CN106520715B - A kind of short-chain dehydrogenase and its gene, recombinant expression carrier, genetic engineering bacterium and its application in the synthesis of astaxanthin chiral intermediate - Google Patents
A kind of short-chain dehydrogenase and its gene, recombinant expression carrier, genetic engineering bacterium and its application in the synthesis of astaxanthin chiral intermediate Download PDFInfo
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- CN106520715B CN106520715B CN201610900528.3A CN201610900528A CN106520715B CN 106520715 B CN106520715 B CN 106520715B CN 201610900528 A CN201610900528 A CN 201610900528A CN 106520715 B CN106520715 B CN 106520715B
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- 102000009105 Short Chain Dehydrogenase-Reductases Human genes 0.000 title claims abstract description 38
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- 238000003259 recombinant expression Methods 0.000 title claims abstract description 17
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- 229940022405 astaxanthin Drugs 0.000 title abstract description 24
- 239000001168 astaxanthin Substances 0.000 title abstract description 24
- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 title abstract description 23
- 235000013793 astaxanthin Nutrition 0.000 title abstract description 23
- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 title abstract description 23
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- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
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- 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.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- 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
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01184—Carbonyl reductase (NADPH) (1.1.1.184)
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Abstract
The present invention provides a kind of short-chain dehydrogenase, and its gene, recombinate short-chain dehydrogenase, recombinant expression carrier containing the gene, and genetic engineering bacterium, the preparation method of the recombinase and the short-chain dehydrogenase or application of the genetic engineering bacterium in asymmetric reduction prochiral ketone synthesizing astaxanthin chiral intermediate containing the enzyme.Heretofore described short-chain dehydrogenase asymmetric reduction prepares chiral alcohol with remarkable advantage, can synthesize high-optical-purity astaxanthin chiral intermediate (ee > 99%).Catalyst is easily prepared, reaction condition is mild, substrate wide adaptability, environmental-friendly, and its recombinant cell can in not outer plus any coenzyme system containing isopropanol reaction efficient catalytic prochiral ketone asymmetric reduction, there is good industrial applications development prospect.
Description
(1) technical field
The invention belongs to technical field of biochemical industry, and in particular to a kind of short-chain dehydrogenase and its gene, and containing should
The recombinant expression carrier and recombinant expression transformants and the short-chain dehydrogenase of gene or recombinant cell containing the enzyme are in shrimp blueness
Application in plain chiral intermediate synthesis.
(2) background technique
Astaxanthin (Astaxanthin, 3,3 '-dihydroxy -4,4 '-diketo-β, β '-carrotene) is that nature is extensive
A kind of existing novel carotenoid.Astaxanthin is that one of strongest natural, oxidation resistance are in the world
10 times or more of other carotenoid are 300~500 times of vitamin E；With antitumor, anti-radiation, anti-aging, raising
Therefore the multiple biological functions such as immunity, anti-photosensitization and body colour developing are widely used in health care product, drug, makeup
In the production of product, food and feed etc..Astaxanthin is there are mainly three types of different isomers kenels, and antioxidant activity is strongest is
The astaxanthin of (3S, 3 ' S)-configuration.
Currently, the main acquisition methods of astaxanthin include chemical synthesis, extraction and microorganism hair from the shell of Crustaceans
Ferment etc..Wherein, chemically synthesized astaxanthin is approved by the fda in the United States for feeding additive aquatic animal the eighties in last century；But
Exist while chemical synthesis astaxanthin a variety of isomers, is unfavorable for digestion and absorption and the internal deposition of animal；In addition, people couple
The doubt of chemical synthesis astaxanthin edible safety is not eliminated always, is also only limitted to feed additive industry to its use.Phase
For chemically synthesized astaxanthin, natural astaxanthin has that production process is relatively simple, few non-functional isomeries in product
Body, it is feature-rich, be easy to be absorbed the advantages that strong with antioxidant activity, therefore, natural astaxanthin efficiently to be produced into be important
Selection.However, due to containing higher chitin and ash content, the protein of low concentration and other battalion in the crust of crustacean
It forms point, limits the extraction and recycling of crustacean astaxanthin.On the other hand, although strain improvement, fermentation item can be passed through
The number of ways such as piece optimization improve the yield of the astaxanthin of microorganism (red phaffia rhodozyma and haematococcus pluvialis etc.), but due to pairing
Not deep enough at the cometabolism Mechanism Study of astaxanthin, the yield of astaxanthin does not obtain always breakthrough raising.
