CN102286556B - Application of sorbose reductase in preparation of -4-chloro-3-hydroxy ethyl butyrate by biological method - Google Patents
Application of sorbose reductase in preparation of -4-chloro-3-hydroxy ethyl butyrate by biological method Download PDFInfo
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Abstract
The invention discloses application of sorbose reductase in preparation of -4-chloro-3-hydroxybutanoate ethyl ester by a biological method. Namely, the amino acid sequence is shown as SEQ ID NO: 2 as a catalyst, 4-chloroacetoacetate ethyl ester as a substrate and NADPH as a cofactor to prepare -4-chloro-3-hydroxybutyric acid ethyl ester by asymmetric reduction. The invention firstly converts the amino acid sequence shown as SEQIDNO: 2, the sorbose reductase has good effect when applied to the asymmetric reduction of 4-chloroacetoacetic acid ethyl ester to prepare -4-chloro-3-hydroxy butyric acid ethyl ester, the enzyme activity is as high as 5.6U/mg, the yield of the sorbose reductase to a substrate is as high as 90%, the enantiomeric excess value of a product is 100%, the yield is high, and the production cost is greatly reduced.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of sorbose reductase, particularly this sorbose reductase is by the application in biological method production (S)-4-chloro-3 3-hydroxyethyl butyrates.
Background technology
(S)-4-chloro-3 3-hydroxyethyl butyrates (Ethyl 4-chloro-3-hydroxybutanoate, (S)-CHBE) be a kind of important organic intermediate, can be used for the synthetic of a lot of active medicines, as statins---hydroxymethyl glutaryl CoA (HMG-CoA) reductase inhibitor and 4-pyridone alkane ketone [1] such as (4-hydroxypyrrolidone).
With the latent chiral substrates of 4-chloroacetyl acetacetic ester (4-chloroacetoacetate Ethyl COBE) as reduction reaction, being easy to syntheticly and cheap, is that substrate carries out asymmetric reduction reaction to obtain (S)-CHBE be very cost-effective preparation approach with it.
Prepare chirality CHBE about the COBE asymmetric reduction up to now and carried out a lot of research reports.Summarize and get up to mainly contain chemical method and biological process.
Chemical catalysis method of asymmetrically reducing, catalyst system therefor comprise metals such as your high rhodium of price, Ruthenium, and the shortcoming that adopts chemical method synthesis of chiral CHBE is that the optical purity of product is not high enough, and the hydrogen-pressure that catalytic reduction reaction need be very high, and power consumption is high, pollutes big.
Biological process is divided into enzyme catalysis and whole-cell catalytic method, Shimizu etc. use from
Sporobolomyces salmonicolorAKU 4429The aldehyde radical reductase enzyme that relies on of NADPH prepare chirality CHBE at single aqueous phase system [2] and water/organic solvent two-phase system [3] catalytic reduction COBE respectively, because using the used enzyme of enzyme catalysis reduction reaction will obtain from microorganism cells in separation and purification, loaded down with trivial details and the easy inactivation of enzyme of process operation, compare with whole-cell catalytic, use less.Full cell rule is divided into the employing wild yeast and genetic engineering bacterium catalysis COBE is two kinds of (S)-CHBE, and Yasohara etc. [4] screen from 400 saccharomycetes and obtained a strain C
Andida magnoliaeIn water/n-butyl acetate system, (S) – CHBE can reach 90 g/L at the accumulated concentrations of organic phase, and the optical purity enantiomeric excess value of product (enantiomeric excess e.e) reaches 96% to need to control under the condition of pH value product in adding glucose, NADP and Hexose phosphate dehydrogenase and reaction process.Owing to adopt and often to contain the multiple reductase enzyme of isomorphism type CHBE not of can catalysis COBE being in the wild yeast, therefore it is often very low to adopt wild yeast to carry out the optical activity of the product that catalysis obtains, the good microorganism strains that need screen highly-solid selectively is very difficult, uses the recombination bacillus coli asymmetric synthesis to have (the S)-CHBE of highly-solid selectively so recent research concentrates on emphatically.Yasohara etc. [5] are from the candida magnoliae bacterium
Candida magnoliaeThe middle carbonyl reductase that has obtained a coenzyme NADP 11 dependent form that separates, this enzyme and glucose dehydrogenase gene are cloned into coexpression in the intestinal bacteria, regularly adding under the condition of an amount of coenzyme NAD P and glucose and portion-wise addition substrate, the asymmetric reduction of catalysis COBE (S)-CHBE, its yield and optical purity are respectively 85% and 100%e.e. [6].
