h.By 200 μ
L bacterium solution is added on the plate of the kanamycins containing 30 μ g/mL, and 37 DEG C are incubated overnight 12-16h, obtain recombinant bacterium
E.coliBL21 (contains pET-28a-sou2)。
Embodiment 5: the fermentation of recombinant bacterium and the preparation of lyophilized cells
Cultivate recombinant bacterium (recombinant bacterium constructed in embodiment 2 and embodiment 4) in the fermenter.The seed culture medium of fermentation uses
LB culture medium.Fermentation medium is TB culture medium: yeast extract 25g/L, peptone 15g/L, NaCl10g/L, glucose 2g/
L, lactose 3g/L.PH is adjusted to 7.2 before fermentation medium sterilizes, in 110 DEG C of sterilizing 20min.When fermentation starts, by 1.5% inoculation
Amount inoculation OD600About 1.0 fresh seeds liquid is into fermentor.The temperature of fermentation liquid is controlled at 30 DEG C and maintains dissolved oxygen in 10-
30%.8000rpm is centrifuged 10min and collects thallus after fermentation, weighs wet cell weight.By 0.85% physiological saline of wet cell
It places after washing time and is freezed in -80 DEG C of refrigerators, then be placed in freeze dryer be freeze-dried and can be prepared by lyophilized cells.
Lyophilized cells are sufficiently suspended with buffer, after ultrasonication, 10000rpm/min, 4 DEG C of centrifugation 10min are taken
Clearly.The measurement of gluconate dehydrogenase enzyme activity: enzyme reaction system includes 100mMTris-HCl buffering (pH8.0), 10mM glucose
Sour sodium, 1mMNAD (P)+, 30 DEG C, the rising of light absorption value is measured at 340nm.Enzyme activity is defined as generating 1 μm of olNADPH in per minute
Required enzyme amount is an enzyme-activity unit U.Albumen is measured using Brandford method.The results show that control bacteriumE.coliThe specific enzyme activity of BL21 (containing pET-28a) is 0, and recombinant bacterium E.coliBL21(contains pET-28a-gdh) specific enzyme activity be
20U/mg。
The measurement of carbonyl acyl reductase enzyme activity: enzyme reaction system includes 100mMTris-HCl buffering (pH8.0),
0.2mMNADPH, measures the decline of light absorption value by 30 DEG C at 340nm.Enzyme activity is defined as consuming 1umolNAD (P) H institute in per minute
The enzyme amount needed is an enzyme-activity unit U.Albumen is measured using Brandford method.As the result is shown, bacterium is compareedE.coliThe specific enzyme activity of BL21 (containing pET-28a) is 0.2U/mg, and recombinant bacteriumE.coliBL21(contains pET-28a-sou2) ratio
Enzyme activity is 15U/mg.
Embodiment 6: aqueous phase system utilizes crude enzyme liquid coproduction 5- keto-D-gluconic acid and (S)-CHBE
In the pH8.0Tris-HCl buffering of each 10g of lyophilized cells in Example 5, the 20mL being suspended in.Use high pressure homogenizer
(-20℃、8.0×107Pa) cell is crushed.For clasmatosis liquid after 4 DEG C, 10000rpm/min centrifugation 30min, it is heavy to abandon
It forms sediment, supernatant is the crude enzyme liquid containing gluconate dehydrogenase and carbonyl acyl reductase.Reaction system are as follows: 50U gluconic acid dehydrogenation
Enzyme, 100U carbonyl acyl reductase, the chloro- 3- acetoacetate second of sodium gluconate 100mmol/L, NAD (P)+0.2mmol/L and 4-
Ester 150mmol/L reacts 12h under the conditions of 30 DEG C of reaction temperature, revolving speed 200rpm in the Tris-HCl of 10 mL pH 8.0,
And it is sampled on demand during the reaction.After reaction, centrifugation goes to precipitate, and isometric ethyl acetate is added in supernatant and is extracted
It takes, (S)-CHBE is obtained after organic phase is spin-dried for, appropriate calcium chloride is added in water phase, after completely dissolution, 5- ketone group-D- is obtained after being spin-dried for
Calcium gluconate.Wherein sampling sample heats 5min in 95 DEG C, and 10000rpm/min centrifugation 5min is taken supernatant, examined using HPLC
The content for surveying sodium gluconate and 5- keto-D-gluconic acid utilizes the chloro- 3- ethyl acetoacetate of GC detection 4- and (S)-
CHBE.In the reaction system of double enzymes coupling, 5- keto-D-gluconic acid concentration is continuously increased at any time, final gluconic acid
Sodium conversion ratio is greater than 90%, and (S)-CHBE yield is 70%.
