CN104846025B - A kind of method for preparing (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters - Google Patents
A kind of method for preparing (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters Download PDFInfo
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Abstract
The invention discloses a kind of method for preparing 3 beta-hydroxymethyl butyrate of (2S, 3R) 2 benzoyl aminomethyl, this method includes：Prepare the engineering bacteria of the enzyme gene containing carbonyl reduction and the engineering bacteria containing glucose dehydrogenase gene；The resting cell suspension of two kinds of engineering bacterias is prepared respectively；It is mixed after two kinds of resting cell suspensions mixing, then with 2 benzoyl aminomethyl of substrate racemic, 3 carbonyl methyl butyrate, hydrogen donor and co-factor, carries out asymmetric reduction reaction, 3 beta-hydroxymethyl butyrate of (2S, 3R) 2 benzoyl aminomethyl is made；The base sequence of carbonyl reduction enzyme gene is as shown in SEQ ID NO.1, and the base sequence of the glucose dehydrogenase gene is as shown in SEQ ID NO.2.This method can be catalyzed 2 benzoyl aminomethyl of substrate racemic, 3 carbonyl methyl butyrate reaction generation 3 beta-hydroxymethyl butyrate of (2S, 3R) 2 benzoyl aminomethyl, improve the conversion ratio and purity of product.
Description
Technical field
The present invention relates to biopharmaceutical technology more particularly to a kind of prepare (2S, 3R) -2- benzoyl aminomethyls -3-
The method of beta-hydroxymethyl butyrate.
Background technology
(2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters is a kind of beta-hydroxy esters object with optical activation
One kind of matter and chiral alcohol, shown in chemical structural formula such as formula (II).(2S, 3R) -2- benzoyl aminomethyl -3- hydroxyl fourths
Sour methyl esters is synthesis (3R, 4R) -3- [(R) -1- tert-butyl dimethyl silica ethyls] -4-AA (letter
Referred to as 4-AA, shown in chemical structural formula such as formula (III)) key starting material, 4-AA is a kind of important medical fine chemistry
Product are mainly used for synthesizing the antibiotic of all kinds of training south class, such as Imipenem, Biapenem, Meropenem and faropenem.This
A little medicinal usages are extensive, and broad spectrum high-effect antibacterial action is respectively provided with to Grain-negative and positive bacteria, aerobic bacteria, anaerobic bacteria etc., thus
People is subject to greatly to pay attention to.At present, in terms of the synthesis technology of 4-AA, with racemic 2- benzoyl aminomethyls -3- carbonyl fourths
Sour methyl esters is comparatively superior for the synthetic route of raw material, therefore, how to be established with high selectivity in chirality by reacting
The heart is the key point of reaction process.
Effect is preferably using chiral catalyst (R)-BINAP-Ru, but the method needs to use in current existing report
To precious metal ruthenium as catalyst, and need to carry out under high-temperature and high-pressure conditions, it is more demanding to reactor, therefore the route
Limit the large-scale industrial production of 4-AA.In addition, also have some reports that asymmetric reaction is carried out using biological catalysis,
Such as Peter Schneider (United States Patent (USP), US 4927507) once reported a kind of utilization Saccharomyces cerevisiae reductase 12-benzoyl
The method of aminomethyl -3- carbonyl butyrates, obtained product configuration are (2S, 3S) -2- benzoyl aminomethyls -3-hydroxybutyrate ester
(2R, 3S) -2- benzoyl aminomethyls -3-hydroxybutyrate ester, thus need to carry out configuration reversal using chemical method, operation
Cumbersome, the rate of recovery is low.
Codexis companies of the U.S. have screened a kind of carbonyl reductase AdhR, by molecular biology method to it
After having carried out a series of improvement, realize regenerating coenzyme by the use of isopropanol as cosubstrate, AdhR directly catalysis is made to prepare (2S, 3R)
The product of configuration.But profit is difficult to that coenzyme consumption and regenerated vigor is made to match in this way, so as to influence the catalysis of AdhR
Effect；Reaction is easily limited be subject to two substrates and product thermodynamical equilibrium, it is more difficult to obtain the thermodynamic condition of optimal reaction；
Excessive cosubstrate also can generate inhibitory action to enzyme and reduce catalytic efficiency, and the addition of cosubstrate can increase product separation
The complexity of purifying increases cost.
