CN105695519A - Novel biological preparation method of optically pure 2-hydroxy butyric acid - Google Patents
Novel biological preparation method of optically pure 2-hydroxy butyric acid Download PDFInfo
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Abstract
The invention relates to a novel production and synthesis method of (R)-2-hydroxy butyric acid and (S)-2-hydroxy butyric acid, and particularly provides a method for producing (R)-2-hydroxy butyric acid and (S)-2-hydroxy butyric acid by catalyzing threonine through threonine dehydrase, lactic dehydrogenase and formate dehydrogenase in a one-pot mode. The (R)-2-hydroxy butyric acid and the (S)-2-hydroxy butyric acid can be widely applied to medicine synthesis, biodegradable material synthesis and fine chemicals synthesis. The method has the remarkable advantages that the reaction condition is mild, pollution is avoided, and the technological path is simple.
Description
Technical field
The invention belongs to living things catalysis and prepare pharmaceutical intermediate and Green Chemistry field, relate to a kind of method being prepared optical voidness 2-hydroxybutyric acid by threonine one kettle way。Relate generally to the biological novel preparation method of one (R)-2-hydroxybutyric acid and (S)-2-hydroxybutyric acid。
Background technology
Chirality 2-hydroxybutyric acid can be widely applied to the synthesis of pharmaceutical synthesis, Biodegradable material and fine chemicals。As (R)-2-hydroxybutyric acid can be used for the synthesis of antiepileptic levetiracetam and Bu Waxitan, (S)-2-hydroxybutyric acid then can be used for the synthesis of PPAR alpha-2 antagonists (R)-K-13675 and PPAR γ antagonist MK-0533。Additionally, optically pure 2-hydroxybutyric acid also has huge application prospect in Biodegradable material field。
The production of bibliographical information optical voidness 2-hydroxybutyric acid mainly has the enzyme catalysis Split Method of the enzyme catalysis reducing process of 2-butanone acid, 2-hydroxybutyric acid, reports, such as (CN101974603) such as Ma Cuiqing, the microorganism intact cell utilized containing NAD (nicotinamide adenine dinucleotide) dependent/non-dependent l-hydroxy acid dehydrogenase or crude enzyme liquid splits 2-hydroxybutyric acid and generates (R)-2-hydroxybutyric acid and the method for 2-butanone acid。Owing to 2-butanone acid and 2-hydroxybutyric acid production cost are higher, optics alcohol 2-hydroxybutyric acid production cost is caused to remain high。Literature survey finds that threonine dehydratase catalysis threonine dehydration can generate 2-butanone acid, but, owing to this enzyme is had bigger inhibitory action by 2-butanone acid, cause the production concentration of accumulation to be not as high。
We have found that in the technique of current living things catalysis synthesis (R)-2-hydroxybutyric acid or (S)-2-hydroxybutyric acid, still do not utilize biocatalyzer by the method for threonine one pot process (R)-2-hydroxybutyric acid or (S)-2-hydroxybutyric acid。This method utilize threonine dehydratase, lactic acid dehydrogenase and hydrogenlyase carry out cascade reaction or in genetic engineering bacterium coexpression threonine dehydratase, lactic acid dehydrogenase and hydrogenlyase, threonine one kettle way prepare (R)-2-hydroxybutyric acid or (S)-2-hydroxybutyric acid。This process has that reaction condition gentleness, response speed be fast, pollution-free and the distinguishing feature such as process route is simple。
Summary of the invention
For reporting in preparation method (R)-2-hydroxybutyric acid or the problem such as (S)-2-hydroxybutyric acid production concentration is low, optical purity is not high, production cost is high, the present invention proposes to utilize threonine dehydratase, lactic acid dehydrogenase and formate dehydrogenase enzyme catalysis threonine one kettle way production (R)-2-hydroxybutyric acid or (S)-2-hydroxybutyric acid。(such as chemical equation 1)。