Embodiment 7: aqueous phase system utilizes whole cell coproduction 5- keto-D-gluconic acid and (S)-CHBE
In the pH8.0Tris-HCl buffering of each 2.5g of lyophilized cells in Example 5, the 20mL being suspended in.Reaction system are as follows:
50U gluconate dehydrogenase, 100U carbonyl acyl reductase, sodium gluconate 100mmol/L, 0.1% Triton X-100, NAD (P)
The chloro- 3- ethyl acetoacetate 150mmol/L of+0.2mmol/L and 4- is in the Tris-HCl of 10 mL pH 8.0, at 30 DEG C,
12h is reacted under conditions of 200 rpm of revolving speed, and is sampled on demand during the reaction.After reaction, centrifugation goes to sink
It forms sediment, isometric ethyl acetate is added in supernatant and is extracted, (S)-CHBE is obtained after organic phase is spin-dried for, appropriate chlorination is added in water phase
Calcium obtains 5- keto-D-gluconic acid calcium after completely dissolution after being spin-dried for.Wherein sampling sample heats 5min in 95 DEG C,
10000rpm/min centrifugation 5min takes supernatant, and the content of sodium gluconate and 5- keto-D-gluconic acid, benefit are detected using HPLC
With the chloro- 3- ethyl acetoacetate of GC detection 4- and (S)-CHBE.In the reaction system of double enzymes coupling, 5- Keto-D-Gluconate
Acid concentration is continuously increased at any time, and final sodium gluconate conversion ratio is greater than 79%, and (S)-CHBE yield is 53%.
Embodiment 8: diphasic system utilizes crude enzyme liquid coproduction 5- keto-D-gluconic acid and (S)-CHBE
In 8.0 Tris-HCl of the pH buffering of each 10g of lyophilized cells in Example 5,200 mL being suspended in.It is equal using high pressure
Matter machine (- 20 DEG C, 8.0 × 107Pa) cell is crushed.Clasmatosis liquid through 4 DEG C, 10000rpm/min centrifugation 30min after,
Precipitating is abandoned, supernatant is the crude enzyme liquid containing gluconate dehydrogenase and carbonyl acyl reductase.Reaction system are as follows: 50U gluconic acid
Dehydrogenase, 100U carbonyl acyl reductase, the chloro- 3- acetoacetate of sodium gluconate 100mmol/L, NAD (P)+0.2mmol/L and 4-
5mL n-butyl acetate is added in the Tris-HCl of 5mL pH8.0 in ethyl ester 150mmol/L, at 30 DEG C of reaction temperature, revolving speed
12h is reacted under the conditions of 200rpm, and is sampled on demand during the reaction.After reaction, centrifugation goes to precipitate, will be organic
(S)-CHBE is obtained after being mutually spin-dried for, appropriate calcium chloride is added in water phase, after completely dissolution, 5- keto-D-gluconic acid is obtained after being spin-dried for
Calcium.Wherein sampling sample heats 5min in 95 DEG C, and 10000rpm/min centrifugation 5min takes supernatant, detects glucose using HPLC
The content of sour sodium and 5- keto-D-gluconic acid utilizes the chloro- 3- ethyl acetoacetate of GC detection 4- and (S)-CHBE.In double enzymes
In the reaction system of coupling, 5- keto-D-gluconic acid concentration is continuously increased at any time, and final sodium gluconate conversion ratio is greater than
99%, (S)-CHBE yield is 83%.
The detection method of product:
Sodium gluconate and 5- keto-D-gluconic acid are measured using high performance liquid chromatography (HPLC).High performance liquid chromatograph is adopted
With Japanese Shimadzu (Shimadzu) company, chromatographic column be U.S. Bio-Rad company AminexHPX-87Hcolumn (300 ×
7.8mm) chromatographic column, mobile phase 5mMH2SO4, flow velocity 0.6mL/min, column temperature is 35 DEG C；Using UV detector.
Using the chloro- 3- ethyl acetoacetate of gas-chromatography (GC) measurement 4- and (S)-CHBE.Gas chromatograph is using Japan
Shimadzu (Shimadzu) company GC-2010, chromatographic column Rtx-VMS gasifies 240 DEG C of room temperature, 60 DEG C of chromatographic column initial temperature, retains
2min, heating rate are 15 DEG C/min, 240 DEG C of final temperature, retain 4min, 240 DEG C of temperature of detector (FID).Carrier gas: helium, stream
Speed: 0.43mL/min.
The foregoing is merely the preferred embodiments of the invention, it is noted that for the ordinary skill people of the art
For member, without departing from the principle of the present invention, also it can make several improvements and retouch, these improvements and modifications should also regard
For protection scope of the present invention.
Sequence table
<110>Nanjing University of Technology
<120>a kind of method of coproduction 5- keto-D-gluconic acid and 4- chloro-3-hydroxyl ethyl butyrate
<141> 2018-10-25
<160> 2
<170> SIPOSequenceListing 1.0
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<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
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ccggaattca tgagtacctc tctttttg 28
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<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
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Claims (8)
1. a kind of method of coproduction 5- keto-D-gluconic acid and 4- chloro-3-hydroxyl ethyl butyrate, which is characterized in that it is with Portugal
Grape saccharic acid and the chloro- 3- ethyl acetoacetate of 4- are raw material, with the whole cell containing gluconate dehydrogenase, carbonyl acyl reductase or are contained
The resolvase for having gluconate dehydrogenase, the immobilized cell of carbonyl acyl reductase or gluconate dehydrogenase, carbonyl acyl reductase is
Catalyst, in the reaction system, with NAD (P)+or NAD+For hydrogen acceptor, catalysis preparation 5- keto-D-gluconic acid and 4- are chloro-
Ethyl 3-hydroxybutanoate.