The content of the invention
The present invention provides a kind of method for preparing (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters, the party
Method can be catalyzed substrate racemic 2- benzoyl aminomethyl -3- carbonyls methyl butyrate reaction generation (2S, 3R) -2- benzene carbon amide first
Base -3-hydroxybutyrate methyl esters improves the conversion ratio and purity of product.
A kind of method for preparing (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters, including：
(1) engineering bacteria of the enzyme gene containing carbonyl reduction and the engineering bacteria containing glucose dehydrogenase gene are prepared；
(2) the resting cell suspension of two kinds of engineering bacterias is prepared respectively；
(3) after two kinds of resting cell suspensions are mixed, then with substrate racemic 2- benzoyl aminomethyl -3- carbonyl butyric acid first
Ester, hydrogen donor and co-factor mixing, carry out asymmetric reduction reaction, (2S, 3R) -2- benzoyl aminomethyl -3- hydroxyl fourths are made
Sour methyl esters；
The base sequence of the carbonyl reduction enzyme gene as shown in SEQ ID NO.1, the glucose dehydrogenase gene
Base sequence is as shown in SEQ ID NO.2.
The carbonyl reduction enzyme gene (referred to as LbADH genes), clones from wild mushroom Lactobacillus brevis,
The amino acid sequence of the protein of carbonyl reductase gene code is as shown in SEQ ID NO.7；The glucose dehydrogenase gene
(referred to as GdhBM genes) is cloned from Bacillus Megaterium, the ammonia of the protein of glucose dehydrogenase gene coding
Base acid sequence is as shown in SEQ ID NO.8.
Wherein, the engineering bacteria contains expression vector pET-30a (+), and host cell is e. coli bl21 (DE3).
The carbonyl reduction enzyme gene and the glucose dehydrogenase gene expression in e. coli bl21 (DE3) respectively,
Obtain carbonyl reductase and glucose dehydrogenase.
The carbonyl reductase and glucose dehydrogenase are in the form of the resting cell of genetic engineering bacterium as asymmetry
Catalyst in reduction reaction.The preparation method of resting cell suspension, including：The engineering bacteria is inoculated into and receives mycin containing card
Culture medium in, shaking table activation after, expand culture to OD600 values reach 0.8~1.2 when, addition derivant, continue to cultivate, from
The heart collects cell, is resuspended with buffer solution, obtains the resting cell suspension.
Preferably, the derivant is IPTG, concentration is 0.5~0.8mM.Preferably, after adding in derivant
Condition of culture is：Cultivation temperature is 16~25 DEG C, and incubation time is 10~20h.The buffer solution is sodium dihydrogen phosphate-phosphoric acid
Disodium hydrogen, preferably, the concentration of the buffer solution is 100~150mM；Also containing 1~10mM's in the buffer solution
MgCl2。
The reaction equation of asymmetric reduction reaction of the present invention, it is as follows：
In entire reaction process, on the one hand, carbonyl reductase LbADH is catalyzed racemic 2- benzoyl aminomethyl -3- carbonyls
Pure (2S, the 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters of base methyl butyrate asymmetric reduction generation alloisomerism, together
When with reduced form co-factor NADPH be converted into oxidized form co-factor NADP+Process, on the other hand, glucose dehydrogenase
Glucose is oxidized to gluconolactone, while has regenerated reduced form co-factor NADPH, forms a co-factor consumption and regeneration
Closed circuit, promote the progress of main reaction.
In asymmetric reduction reaction, the hydrogen donor is glucose, and the co-factor is NADP+/NADPH。
In reaction process, the concentration of carbonyl reductase and glucose dehydrogenase has an impact the yield of final product.As
It is preferred that in the mixed liquor of two kinds of resting cell suspensions, the resting cell containing carbonyl reductase and the tranquillization containing glucose dehydrogenase
The mass ratio of cell is 4: 1~1: 2.It is further preferred that the resting cell containing carbonyl reductase and the tranquillization containing glucose dehydrogenase are thin
The mass ratio of born of the same parents is 1: 1.