Chemical equation 1 synthetic route。
The present invention specifically comprises the following steps that
1. the structure of single expression vector
The heretofore described genetic engineering bacterium producing threonine dehydratase, concrete construction method is: with e. coli k12 genomic DNA for template, primer ilvA-F(CGCGGATCCGATGGCTGACTCGCAAC according to pre-designed) and pcr amplification condition and amplification system ilvA-R(CCCAAGCTTTCACTAACCCGCCAAAAAG) carry out PCR, obtain ilvA gene, it is connected with the pRSFduet carrier after BamHI and HindIII double digestion after BamHI and HindIII double digestion, convert BL21 (DE3) competence, picking monoclonal shakes bacterium, upgrading grain carries out double digestion checking, the plasmid that endonuclease bamhi size is correct carries out checking order and verifies further。
The genetic engineering bacterium of heretofore described lactic acid producing dehydrogenase, concrete construction method is: by deriving from D type lactate dehydrogenase gene (D-LDH) of staphylococcus epidermidis (Staphylococcusepidermidis) ATCC12228 and deriving from the L-type lactate dehydrogenase gene (L-LDH) of rabbit (Oryctolaguscuniculus) and synthesized by Shanghai Xu Guan company, be connected on pRSFduet and pET32a carrier。Then expression vector proceeds to BL21 (DE3) competence respectively, and picking monoclonal shakes bacterium, and genetic engineering bacterium is carried out fermentation culture, it is achieved that the efficient heterogenous expression of D type lactic acid dehydrogenase and L-type lactic acid dehydrogenase。
2. the structure of pair enzyme coexpression vector
Respectively by pRSFduet-D-LDH and pET32a-L-LDH NdeI and XhoI enzyme action, glue reclaims D-LDH and L-LDH genetic fragment, it is connected respectively to second expression cassette of pRSFduet-ilvA carrier, convert BL21 (DE3) competence, picking monoclonal shakes bacterium, upgrading grain carries out double digestion checking, and the plasmid that endonuclease bamhi size is correct carries out checking order and verifies further, obtains the double; two enzyme coexpression vector of pRSFduet-ilvA-D-LDHandpRSFduet-ilvA-L-LDH。
3. the structure of three enzyme coexpression vectors
Preserve amplification plasmid pET21d-FDH from this laboratory and obtain deriving from the whole expression cassette (comprising RBS sequence and promoter) of the hydrogenlyase of Candida boidinii (Candidaboidinii), by complementary series one-step cloning test kit, fragment is connected on pRSFduet-ilvA-L-LDHandpRSFduet-ilvA-D-LDH dual-expression vector, convert BL21 (DE3) competence, picking monoclonal shakes bacterium, upgrading grain carries out double digestion checking, the plasmid that endonuclease bamhi size is correct carries out checking order and verifies further, obtain pRSFduet-ilvA-L-LDH-FDH and pRSFduet-ilvA-D-LDH-FDH three enzyme coexpression vector。Then expression vector respectively proceeds to BL21 (DE3) competence, and picking monoclonal shakes bacterium, and genetic engineering bacterium is carried out fermentation culture, it is achieved efficiently heterogenous expression。
4. the expression of enzyme and microorganism collection
E. coli bl21 (DE3) containing plasmid is cultivated seed liquor at 37 DEG C of incubator overnight, 1% inoculation LB fluid medium is cultivated, 37 DEG C be cultured to OD600 about 0.8 after, the isopropyl-beta D-thio galactopyranoside (IPTG) being added thereto to final concentration 0.1mM carries out abduction delivering, inducing temperature is 25 DEG C, and induction time is 20 hours。After abduction delivering terminates, within centrifugal 20 minutes, collect thalline in 6000rpm。