In sum, existing catalysis COBE for the technology of (S)-CHBE exists that the substrate yield is low, at the bottom of the product optical activity, problem such as cost height.
The reductase enzyme that relates in this patent is a kind of of sorbose reductase, it comprises 281 amino acid, its number of including in Genbank is XP_715552.1, (http://www.ncbi.nlm.nih.gov/protein/XP_715552.1), its aminoacid sequence is shown in SEQ ID NO:2.The gene of this albumen of encoding contains the 843bp base, and its number of including in Genbank is EAK96529.1, and (http://www.ncbi.nlm.nih.gov/protein/EAK96529), its gene order is shown in SEQ ID NO:1.So far do not find that this sorbose reductase is used for the report of COBE asymmetric reduction preparation (S)-CHBE.
[reference]:
[1] Karanewsky DS, Badia MC, Ciosek CP Jr, Robl JF, Sofia MJ, Simpkins LM, DeLange B, Harrity TW, Biller SA, Gorden EM (1990) Phosphorus-containing inhibitors of HMG-CoA reductase. 1. 4-[2-arylethyl]-hydroxyphosphinyl]-3-hydroxybutanoic acids: a new class of cell-selective inhibitors of cholesterol biosynthesis. J Med Chem 33:2925–2956。
[2] Shimizu S, Kataoka M,Morishita A Katoh M, Morikawa T, Miyoshi T,Yamada H(1990a) Microbial asymmetric reduction of ethyl 4-chloro-3-oxobutaoate to optically active ethyl 4-4-chloro-3-hydroxybutanoate. Biotechnol lett 12:593-596。
[3] Shimizu S, Kataoka M, Katoh M, Morikawa T,Miyoshi T,Yamada H(1990b) Stereoselective reduction of ethyl 4-chloro-3-oxobutaoate by a microbial aldehyde reductase in an organic solvent-water diphasic system. Appl Environ Microbiol 56: 2374-2377。
[4] Yasohara Y, Kizaki N, Hasegawa J, Takahashi S, Wada M, Kataoka M, Shimizu S (1999) Synthesis of optically activeethyl 4-chloro-3- hydroxybutanoate by microbial reduction. Appl Microbiol Biotechnol 51:847–851。
[5] Wada M, Kataoka M, Kawabata H, Yasohara Y, Kizaki N, Hasegawa J, Shimizu S (1998) Purification and characterization of NADPH-ependent carbonyl reductase involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate, from Candida magnoliae. Biosci Biotechnol Biochem 62:280–285。
[6] Yasohara Y, Kizaki N, Hasegawa J, Wada M, Kataoka M, Shimizu S (2000) Molecular cloning and overexpression of the gene encoding an NADPH-dependent carbonyl reductase, involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate, from Candida magnoliae. Biosci Biotechnol Biochem 64:1430–1436。
Summary of the invention
Technical problem to be solved by this invention provides the application of a kind of sorbose reductase in COBE asymmetric reduction preparation (S)-CHBE.
For solving the problems of the technologies described above, the technical solution adopted in the present invention is as follows:
The sorbose reductase of a kind of aminoacid sequence shown in SEQ ID NO:2 is in (the application in (S)-CHBE) of 4-chloroacetyl acetacetic ester (COBE) asymmetric reduction preparation (S)-4-chloro-3 3-hydroxyethyl butyrates.
Being catalyzer with the sorbose reductase of aminoacid sequence shown in SEQ ID NO:2 namely, is substrate with the 4-chloroacetyl acetacetic ester, asymmetric reduction preparation (S)-4-chloro-3 3-hydroxyethyl butyrates.
Concrete reaction is with the reorganization bacterium of expressing gene sequence shown in SEQ ID NO:1,1.5 the 4-chloroacetyl acetacetic ester of ~ 300g/L, under pH6.0 ~ 7.5,20 ~ 30 ℃, 180 ~ 280rpm condition, respectively with 200mmol/L ~ 2mol/L sorbyl alcohol, N.F,USP MANNITOL, Xylitol, reaction 16 ~ 20h obtains (S)-4-chloro-3 3-hydroxyethyl butyrates.Wherein, add sugar alcohol and can generate NADPH, and reaction cycle is carried out, reduced add-on to have reduced production cost.