Specifically, in asymmetric reduction reaction, the concentration of substrate be 2.5~500g/L, the tranquillization containing carbonyl reductase
The concentration of cell is 0.1gDry weight/ L~25gDry weight/ L, the concentration of the resting cell containing glucose dehydrogenase is 0.1gDry weight/ L~
25gDry weight/ L, the concentration of hydrogen donor is 5~750g/L, and the concentration of co-factor is 0~0.5mM.
Preferably, the temperature of the asymmetric reduction reaction is 25~40 DEG C, reaction buffer pH is 6.0~8.0.More
It is preferred that reaction temperature is 30~37 DEG C, reaction buffer pH is 6.5~7.5.
In entire reaction process, after reaction is completely depleted to HPLC detection substrates, with isometric organic solvent extraction 2
~4 times, merge extraction phase, anhydrous sodium sulfate drying, vacuum distillation removes organic solvent, obtains target product.
Compared with prior art, the invention has the advantages that：
(1) it is of the invention by the engineering bacteria of the LbADH containing carbonyl reductase of structure and the engineering of the GdhBM containing glucose dehydrogenase
Bacterium is applied to catalysis substrate racemic 2- benzoyl aminomethyl -3- carbonyls methyl butyrate generation (2S, 3R) -2- benzene carbon amide first
In the reaction of base -3-hydroxybutyrate methyl esters, provided for the production of (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters
A kind of new preparation approach；
(2) present invention is combined using carbonyl reductase LbADH and glucose dehydrogenase GdhBM, is made in reaction process
Coenzyme consumes and regenerated vigor matches, and obtains optimal preparation (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate
The method of methyl esters improves the conversion ratio and purity of product.
Description of the drawings
Fig. 1 is the electrophoretogram of gene Lbadh of the present invention；
M：Nucleic acid Marker, 1,2：Gene Lbadh samples.
Fig. 2 is the electrophoretogram of gene GdhBM of the present invention；
M：Nucleic acid Marker, 1,2：Gene GdhBM samples.
Fig. 3 is the collection of illustrative plates of plasmid pET30-LbADH.
Fig. 4 is the collection of illustrative plates of plasmid pET30-GdhBM.
Fig. 5 is the protein s DS-PAGE electrophoretograms of genetic engineering bacterium EcoLbADH induced expressions；
M：Protein Marker, 1：The broken born of the same parents' supernatant of pET-30a (+) empty plasmid control, 2：Genetic engineering bacterium EcoLbADH
Induction thalline breaks born of the same parents' supernatant, and 3：Genetic engineering bacterium EcoLbADH induction thalline break born of the same parents' precipitation.
Fig. 6 is the protein s DS-PAGE electrophoretograms of genetic engineering bacterium EcoGdhBM induced expressions；
M：Protein Marker, 1：Genetic engineering bacterium EcoGdhBM induction thalline break born of the same parents' supernatant, and 2：Genetic engineering bacterium
EcoGdhBM induction thalline break born of the same parents' precipitation.
Fig. 7 is that the HPLC of 2- benzoyl aminomethyl -3- carbonyl methyl butyrate standard items analyzes collection of illustrative plates.
Fig. 8 is that the HPLC of (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methacrylate calibration analyzes collection of illustrative plates.
Fig. 9 is the HPLC analytical standard collection of illustrative plates that (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters surveys ee values.
Figure 10 is 2- benzoyl aminomethyl -3- carbonyl methyl butyrates1H NMR spectras.
Figure 11 is reaction product (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters1H NMR spectras.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and detailed description.
The structure of 1 plasmid pET30-LbADH of embodiment
With primers F _ LbADH/R_LbADH clone's Lbadh genes, Lbadh genes (such as SEQ that length is 759bp is obtained
Shown in ID NO.1).Nucleic acid electrophoresis verifies gene size, such as Fig. 1.