2. the side of a kind of coproduction 5- keto-D-gluconic acid according to claim 1 and 4- chloro-3-hydroxyl ethyl butyrate
Method, which is characterized in that the gluconate dehydrogenase is described as expressed by the recombinant bacterium containing gluconate dehydrogenase gene
Gluconate dehydrogenase gene derive from Gluconobacter oxvdans (Gluconobacter oxydans), or weak oxide Portugal
Saccharobacillus (Gluconobacter suboxydans) or Pasteur's acetobacter (Acetobacter pasteurianus), or
Climb film acetobacter (Acetobacter ascendens) or Streptococcus suis (Streptococcus suis) or citric acid
Bacterium (Klebsiellasp) or penicillium chrysogenum (Penicillium chrysogenum) etc. other contain gluconic acid dehydrogenation
The strain of enzyme.
3. the side of a kind of coproduction 5- keto-D-gluconic acid according to claim 1 and 4- chloro-3-hydroxyl ethyl butyrate
Method, which is characterized in that the carbonyl acyl reductase is as expressed by the recombinant bacterium containing carbonyl acyl reductase gene, and the carbonyl acyl is also
Nitroreductase gene derives from Streptomyces Syancus (Streptomyces coelicolor) or Candida albicans (Candida
) or candida magnoliae (Candida magnoliae) or ooze kluyveromyces (Kluyveromyces albicans
Aestuarii), saccharomyces cerevisiae (Saccharomyces cerevisiae) puts down nearly sliding Candida (Candida
Parapsilosis), flat Rhodococcus sp (Rhodococcus erythropolis) etc. other contain the species of carbonyl acyl reductase.
4. the side of a kind of coproduction 5- keto-D-gluconic acid according to claim 1 and 4- chloro-3-hydroxyl ethyl butyrate
Method, which is characterized in that catalystic converter system is aqueous phase system or water phase and organic heterogeneous system mixed.
5. the side of a kind of coproduction 5- keto-D-gluconic acid according to claim 4 and 4- chloro-3-hydroxyl ethyl butyrate
Method, which is characterized in that reaction system composition are as follows: water phase volume is 1-10mL, pH 6.0-10.0, and organic phase volume is
0-9mL, 1.5-150g/L sodium gluconate, the chloro- 3- ethyl acetoacetate of 4- of 3-300g/L, the gluconic acid of 50-5000U are de-
The NAD (P)+or NAD of hydrogen enzyme, the carbonyl acyl reductase of 70-8000U and 0.05-0.8mmol/L+。
6. the side of a kind of coproduction 5- keto-D-gluconic acid according to claim 1 and 4- chloro-3-hydroxyl ethyl butyrate
Method, which is characterized in that the reaction system, reaction condition are as follows: 25-45 DEG C of reaction temperature, speed of agitator 180-280rpm, reaction
Time 1-24h.
7. the side of a kind of coproduction 5- keto-D-gluconic acid according to claim 4 and 4- chloro-3-hydroxyl ethyl butyrate
Method, which is characterized in that the water phase can be water or phosphate buffer or Tris-HCl buffer or Glycine-NaOH
Buffer；The organic phase is one or more of esters, alkane, halogenated hydrocarbons or ethers；Water phase and organic Phase Proportion can
For 10:0-1:9.
8. the side of a kind of coproduction 5- keto-D-gluconic acid according to claim 7 and 4- chloro-3-hydroxyl ethyl butyrate
Method, which is characterized in that the esters include but is not limited to ethyl acetate, butyl acetate, ethyl caprilate, neck two fourth of phthalic acid
Ester；The alkane includes but is not limited to n-hexane, heptane, octane；The halogenated hydrocarbons includes but is not limited to methylene chloride, three chloromethanes
Alkane；The ethers includes but is not limited to ether, isopropyl ether.
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| CN101613672A (en) * | 2009-08-04 | 2009-12-30 | 南京工业大学 | Recombinant escherichia coli for preparing -4-chloro-3-hydroxybutanoate through asymmetric transformation and construction method thereof |
| CN107954959A (en) * | 2017-11-22 | 2018-04-24 | 南京工业大学 | Method for preparing 2, 5-furandicarboxylic acid and precursor substance thereof |
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| CN101613672A (en) * | 2009-08-04 | 2009-12-30 | 南京工业大学 | Recombinant escherichia coli for preparing -4-chloro-3-hydroxybutanoate through asymmetric transformation and construction method thereof |
| CN107954959A (en) * | 2017-11-22 | 2018-04-24 | 南京工业大学 | Method for preparing 2, 5-furandicarboxylic acid and precursor substance thereof |
Non-Patent Citations (1)
| Title |
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| QI YE: "Construction and co-expression of a polycistronic plasmid encoding carbonyl Construction and co-expression of a polycistronic plasmid encoding carbonyl (S)-4-chloro-3-hydroxybutanoate", 《BIORESOURCE TECHNOLOGY》 * |
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