Carrier families used in heretofore described threonine dehydratase, lactic acid dehydrogenase and hydrogenlyase recombinant bacterium and three enzyme coexpression recombinant bacteriums includes: pET series plasmids, pTXB1 series, pGEX series, pETduet series, pTYB series。
Heretofore described threonine dehydratase, lactic acid dehydrogenase and hydrogenlyase recombinant bacterium and three enzyme coexpression recombinant bacteriums, it is characterised in that described can the Host Strains of efficiently expressing exogenous gene be one of following: BL21 series, Rosetta series, Origami series, Tuner series。
In the present invention, the threonine dehydratase plasmid built, D-lactic acid dehydrogenase plasmid, LDH plasmid, hydrogenlyase plasmid and three enzyme co-expression plasmids conversion host the transformant obtained can grow based on Given information and produce threonine dehydratase of the present invention, D-lactic acid dehydrogenase, LDH and hydrogenlyase。Any artificial or natural containing suitable carbon source, nitrogenous source, inorganic and other nutrient substance medium, as long as the growth of host bacterial can be met and can give expression to destination protein and all can use。Cultural method and condition of culture do not have clear and definite restriction, suitable selection can be carried out, as long as host growth can be met and can produce the threonine dehydratase of corresponding activity, D-lactic acid dehydrogenase, LDH and hydrogenlyase according to the difference of cultural method and type etc.。
The invention still further relates to and utilize threonine dehydratase, hydrogenlyase and D-lactic acid dehydrogenase or LDH conversion threonine to prepare (R)-2-hydroxybutyric acid or the method for (S)-2-hydroxybutyric acid, described method is:
1) by described product threonine dehydratase, hydrogenlyase, the genetic engineering bacterium of D-lactic acid dehydrogenase or LDH is cultivated through seed culture medium, it is inoculated into fermentation medium by a certain percentage, after cultivating certain time, add derivant IPTG or lactose or the two mixture inducing culture certain time, centrifugal collection thalline, then by threonine dehydratase, hydrogenlyase and D-lactic acid dehydrogenase or LDH join buffer that pH value is 7.0 ~ 8.5 (containing 35.7～89.3g/L threonine, 62.4 ~ 156g/L sodium formate, 0 ~ 0.1mM pyridoxal 5-phosphate (PLP), 0.5g/LNAD+), utilizing the online regulator control system of pH, stream adds 2MHCl, and the pH controlling reaction system maintains 7.0～8.5, obtaining (R)-2-hydroxybutyric acid or (S)-2-hydroxybutyric acid after centrifugal, acidifying, extraction, precipitation after reacting completely at 25～30 DEG C, yield is more than 90%。
2) the co-expression gene engineering bacteria of described product threonine dehydratase, hydrogenlyase, D-lactic acid dehydrogenase or LDH is cultivated through seed culture medium, it is inoculated into fermentation medium by a certain percentage, after cultivating certain time, add derivant IPTG or lactose or the two mixture inducing culture certain time, centrifugal thalline of collecting, addition pH value is that the buffer of 7.0～8.5 is (containing 35.7～89.3g/L threonine, 62.4 ~ 156g/L sodium formate, 0 ~ 0.1mMPLP, 0.5g/LNAD+), utilizing the online regulator control system of pH, stream adds 2MHCl, and the pH controlling reaction system maintains 7.0～8.5, obtaining (R)-2-hydroxybutyric acid or (S)-2-hydroxybutyric acid after centrifugal, acidifying, extraction, precipitation after reacting completely at 25～30 DEG C, yield is more than 90%。