The sequence of primers F _ LbADH is：5’CGCGGATCCATGTCTAACCGTTTGGATG-3’；
The sequence of primer R_LbADH is：5’CCGCTCGAGCTATTGAGCAGTGTAGCCAC-3’。
BamHI and XhoI double digestion Lbadh genes recycle the gene band after digestion, BamHI and XhoI double digestions pET-
30a (+) plasmid recycles the plasmid band after digestion, by the Lbadh genes after digestion and pET-30a (+) plasmid after digestion,
It is connected with ligase, transformed clone host Escherichia coli DH5 α.Bacterium colony is carried out with primers F _ LbADH/R_LbADH
PCR, verification conversion recon, then extracts recombinant plasmid, is sequenced.The errorless recombinant plasmid of sequencing result, as recombinates
Plasmid pET30-LbADH, plasmid map is as shown in figure 3, -20 DEG C save backup.
The structure of 2 plasmid pET30-GdhBM of embodiment
With primers F _ GdhBM/R_GdhBM clone's GdhBM genes, GdhBM genes (such as SEQ that length is 786bp is obtained
Shown in ID NO.2).Nucleic acid electrophoresis verifies gene size, such as Fig. 2.
The sequence of primers F _ GdhBM is：5’-GGAAGATCTGATGTATAAAGATTTAGAAGGA-3’；
The sequence of primer R_GdhBM is：5’CCGCTCGAGTTATCCGCGTCCTGCTTGGAA-3’。
GlII and XhoI double digestion GdhBM genes recycle the gene band after digestion, BglII and XhoI double digestions pET-
30a (+) plasmid recycles the plasmid band after digestion, by the GdhBM genes after digestion and pET-30a (+) plasmid after digestion,
It is connected with ligase, transformed clone host Escherichia coli DH5 α.Bacterium colony is carried out with primers F _ GdhBM/R_GdhBM
PCR, verification conversion recon, then extracts recombinant plasmid, is sequenced.The errorless recombinant plasmid of sequencing result, as recombinates
Plasmid pET30-GdhBM, plasmid map is as shown in figure 4, -20 DEG C save backup.
The structure and induced expression of 3 genetic engineering bacterium of embodiment
With the plasmid pET30-LbADH and pET30-GdhBM built in example 1 and 2, expressive host is converted respectively
Escherichia coli BL21(DE3).Bacterium colony is done respectively with primers F _ LbADH/R_LbADH and F_GdhBM/R_GdhBM
PCR verifies the recon of conversion.It is EcoLbADH and EcoGdhBM to verify errorless genetic engineering bacterium.By EcoLbADH and
EcoGdhBM be inoculated into respectively received containing card chloramphenicol resistance 3~5mL liquid LB Tube propagation bases in, at 35 DEG C shaking table activation 12
Hour, the culture obtained after activation is transferred to by 1% switching amount received containing card chloramphenicol resistance liquid LB Shake flask mediums in,
Isothermal vibration culture 3h in fermentation medium, condition of culture are 37 DEG C, 200rpm.Treat that cell concentration grows to OD600When=0.8, add
Enter 0.5mM IPTG (final concentration), 16 DEG C of induction 16h, 10,000g centrifugation 5min collect cell, with pH7.0 sodium phosphate buffers
After washing 1 time, supernatant is abandoned to get resting cell, -80 DEG C is placed in and freezes.Protein expression situation is detected with SDS-pAGE, such as Fig. 5
Shown in 6.
The structure and induced expression of 4 genetic engineering bacterium of embodiment
With the plasmid pET30-LbADH and pET30-GdhBM built in example 1 and 2, expressive host is converted respectively
Escherichia coli BL21(DE3).Bacterium colony is done respectively with primers F _ LbADH/R_LbADH and F_GdhBM/R_GdhBM
PCR verifies the recon of conversion.It is EcoLbADH and EcoGdhBM to verify errorless genetic engineering bacterium.By EcoLbADH and
EcoGdhBM be inoculated into respectively received containing card chloramphenicol resistance 3~5mL liquid LB Tube propagation bases in, at 40 DEG C shaking table activation 8
Hour, the culture obtained after activation is transferred to by 1% switching amount received containing card chloramphenicol resistance liquid LB Shake flask mediums in,
Isothermal vibration culture 3h in fermentation medium, condition of culture are 35 DEG C, 200rpm.Treat that cell concentration grows to OD600When=1.2, add
Enter 0.8mM IPTG (final concentration), 25 DEG C of induction 10h, 10,000g centrifugation 5min collect cell, with pH7.0 sodium phosphate buffers
After washing 1 time, supernatant is abandoned to get resting cell, -80 DEG C is placed in and freezes.