The contriver is based on modern information biology thought, and the binding molecule biology techniques adopts engineered means from Candida albicans
Candida albicansThe gene of clone's sorbose reductase, behind expression in escherichia coli, find its aqueous phase efficiently catalysis COBE be (S)-CHBE, the e.e value is 100%.Simultaneously, by modes such as reaction, portion-wise addition substrate COBE in water/organic phase, remove substrate and product to the restraining effect of cell and enzyme, improved changing effect significantly.Undertaken by the gene to sorbose reductase recombinant expressed, the zymoprotein that has obtained to have the new catalytic function, developed this gene new function---catalysis non-natural substrates COBE is (the S)-CHBE of highly-solid selectively.
Beneficial effect: the present invention is applied to the sorbose reductase of aminoacid sequence shown in SEQ ID NO:2 obtain good effect among COBE asymmetric reduction preparation (S)-CHBE first, and its enzyme work is up to 5.6U/mg.The sorbose reductase of aminoacid sequence shown in SEQ ID NO:2 is to the yield height (greater than 90%) of substrate COBE, the optical activity height of product C HBE (e.e% is 100%), and the output height, greatly reduces production cost.
Description of drawings:
Fig. 1 is the design of graphics of sorbose reductase gene.
Embodiment:
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used for explanation the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1:
Obtaining of step 1, sorbose reductase gene
Candida albicans
Candida albicans(purchasing the Centre in Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversiry), substratum YPD(gL
-1): yeast extract 10g, peptone 20g, glucose 20g mends distilled water to 1L.
With Candida albicans
Candida albicansBe inoculated in the 5mLYPD liquid nutrient medium 30 ℃ and be cultured to logarithmic phase, use genome DNA extracting reagent kit (sky, Beijing is that biological Engineering Co., Ltd yeast genes group is extracted test kit) to extract genome.
The used primer of construction of expression vector adds restriction enzyme site, and primer sequence is as follows:
Upstream primer (SOU1-sense contains NdeI) is: 5'-GGAATTCCATATGATGAGTGAAGAAATCATTTCA-3'
Downstream primer (SOU1-anti contains EcoRI) is: 5'-CCGGAATTCTTATGGACATGTATAACCCCCAT-3'
All primers are synthetic by Mei Ji biotech firm.
The PCR condition of gene:
94 ℃ of sex change 7 min, circulate 30 times by following parameter: 94 ℃ of sex change 1 min, 60 ℃ of annealing 50 s, 72 ℃ are extended 1.5 min.Last 72 ℃ are extended 10 min.
Step 2, expression of gene
With Nde I and EcoRI respectively enzyme cut pET-22b (pET-22b purchases in Novagen (Merck China)) and increase and contain the goal gene of two restriction enzyme sites, glue reclaims purpose fragment and the expression vector of double digestion respectively, the expression vector pET-22b of double digestion is connected with the T4 ligase enzyme with goal gene spends the night, the connection product pET-22b-SOU1 of 10uL is added in Rosetta (DE3) competent cell of 100uL, place 30min on ice, 42 ℃ of heat shock 90sec.Place 2min on ice.The 0.45mL substratum that adds preheating.220rpm 37℃ 1h。200uL bacterium liquid is added on the LB flat board of the penbritin contain 100 μ g/mL respectively and paraxin 37 ℃ of incubated overnight 12-16h.Make up collection of illustrative plates and see Fig. 1.
The mensuration that step 3, enzyme are lived
Picking reorganization bacterium
E.coliRosseta(pET-22b-SOU1) and the intestinal bacteria Rosseta(DE3 that sets out) to containing in the antibiotic LB liquid nutrient medium, 37 ℃ of shaking culture are spent the night.Be inoculated into respectively in the fresh medium by 2 % inoculum sizes then, 37 ℃ are cultured to OD
600Be about at 0.6 o'clock, add IPTG to final concentration 0.8 mmolL
-1, 25 ℃, 220rpm, behind abduction delivering 10 h, centrifugal (4 ℃, 5000rpm, 15min), bacterium mud is resuspended with 100mM potassiumphosphate buffering (pH7.0), ultrasonication cell (power 300W, ultrasonic 5s, intermittently 5s, be total to 5min), centrifugal (4 ℃, 12000rpm, 15min), the enzyme of measuring in the supernatant is lived.
The enzyme reaction system comprises 100mM potassium phosphate buffer (pH6.0), 5mM NADPH, and 20mM COBE, 30 ℃, the 340nm place measures the decline of light absorption value.It is the enzyme U of unit alive that enzyme work is defined as the interior needed enzyme amount of oxidation 1 μ mol NADPH of per minute.Albumen adopts the Brandford method to measure.