The medium being suitable in reaction can be water or the aqueous medium containing different buffer, and its buffer used can be add one or more phosphate or Tris sulfate in water。
The preferred threonine dehydratase of pH value of the present invention, hydrogenlyase, D-lactic acid dehydrogenase or LDH can be expressed within the scope of the pH of its activity, it is preferable that pH value is 7.0～8.5。The preferred threonine dehydratase of reaction temperature, hydrogenlyase, D-lactic acid dehydrogenase or LDH can be expressed in the temperature range of its activity, it is preferable that 20～40 DEG C。
Catalyst of the present invention is can be full cell, broken bacterium solution, thick enzyme and pure enzyme。
Detailed description of the invention
Further illustrate below by way of specific embodiment, its object is to be better understood from summary of the invention, but these embodiments are not construed as limiting the invention。
The cultivation of embodiment 1 genetic engineering bacterium and the preparation of resting cell
On picking flat board, single colony inoculation is to 5ml containing in corresponding antibiotic fermentation medium, and about 15h is as seed liquor in cultivation, is seeded in the fermentation medium containing 600ml according to the inoculum concentration of 1%, at 37 DEG C, the shaking table of 200rpm is cultured to OD600About=0.6～0.8, the IPTG adding final concentration of 0.1mM carries out induction more than 10h in 25 DEG C, collects thalline with 8000rpm centrifugation medium。
Embodiment 2 threonine dehydratase, hydrogenlyase, D-lactic acid dehydrogenase recombinant bacterium resting cell one pot process (R)-2-hydroxybutyric acid
In 250mL there-necked flask, it is sequentially added into 89mL water, 8.93g threonine, 3.58g disodium hydrogen phosphate, 15.6g sodium formate, 1mL10mMPLP and 50mgNAD+Regulate pH to 7.0, it is subsequently adding the resting cell (weight in wet base of 0.6g threonine dehydratase engineering bacteria, 240U), the resting cell (weight in wet base of 10mL hydrogenlyase enzyme liquid (10U) and 1.6gD-lactic dehydrogenase enzyme engineering bacteria, 1493U), utilize the online regulator control system of pH, add 2MHCl solution to reaction system stream, the pH controlling reaction system maintains about pH7.0, and reaction temperature controls at 25 ~ 30 DEG C。After reacting 24 hours, high performance liquid chromatography detection display reacts completely (Agilent1200seriesHPLC, Acclaim120C18column (4.6 × 250mm, 5.0 μm)), 6M salt acid for adjusting pH, to 1 ~ 2, is centrifuged and collects the supernatant, uses 200ml extraction into ethyl acetate 3 times respectively, merge organic facies, anhydrous sodium sulfate dries, and filters, and rotation is steamed recycling design and obtained pale yellowish oil product (R)-2-hydroxybutyric acid 7.41g, yield 95%, ee value is more than 99%。1HNMR(400MHz,CDCl3):δ=4.26(dd,J=6.8,4.6Hz,1H),1.83-1.99(m,1H),1.76(dquin,J=14.3,7.2,7.2,7.2,7.2Hz,1H),1.01(t,J=7.4Hz,3H).13CNMR(100MHz,CDCl3):δ=179.55,71.32,27.26,8.95.MS(ESI):m/z:calcdforC4H7O3 -:103.0395[M-H]-,found:103.0401.
Embodiment 3 threonine dehydratase, hydrogenlyase, LDH recombinant bacterium resting cell catalysis threonine one pot process (S)-2-hydroxybutyric acid
The resting cell of D-lactic acid dehydrogenase engineering bacteria in embodiment 2 is changed into the resting cell (weight in wet base of 1.6gL-lactic dehydrogenase enzyme engineering bacteria, 133U), after reacting 36 hours, treated obtain pale yellowish oil product (S)-2-hydroxybutyric acid 7.57g, yield 97%, ee value is more than 99%。1HNMR(400MHz,CDCl3):δ=4.26(dd,J=6.7,4.5Hz,1H),1.84-1.97(m,1H),1.76(dquin,J=14.3,7.2,7.2,7.2,7.2Hz,1H),1.02(t,J=7.5Hz,3H).13CNMR(100MHz,CDCl3):δ=179.59,71.31,27.27,8.95.MS(ESI):m/z:calcdforC4H7O3-:103.0395[M-H]-,found:103.0405.