5 reaction process monitoring method of embodiment
2- benzoyl aminomethyls -3- carbonyls methyl butyrate is measured to (2S, 3R) -2- benzene carbon amides using HPLC detection methods
The conversion situation of methyl -3-hydroxybutyrate methyl esters.Sample treatment：Take 50 μ L of different time points reaction solution, add in acetonitrile 150~
950 μ L, mixing centrifuge 5min after 12,000g, supernatant are taken to treat sample detection with 0.45 μm of filtering with microporous membrane.Chromatographic condition
For：Chromatographic column：Pursuit C18(150*4.6mm)；Mobile phase：10mM ammonium acetate solutions: acetonitrile=55: 45 (v/v)；Stream
Speed：0.5mL/min；Sample size：5μL；Detection wavelength：254nm.HPLC collection of illustrative plates is as shown in Figure 7.2- benzoyl aminomethyl -3- carbonyls
The retention time of base methyl butyrate is 9.811min, and the H spectrums of Structural Identification are as shown in Figure 10；(2S, 3R) -2- benzene carbon amide first
The retention time of base -3-hydroxybutyrate methyl esters is 7.438min, and the H spectrums of Structural Identification are as shown in figure 11.
6 product ee values determination methods of embodiment
The ee values of target product (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters are measured using chirality HPLC's
Method measures.Sample treatment：Take 100 μ L of different time points reaction solution, ethyl acetate extracts three times, combining extraction liquid, with full
And NaHCO3It respectively washed once with saturation NaCl, anhydrous sodium sulfate drying water removal, filtering, revolving is dissolved in 2mL except solvent, residue
Chromatographic grade absolute ethyl alcohol treats that HPLC is detected.
Chiral HPLC condition is as follows：Chromatographic column：Chiralpak ID(4.6×250mm)；Mobile phase：N-hexane: different
Propyl alcohol: trifluoroacetic acid=80: 20: 0.1；Flow velocity：0.5ml/min；Sample size：5μL；Detection wavelength：254nm.Four kinds of configuration productions
The HPLC collection of illustrative plates of object is as shown in Figure 9.The retention time of (2S, 3R) configuration is 38.34min, and the retention time of (2R, 3S) configuration is
49.31min, the retention time of (2R, 3R) configuration is 54.14min, and the retention time of (2S, 3S) configuration is 57.97min.
The preparation of embodiment 7 (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters
The resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM are taken, with the 100mM's of pH6.0
Sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution is resuspended.0.1g is added in the three neck round bottom flask of 50mLDry weightThe LbADH tranquillization of/L
Cell and 0.1gDry weightThe GdhBM resting cells of/L in addition it is 20mL to state buffer solution and mend to total volume, then add substrate racemic 2- benzene
The NADP of formyl aminomethyl -3- carbonyls methyl butyrate 50mg, cosubstrate glucose 100mg and final concentration of 0.2mM+, then
It is placed in 37 DEG C of constant temperature water baths and is stirred to react for 24 hours, during which adjusting pH value of reaction system with 1M sodium hydrate aqueous solutions maintains
6.0.After reaction, reaction solution is centrifuged removal somatic cells, 0.45 μm of micro-filtrate membrane filtration of supernatant, with HPLC points
Analysis, the yield of product (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters is 90.3%, ee 86.9%.
The preparation of embodiment 8 (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters
The resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM are taken, with the 100mM's of pH7.0
Sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution is resuspended.1g is added in the three neck round bottom flask of 50mLDry weightThe LbADH tranquillization of/L is thin
Born of the same parents and 1gDry weightThe GdhBM resting cells of/L in addition it is 20mL to state buffer solution and mend to total volume, then add substrate racemic 2- benzoyls
The NADP of aminomethyl -3- carbonyls methyl butyrate 500mg, cosubstrate glucose 1g and final concentration of 0.2mM+, it is subsequently placed in 30
It is stirred to react for 24 hours in DEG C constant temperature water bath, during which adjusting pH value of reaction system with 1M sodium hydrate aqueous solutions maintains 7.0.Instead
After answering, reaction solution is centrifuged removal somatic cells, and 0.45 μm of micro-filtrate membrane filtration of supernatant is analyzed with HPLC, is produced
The yield of object (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters is 80.5%, ee 89.6%.