Biological catalysis is since stereoselectivity is high, reaction condition is mild, advantages of environment protection becomes and attracts most attention
One of medicine and fine chemicals green synthesis techniques.Therefore, using the synthesis of biological catalysis highly-solid selectively (3S, 3 '
S the important chiral intermediate of)-astaxanthin, and then the strongest chiral astaxanthin monomer of acquisition antioxidant activity will have important meaning
Justice.
(3) summary of the invention
It is an object of that present invention to provide a kind of pseudomonas putida Pseudomonas putida short-chain dehydrogenase enzyme gene and
It recombinates short-chain dehydrogenase, and the recombinant expression carrier containing the gene, recombinant expression transformants, the preparation side of the recombinase
Method and the short-chain dehydrogenase or recombinant cell containing the enzyme answering in the synthesis of (3S, 3 ' S)-astaxanthin chiral intermediate
With.
The technical solution adopted by the present invention is that:
The first aspect of the present invention provides a kind of short-chain dehydrogenase, SEQ ID NO:2 in amino acid sequence such as sequence table
It is shown.
Short-chain dehydrogenase of the invention derives from (the Pseudomonas putida of pseudomonas putida ATCC 12633
ATCC 12633)。
Amino acid is by lacking, being inserted into or replace one or several in amino acid sequence shown in any couple of SEQ ID NO:2
Amino acid and have short-chain dehydrogenase it is active, still fall within protection scope of the present invention.
The second aspect of the present invention provides a kind of short-chain dehydrogenase enzyme gene, SEQ in (1) nucleotide sequence such as sequence table
Shown in ID NO:1；Or (2) encoding amino acid sequence protein as shown in sequence table SEQ ID NO:2.
The gene is by round pcr from (the Pseudomonas putida ATCC of pseudomonas putida ATCC 12633
12633) it clones and obtains in genome.Specifically the preparation method comprises the following steps: according to the gene order for the short-chain dehydrogenase included in Genbank
Synthetic primer is designed, preferably, upstream primer are as follows: GCTGAGGATCCATGGCTAATGCAAAAACCGC；Downstream primer are as follows:
GCATCCTCGAGTCACCAGACCAAGGGTTCGC；Then with 12633 genomic DNA of Pseudomonas putida ATCC
For template, gene magnification is carried out using polymerase chain reaction (PCR), obtains the short-chain dehydrogenase gene order of overall length 687bp.
The short-chain dehydrogenase gene nucleotide series are as shown in SEQ ID No:1 in sequence table.The amino of the protein of the sequential coding
Acid sequence is as shown in SEQ ID No:2 in sequence table.
As it is known by the man skilled in the art, the nucleotide sequence of short-chain dehydrogenase enzyme gene of the invention is also possible to code sequence
The other any nucleotide sequences for the protein that amino acid sequence shown in SEQ ID No:2 forms in list.
Nucleotide sequence shown in any couple of SEQ ID NO:1 carries out substitution, missing or the insertion of one or more nucleotide
The nucleotide sequence obtained is handled, as long as it has 90% or more homology with nucleotide, belongs to protection model of the invention
It encloses.
The third aspect of the present invention provides a kind of recombination of nucleotide sequence comprising short-chain dehydrogenase enzyme gene of the invention
Expression vector.Short-chain dehydrogenase nucleotide sequence of the invention can be connected to by these recombinant vectors by conventional method in that art
It is built-up on various carriers.The carrier can be the various carriers of this field routine, and such as various plasmids, bacteriophage or virus carry
Body etc., preferably pET-30a.Preferably, recombinant expression carrier of the invention can be obtained by following methods: PCR amplification will be passed through
Resulting short-chain dehydrogenase gene product Ppysdr is connect with carrier pET-30a constructs short-chain dehydrogenase genetic recombination of the invention
Expression plasmid pET30a-Ppysdr.