The result shows that intestinal bacteria Rosseta(DE3 sets out) the ratio enzyme live and be 0.12U/mg, and reorganization bacterium E.coli Rosseta(pET22b-SOU1) the ratio enzyme live and be 5.6U/mg.
Step 4, recombination bacillus coli
E.coliRosseta(pET22b-SOU1) fermentation
Picking reorganization bacterium
E.coliRosseta(pET-22b-SOU1) to containing antibiotic LB nutrient solution, 37 ℃ of shaking culture are spent the night.Be inoculated into respectively in the fresh medium by 2 % inoculum sizes then, 37 ℃ are cultured to OD
600Be about at 0.6 o'clock, add IPTG to final concentration 0.8 mmolL
-1, 25 ℃, 220rpm, behind abduction delivering 10 h, 8000 rpm, 4 ℃ of centrifugal 10 min abandon supernatant, precipitate standby.
Step 5, get above-mentioned precipitation with potassiumphosphate buffering (100 mmolL-1, pH 6.5) washed twice, take by weighing the 0.5g(weight in wet base) intestinal bacteria bacterium mud, be suspended in (pH 6.5 potassiumphosphates buffering is 1:1 with the N-BUTYL ACETATE volume ratio) in the reaction solution of total system 25 mL.Add sorbyl alcohol 1300mmol/L, COBE 200g/L, 30 ℃, 180rpm, 16h.The output of product (S)-CHBE is 180g/L, and the yield of product is: 90%, and optical purity e.e% is 100%.
The detection method of product:
For water react: after reaction finished, centrifugal 10 min of 8000 rpm separated organic layer and water layer.The careful upper strata ethyl acetate of drawing is crossed organic membrane, and mark in adding is preserved test sample.
For water/organic two phase reaction: reaction finishes back centrifugal 10 min of 8000 rpm and separates organic layer and water layer.The careful upper strata ethyl acetate of drawing is crossed organic membrane, and mark in adding is preserved test sample.
PEG-20M capillary column, internal standard substance are naphthalene.Program is: detector FID, and 210 ℃ of temperature, 210 ℃ of temperature of vaporization chamber, 150 ℃ of column temperatures, column cap is pressed 0.03MPa, hydrogen 0.05MPa, air 0.1MPa, tail blows 0.08MPa.With HPLC the opticity of (S)-4-chloro-ethyl 3-hydroxybutanoate is analyzed (chiral column Chiralcel OB, 4.6 * 250mm; Daicel Chemical Industries, Japan), testing conditions: moving phase is normal hexane: normal hexane (9:1), and wavelength 214nm, flow are 0.8mL/min,, the appearance time of R type and S type CHBE is respectively: 10.5min and 11.6min.
Embodiment 2:
As embodiment 1 step 1 to step 4, it is standby to be prepared into precipitation, get above-mentioned precipitation and cushion (100 mmolL-1 with potassiumphosphate, pH 6.5) washed twice, take by weighing the 0.5g(weight in wet base) intestinal bacteria bacterium mud, be suspended in (pH 6.5 potassiumphosphates buffering is 1:1 with the N-BUTYL ACETATE volume ratio) in the reaction solution of total system 25 mL.Add N.F,USP MANNITOL 1300mmol/L, COBE 200g/L, 30 ℃, 180rpm, 16h.The output of product (S)-CHBE is 185g/L, and the yield of product is: 92.5%, and optical purity e.e% is 100%.
The detection method of product is with embodiment 1
Embodiment 3:
As embodiment 1 step 1 to step 4, it is standby to be prepared into precipitation, get above-mentioned precipitation and cushion (100 mmolL-1 with potassiumphosphate, pH 6.5) washed twice, take by weighing the 0.5g(weight in wet base) intestinal bacteria bacterium mud, be suspended in (pH 6.5 potassiumphosphates buffering is 1:1 with the N-BUTYL ACETATE volume ratio) in the reaction solution of total system 25 mL.Add Xylitol 1300mmol/L, COBE 200g/L, 30 ℃, 180rpm, 16h.The output of product (S)-CHBE is 130g/L, and the yield of product is: 65%, and optical purity e.e% is 100%.
The detection method of product is with embodiment 1.