Embodiment 4 threonine dehydratase, hydrogenlyase, D-lactic acid dehydrogenase three enzyme coexpression recombinant bacterium resting cell catalysis threonine one pot process (R)-2-hydroxybutyric acid (adding PLP)
In 250mL there-necked flask, it is sequentially added into 99mL water, 8.93g threonine, 3.58g disodium hydrogen phosphate, 15.6g sodium formate, 1mL10mM pyridoxal 5-phosphate and 50mgNAD+, regulate pH to 7.0, be subsequently adding 2g threonine dehydratase, hydrogenlyase, D-lactic acid dehydrogenase three enzyme coexpression recombinant bacterium resting cell, utilize the online regulator control system of pH, adding 2M hydrochloric acid to reaction system stream, the pH controlling reaction system maintains about pH7.0, and reaction temperature controls at 25 ~ 30 DEG C。After reacting 8 hours, high performance liquid chromatography detection display reacts completely (Agilent1200seriesHPLC, Acclaim120C18column (4.6 × 250mm, 5.0 μm)), 6M salt acid for adjusting pH, to 1 ~ 2, is centrifuged and collects the supernatant, uses 200ml extraction into ethyl acetate 3 times respectively, merge organic facies, anhydrous sodium sulfate dries, and filters, and rotation is steamed recycling design and obtained pale yellowish oil product (R)-2-hydroxybutyric acid 7.25g, yield 93%, ee value is more than 99%。
Embodiment 5 threonine dehydratase, hydrogenlyase, LDH three enzyme coexpression recombinant bacterium resting cell catalysis threonine one pot process (S)-2-hydroxybutyric acid (adding PLP)
Change threonine dehydratase, hydrogenlyase, D-lactic acid dehydrogenase three enzyme coexpression recombinant bacterium resting cell in embodiment 4 into 2.0g threonine dehydratase, hydrogenlyase, LDH three enzyme coexpression recombinant bacterium resting cell, after reacting 24 hours, treated obtain pale yellowish oil product (S)-2-hydroxybutyric acid 7.57g, yield 97%, ee value is more than 99%。
Embodiment 6 threonine dehydratase, hydrogenlyase, D-lactic acid dehydrogenase three enzyme coexpression recombinant bacterium resting cell catalysis threonine one pot process (R)-2-hydroxybutyric acid (without PLP)
Without pyridoxal 5-phosphate in embodiment 4, after reacting 12 hours, treated obtaining pale yellowish oil product (R)-2-hydroxybutyric acid 7.49g, yield 96%, ee value is more than 99%。
Embodiment 7 threonine dehydratase, hydrogenlyase, LDH three enzyme coexpression recombinant bacterium resting cell catalysis threonine one pot process (S)-2-hydroxybutyric acid (without PLP)
Without pyridoxal 5-phosphate in embodiment 5, after reacting 24 hours, treated obtaining pale yellowish oil product (S)-2-hydroxybutyric acid 7.41g, yield 95%, ee value is more than 99%。
SEQUENCELISTING
<110>Tianjin Institute of Industrial Biotechnology, Chinese Accademy of Sciences
<120>the biological novel preparation method of a kind of optical voidness 2-hydroxybutyric acid
<130>2016
<160>4
<170>PatentInversion3.3
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ctggaacgtctgctgccgttcaacccgaaagaactgctgtactacgactaccaggctctg600
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gctcaggctgacatcgttactgttaacgctccgctgcacgcaggcactaaaggtctgatt720
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ggtggtgacgtatggtttccgcagccggctccgaaagatcacccgtggcgtgatatgcgt900
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cagacccgttacgctgaaggtactaaaaacatcctggaatctttcttcactggtaaattc1020
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Claims (8)
1. the new method of biosynthesis (R)-2-hydroxybutyric acid and (S)-2-hydroxybutyric acid, it is characterized in that utilizing threonine dehydratase, lactic acid dehydrogenase and hydrogenlyase or their coexpression recombinant bacterium catalysis threonine one kettle ways to prepare (R)-2-hydroxybutyric acid or (S)-2-hydroxybutyric acid, comprise the steps:
A) structure of threonine dehydratase recombinant bacterium, hydrogenlyase recombinant bacterium and D/L-lactic acid dehydrogenase recombinant bacterium and three enzyme coexpression recombinant bacteriums；