The fourth aspect of the present invention provides a kind of genetic engineering bacterium of expression recombination short-chain dehydrogenase, can be by will be of the invention
Recombinant expression carrier convert and obtained into host microorganism.The host microorganism can be the various hosts of this field routine
Microorganism, as long as meeting, recombinant expression carrier can stablize self-replacation and entrained short-chain dehydrogenase enzyme gene of the invention can
With effective expression.The preferred Escherichia coli of the present invention, more preferable E. coli BL21 (DE3).By recombinant plasmid
PET30a-Ppysdr is converted into E.coli BL21 (DE3), obtains engineering bacteria E.coli BL21 (DE3)/pET30a-
Ppysdr。
The fifth aspect of the present invention provides a kind of preparation method for recombinating short-chain dehydrogenase, includes the following steps: culture originally
The recombinant expression transformants of invention, induction obtain recombination short-chain dehydrogenase.Wherein, used in the culture recombinant expression transformants
Culture medium can be this field and can make transformants grew and generate the culture medium of short-chain dehydrogenase of the invention, preferably LB culture
Base: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH 7.2.Cultural method and condition of culture are not particularly limited,
As long as enabling transformant to grow and generating short-chain dehydrogenase.It is preferred that following methods: by recombination large intestine of the present invention
Bacillus E.coli BL21 (DE3)/pET30a-Ppysdr is seeded in the LB culture medium containing kanamycins and cultivates, when culture solution
Optical density OD600When reaching 0.5~0.7, in final concentration of 0.1~1.0mM isopropyl-beta D-thio galactopyranoside
It (IPTG), can high efficient expression recombination short-chain dehydrogenase of the invention under induction.
The sixth aspect of the present invention provides the short-chain dehydrogenase or its recombinant cell at asymmetry catalysis prochiral ketone (I)
Prepare the application in (3S, 3 ' S)-astaxanthin chiral intermediate (II).
Specifically, the application are as follows: with prochiral ketone (I) for substrate, with the short-chain dehydrogenase or its recombinant cell
For catalyst, using NADH or NADPH as coenzyme, at 20~50 DEG C, in the conversion reaction that the buffer of pH 5.5~10.5 is constituted
It is reacted in system a, after fully reacting, reaction solution is isolated and purified to obtain corresponding product.
The reaction condition can be selected by the normal condition used in this field.
Further, initial substrate concentration is 5~1000mmol/L in the transformation system a.
Further, recombinated in the transformation system a the pure enzyme of short-chain dehydrogenase in reaction solution preferable concentration for 0.1~
2.0mg/mL.The quality dosage of thallus is calculated as 10~400g/L with thallus weight in wet base in the transformation system a.
Further, the transformation system a further includes organic solvent, by substrate, catalyst, organic solvent and pH 5.5~
10.5 buffer constitutes transformation system b, and organic solvent accounts for transformation system b total volume 1~20%, in transformation system b at the beginning of substrate
Beginning concentration is 5~1000mmol/L, and the quality dosage of thallus is calculated as 10~400g/L with thallus weight in wet base.
Further, the reaction carries out in the buffer of pH 7.5.
Further, reaction system can also add 1~15% alcohol or sugar is used as auxiliary substrate, can significantly improve the work of reaction
Power.The auxiliary substrate includes but is not limited to one of following: 1. ethyl alcohol, 2. isopropanol, 3. glucose, 4. sucrose etc..
Further, the auxiliary substrate in reaction system is isopropanol.
Further, the concentration of isopropanol is 10% in reaction system.
Further, the conversion reaction solution isolation and purification method are as follows: after reaction, conversion reaction solution is centrifuged, is taken
Clear liquid is extracted with isometric ethyl acetate, and organic layer is the crude product containing corresponding chiral alcohol, crude product purification is obtained corresponding
Chiral alcohol.The method of the crude product purification is techniques well known, usually organic solvent extraction, chromatographic isolation and absorption point
From etc..
Transformation system a and transformation system b of the present invention are transformation system, for convenient for differentiation different step transformation system
Composition it is different and name, letter itself is without meaning.
The beneficial effects are mainly reflected as follows: it provides a kind of from Pseudomonas putida ATCC
12633 short-chain dehydrogenase and its gene, and recombinant expression carrier and recombinant expression transformants containing the gene, by this
Short-chain dehydrogenase or recombinant cell asymmetric reduction containing the enzyme can prepare the astaxanthin chiral intermediate of high-optical-purity；This
Short-chain dehydrogenase described in invention or the recombinant cell asymmetric reduction containing the enzyme, which prepare chiral alcohol, has remarkable advantage, can
It synthesizes high-optical-purity astaxanthin chiral intermediate (ee > 99%).Catalyst is easily prepared, stereoselectivity is high, reaction condition
Mildly, environmental-friendly, there is good application and development prospect.
(4) Detailed description of the invention
Fig. 1 is short-chain dehydrogenase gene PCR amplified production agarose gel electrophoresis figure；
Fig. 2 is short-chain dehydrogenase enzyme purification SDS-PAGE figure.