Sequence table
＜110〉Nanjing University of Technology
＜120〉application of a kind of sorbose reductase in producing (S)-4-chloro-3 3-hydroxyethyl butyrates
<130> njut080822
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<170> PatentIn version 3.3
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<211> 849
<212> DNA
＜213〉Candida albicans (Candida albicans)
<220>
<221> CDS
<222> (1)..(849)
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acgggatcat ctggaggaat tggttgggct gtcgccgaag catttgctca agctggtgct 180
gatgttgcca tctggtataa ttcgaaacca gcagatgcca aagctgaata tttaactgaa 240
aaatatggtg tcaaagccaa agcttataaa tgtaatgtaa ctgatcctaa tgatgtttct 300
aaagtgatta atgaaattga aaaagatttc ggtactattg atatatttgt tgctaatgct 360
ggagttgcat ggactgatgg accagaaatt gatgttcaag gctatgatca atggaaaaag 420
atcgttgatt gtgatttaaa tggagtttat tattgtgctc ataccgtggg acaaatcttt 480
aaaaagaata aatctggttc attaattatt acttcatcaa tgtcagggac aattgttaat 540
atccctcaat tacaagctcc ttataatgct gctaaagctg catgtactca tttagccaaa 600
tcattgagtg tggaatgggc tagttttggt gctagagtaa attcaatttc tccagggtat 660
atattgactg atattgctga ttttgctgat ccagaaatga aaaagaaatg gtggcaattg 720
acacctttgg gaagagaagg attaccacaa gaattagtgg gggcatattt atacttggcc 780
tcaaatgcat caacttatac tactggttca aatattgctg ttgatggggg ttatacatgt 840
ccataa 846
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<211> 282
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＜213〉Candida albicans (Candida albicans)
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MSEEIISFTNPALGPLPTKAPQLPSNVLDLFSLKGKVASVTGSSGGIGWAVAEAFAQAGADVAIWYNSKPADAKAEYLTEKYGVKAKAYKCNVTDPNDVSKVINEIEKDFGTIDIFVANAGVAWTDGPEIDVQGYDQWKKIVDCDLNGVYYCAHTVGQIFKKNKSGSLIITSSMSGTIVNIPQLQAPYNAAKAACTHLAKSLSVEWASFGARVNSISPGYILTDIADFADPEMKKKWWQLTPLGREGLPQELVGAYLYLASNASTYTTGSNIAVDGGYTCP
Claims (1)
1. the application of sorbose reductase in biological process preparation (S)-4-chloro-3 3-hydroxyethyl butyrates, it is characterized in that with the sorbose reductase of aminoacid sequence shown in SEQ ID NO:2 be catalyzer, be substrate with the 4-chloroacetyl acetacetic ester, be cofactor with NADPH, asymmetric reduction preparation (S)-4-chloro-3 3-hydroxyethyl butyrates, be about to express the reorganization bacterium of the gene shown in SEQ ID NO:1, with 200mmol/L ~ 2mol/L sorbyl alcohol, 1.5 the 4-chloroacetyl acetacetic ester of ~ 300g/L, in pH6.0 ~ 7.5,20 ~ 30 ℃, react 16 ~ 20h under 180 ~ 280rpm condition, obtain (S)-4-chloro-3 3-hydroxyethyl butyrates.
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Non-Patent Citations (8)
| Title |
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| Candida albicans SOUI encodes a sorbose reductase required for L-sorbose utilization;Jay R.Greenberg等;《Yeast》;20051231;第22卷;957-969 * |
| EAK96529.1.hypothetical protein CaO19.2896[Candida albicans SC5314].《GenBank》.2004,1. |
| hypothetical protein CaO19.2896[Candida albicans SC5314];EAK96529.1;《GenBank》;20040806;1 * |
| Jay R.Greenberg等.Candida albicans SOUI encodes a sorbose reductase required for L-sorbose utilization.《Yeast》.2005,第22卷957-969. |
| Molecular cloning and overexpression of the gene encoding an NADPH-dependent carbonyl reductase from Candida magnoliae,involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate;Yoshihiko Yasohara等;《Biosci.Biotechnol.Biochem.》;20001231;第64卷(第7期);1430-1436 * |
| N.Kizaki等.Synthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate by Escherichia coli transformant cells coexpressing the carbonyl reductase and glucose dehydrogenase genes.《Appl Microbiol Biotechnol》.2001,第55卷590-595. |
| Synthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate by Escherichia coli transformant cells coexpressing the carbonyl reductase and glucose dehydrogenase genes;N.Kizaki等;《Appl Microbiol Biotechnol》;20010407;第55卷;590-595 * |
| Yoshihiko Yasohara等.Molecular cloning and overexpression of the gene encoding an NADPH-dependent carbonyl reductase from Candida magnoliae,involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate.《Biosci.Biotechnol.Biochem.》.2000,第64卷(第7期),1430-1436. |
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