B) by threonine dehydratase recombinant bacterium, hydrogenlyase recombinant bacterium and D/L-lactic acid dehydrogenase recombinant bacterium and three enzyme coexpression recombinant bacteriums amplification cultivation in the fermentation medium respectively, and carry out after induction produces destination protein, centrifugal collect thalline；
C) by the threonine dehydratase recombinant bacterium collected, hydrogenlyase recombinant bacterium and D/L-lactic acid dehydrogenase recombinant bacterium or three enzyme coexpression recombinant bacteriums, moisture, threonine, disodium hydrogen phosphate, sodium formate, pyridoxal 5-phosphate (PLP) and nicotinamide adenine dinucleotide (NAD are joined+), regulating pH to 7.0, react certain time under proper condition, substrate, through the acid of intermediate product 2-butanone, is eventually converted into (R)-2-hydroxybutyric acid or (S)-2-hydroxybutyric acid；
D) reaction collects the supernatant after stopping with hcl acidifying from reaction system, and is extracted with ethyl acetate repeatedly, merges organic facies, and anhydrous sodium sulfate dries, and filters, and rotation is steamed recycling design and obtained target product。
2. a) step as claimed in claim 1 produces the co-expression gene engineering bacteria of threonine dehydratase, lactic acid dehydrogenase and hydrogenlyase, it is characterized in that threonine dehydratase, D/L-lactic acid dehydrogenase and hydrogenlyase being building up on same carrier simultaneously, and be transformed into Host Strains and carry out abduction delivering。
3. a) step as claimed in claim 1 produces the genetic engineering bacterium of threonine dehydratase, D/L-lactic acid dehydrogenase or hydrogenlyase, it is characterized in that being building up on a carrier threonine dehydratase, D/L-lactic acid dehydrogenase or hydrogenlyase, and be transformed into Host Strains and carry out abduction delivering。
4. a) step as claimed in claim 1 produces the co-expression gene engineering bacteria of threonine dehydratase, D/L-lactic acid dehydrogenase and hydrogenlyase, it is characterised in that described can the plasmid of efficiently expressing exogenous gene be one of following: pET series plasmids, pETduet series pET series plasmids, pTXB1 series, pGEX series, pETduet series, pTYB series；The Host Strains of efficiently expressing exogenous gene is one of following: BL21 series, Rosetta series, Origami series, Tuner series。
5. a) step as claimed in claim 1 produces the genetic engineering bacterium of threonine dehydratase, D/L-lactic acid dehydrogenase or hydrogenlyase, it is characterised in that described can the plasmid of efficiently expressing exogenous gene be one of following: pET series plasmids, pETduet series pET series plasmids, pTXB1 series, pGEX series, pETduet series, pTYB series；The Host Strains of efficiently expressing exogenous gene is one of following: BL21 series, Rosetta series, Origami series, Tuner series。
6., in the method for claim 1 c) in step, wherein said enzyme can be complete microbial cell, cell breakage liquid, thick enzyme or pure enzyme。
7. method for transformation c) step as claimed in claim 1, it is characterised in that described reaction condition: temperature is 20～40 DEG C, it is preferable that 25～30 DEG C；Buffer used is phosphate, Tris sulfate；The pH of buffer used is 7.0～9.0, it is preferable that pH is 7.0；Response time is 8-36 hour。
8. method for transformation c) step as claimed in claim 1, it is characterised in that the concentration of described threonine is 35.7～89.3g/L。
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| CN112852701A (en) * | 2021-02-06 | 2021-05-28 | 江南大学 | Construction and application of single-cell factory for synthesizing (S) -2-hydroxybutyric acid |
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