(5) specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
The acquisition of 12633 short-chain dehydrogenase enzyme gene of embodiment 1:Pseudomonas putida ATCC, recombinant plasmid structure
Build and convert Escherichia coli
The complete genome DNA of 12633 thallus of Pseudomonas putida ATCC is extracted with DNA extraction kit, with
The DNA is template, respectively with upstream primer (GCTGAGGATCC) and downstream primer ATGGCTAATGCAAAAACCGC
(GCATCCTCGAGTCACCAGACCAAGGGTTCGC) pcr amplification reaction is carried out for effect primer.PCR reaction system each component
Additional amount (50 μ L of total volume): 5 × PrimeSTARTM10 μ L, 10mM dNTP of HS DNA polymerase Buffer
Mixture (each 2.5mM of dATP, dCTP, dGTP and dTTP) 4 μ L, concentration be 50 μM upstream primer, each 1 μ L of downstream primer, base
Because of group DNA 1 μ L, PrimeSTARTMHS DNA polymerase 0.5 μ L, seedless 32.5 μ L of sour water.PCR reaction condition are as follows:
98 DEG C of 1min of initial denaturation, subsequently into 98 DEG C of 10s of temperature cycles, 56 DEG C of 15s, 72 DEG C of 1min, totally 30 are recycled, and last 72 DEG C are prolonged
5min is stretched, final temperature is 4 DEG C.Short-chain dehydrogenase gene PCR amplified production agarose gel electrophoresis results are shown in attached drawing 1.Sequencing
Analysis the result shows that, the nucleotide sequence length expanded through the above process be 687bp (its nucleotide sequence such as SEQ ID
Shown in NO:1), one complete open reading frame of the sequential coding.The gene order is analyzed using software, deduces institute
State amino acid sequence shown in short-chain dehydrogenase enzyme gene coding SEQ ID NO:2.
PCR product carries out double digestion by BamHI/XhoI, recycles target fragment through Ago-Gel QIAquick Gel Extraction Kit, then
With T4 ligase by the segment with the commercial carrier pET-30a connection with identical restriction enzyme enzymatic treatment, building recombination table
Up to plasmid pET30a-Ppysdr.
The recombinant expression carrier pET30a-Ppysdr of above-mentioned building is converted into e. coli bl21 (DE3), weight is obtained
Group E. coli BL21 (DE3)/pET30a-Ppysdr, is coated on the plate containing kanamycins, cultivated at 37 DEG C
Night, random picked clones carry out bacterium colony PCR identification, positive colony sequencing verifying, the results showed that recombinant expression carrier pET30a-
Ppysdr successful conversion is into expressive host E.coli BL21 (DE3), and short-chain dehydrogenase enzyme gene has become function and is cloned into pET-
The site BamHI and XhoI of 30a.
Embodiment 2: the inducing expression of short-chain dehydrogenase is recombinated
Embodiment 1 construct engineering bacteria E.coli BL21 (DE3)/pET30a-Ppysdr be seeded to containing 50 μ g/mL cards that
In the LB liquid medium of mycin, 37 DEG C of overnight incubations, then be inoculated into 1% inoculum concentration (v/v) containing 50 μ g/mL kanamycins
In 50mL LB culture medium, 37 DEG C, 200rpm is cultivated to cell concentration OD600To 0.6 or so, it is added final concentration of 0.1mM's
After IPTG, 26 DEG C of Fiber differentiation 6h, 4 DEG C, 8000rpm centrifugation 10min collection thallus are spare in -80 DEG C of storages.
Embodiment 3: isolating and purifying for short-chain dehydrogenase is recombinated
The somatic cells that embodiment 2 is collected are suspended in 10mL Na2HPO4-NaH2PO4In buffer (100mM, pH 8.0),
Oscillation shakes up and is crushed (effective time 8min) under postposition ultrasonic wave.Broken liquid is broken in 12,000rpm centrifugation 10min removal cell
Piece is collected supernatant (crude enzyme liquid) and is isolated and purified for the subsequent of enzyme.Purification column is Ni-NTA, and packed column volume 5mL is first used
Loading equilibration buffer (20mM sodium phosphate, 500mM NaCl and 20mM imidazoles, pH 7.4) balances Ni-NTA column, with 5mL/min
Rate loading crude enzyme liquid, with loading equilibration buffer elute to remove unadsorbed albumen, finally use elution buffer (20mM
T sodium phosphate, 500mM NaCl and 500mM imidazoles, pH 7.4) elution collection target protein.Enzyme solution is carried out with HiTrap desalting column
Desalination, desalination buffer are Na2HPO4-NaH2PO4(100mM, pH 7.5) buffer, the pure enzyme solution of gained are spare in 4 DEG C of storages.
Enzyme solution after purification is analyzed with SDS-PAGE, and SDS-PAGE electrophoresis is shown in Fig. 2, the results showed that through Ni-NTA affinity chromatography, is obtained
To electrophoretically pure recombination short-chain dehydrogenase PpYSDR.
Embodiment 4: recombination short-chain dehydrogenase asymmetric reduction prochiral ketone (I)
Obtained pure enzyme solution 1mL in Example 3, then it is separately added into 10mM prochiral ketone (I) and 5mM NADH conduct
Substrate and coenzyme.16h is reacted in 30 DEG C of constant-temperature table oscillations (200rpm).Reaction solution is extracted through isometric ethyl acetate, is used
The content and enantiomeric excess value (ee) of chiral capillary gas chromatography method analysis substrate and product.Product (3S, 3 ' S)-shrimp is green
Optical purity > 99% of plain chiral intermediate, conversion ratio 65%.
Embodiment 5: recombination short-chain dehydrogenase asymmetric reduction prochiral ketone (I)
Obtained pure 900 μ L of enzyme solution in Example 3 is added 100 μ L isopropanols, then is separately added into 10mM prochiral ketone
(I) and 5mM NADH is as substrate and coenzyme.16h is reacted in 30 DEG C of constant-temperature table oscillations (200rpm).Reaction solution is through isometric
Ethyl acetate extraction, with chiral capillary gas chromatography method analysis substrate and product content and enantiomeric excess value (ee).
Product (3S, 3 ' S)-astaxanthin chiral intermediate (II) optical purity > 99%, conversion ratio 96.2%.
Embodiment 6: recombination bacillus coli asymmetric reduction prochiral ketone (I)
In the Na of 900 μ L, 100mM2HPO4-NaH2PO4In buffer (pH 7.5), 100 μ L isopropanols are added, are added
Obtained wet thallus cell in 0.05g embodiment 2 adds 10mM prochiral ketone (I) as substrate.In 30 DEG C of constant-temperature tables
It vibrates (200rpm) and reacts 12h.Reaction solution centrifugation, takes supernatant, extracts through isometric ethyl acetate, with chiral capillary gas
The content and enantiomeric excess value (ee) of GC headspace analysis substrate and product.Product (3S, 3 ' S)-astaxanthin chiral intermediate
(II) optical purity > 99%, conversion ratio 95.8%.
Claims (3)
1. a kind of short-chain dehydrogenase or genetic engineering bacterium are prepared in (3S, 3 ' S)-astaxanthin chirality in asymmetry catalysis prochiral ketone
Application in mesosome, which is characterized in that the amino acid sequence of the short-chain dehydrogenase is as shown in SEQ ID NO:2；The gene
Engineering bacteria is as nucleotide sequence or encoding amino acid sequence comprising the encoding short-chain dehydrogenase gene as shown in SEQ ID NO:1
The recombinant expression carrier of the nucleotide sequence of protein shown in SEQ ID NO:2 is converted to be obtained into host microorganism；Wherein
The structural formula of prochiral ketone is shown in formula I, and the structural formula of (3S, 3 ' S)-astaxanthin chiral intermediate is as shown in formula II:
The transformation system that the asymmetry catalysis prochiral ketone prepares (3S, 3 ' S)-astaxanthin chiral intermediate is transformation system a,
It is institute in the 0.1~2.0mg/mL or transformation system a that short-chain dehydrogenase concentration in reaction solution is recombinated in transformation system a
The quality dosage for stating genetic engineering bacterium is calculated as 10~400g/L with thallus weight in wet base.
2. application according to claim 1, which is characterized in that using prochiral ketone as substrate, with short-chain dehydrogenase or its
Genetic engineering bacterium is catalyst, using NADH or NADPH as coenzyme, at 20~50 DEG C, what the buffer in pH5.5~10.5 was constituted
It is reacted in conversion reaction system a, after fully reacting, reaction solution is isolated and purified to obtain in product (3S, 3 ' S)-astaxanthin chirality
Mesosome reacts as follows:
3. application according to claim 1, which is characterized in that reaction system can also add isopropanol as auxiliary substrate.
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