The preparation of embodiment 9 (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters
The resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM are taken, with the 100mM's of pH8.0
Sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution is resuspended.5g is added in the three neck round bottom flask of 50mLDry weightThe LbADH tranquillization of/L is thin
Born of the same parents and 5gDry weightThe GdhBM resting cells of/L in addition it is 20mL to state buffer solution and mend to total volume, then add substrate racemic 2- benzoyls
The NADP of aminomethyl -3- carbonyls methyl butyrate 2g, cosubstrate glucose 4g and final concentration of 0.2mM+, it is subsequently placed in 40 DEG C of perseverances
12h is stirred to react in warm water bath, during which adjusting pH value of reaction system with 1M sodium hydrate aqueous solutions maintains 8.0.Reaction knot
Shu Hou, reaction solution are centrifuged removal somatic cells, and 0.45 μm of micro-filtrate membrane filtration of supernatant is analyzed, product with HPLC
The yield of (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters is 73.5%, ee 91.4%.
The preparation of embodiment 10 (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters
The resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM are taken, with the 100mM's of pH7.0
Sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution is resuspended.10g is added in the three neck round bottom flask of 50mLDry weightThe LbADH tranquillization of/L
Cell and 10gDry weightThe GdhBM resting cells of/L in addition it is 20mL to state buffer solution and mend to total volume, then add substrate racemic 2- benzene
The NADP of formyl aminomethyl -3- carbonyls methyl butyrate 5g, cosubstrate glucose 10g and final concentration of 0.5mM+, it is subsequently placed in
It is stirred to react for 24 hours in 25 DEG C of constant temperature water baths, during which adjusting pH value of reaction system with 1M sodium hydrate aqueous solutions maintains 7.0.
After reaction, reaction solution is centrifuged removal somatic cells, and 0.45 μm of micro-filtrate membrane filtration of supernatant is analyzed with HPLC,
The yield of product (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters is 75.2%, ee 90.2%.
The preparation of embodiment 11 (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters
The resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM are taken, with the 100mM's of pH7.0
Sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution is resuspended.20g is added in the three neck round bottom flask of 50mLDry weightThe LbADH tranquillization of/L
Cell and 20gDry weightThe GDHBM resting cells of/L in addition it is 20mL to state buffer solution and mend to total volume, then add substrate racemic 2- benzene
The NADP of formyl aminomethyl -3- carbonyls methyl butyrate 10g, cosubstrate glucose 15g and final concentration of 0.5mM+, it is subsequently placed in
It is stirred to react for 24 hours in 30 DEG C of constant temperature water baths, during which adjusting pH value of reaction system with 1M sodium hydrate aqueous solutions maintains 7.0.
After reaction, reaction solution is centrifuged removal somatic cells, and 0.45 μm of micro-filtrate membrane filtration of supernatant is analyzed with HPLC,
The yield of product (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters is 83.2%, ee 87.6%.
The preparation of embodiment 12 (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters
The resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM are taken, with the 100mM's of pH7.0
Sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution is resuspended.25g is added in the three neck round bottom flask of 50mLDry weightThe LbADH tranquillization of/L
Cell and 25gDry weightThe GdhBM resting cells of/L in addition it is 20mL to state buffer solution and mend to total volume, then add substrate racemic 2- benzene
The NADP of formyl aminomethyl -3- carbonyls methyl butyrate 10g, cosubstrate glucose 15g and final concentration of 0.5mM+, it is subsequently placed in
It is stirred to react for 24 hours in 37 DEG C of constant temperature water baths, during which adjusting pH value of reaction system with 1M sodium hydrate aqueous solutions maintains 7.0.
After reaction, reaction solution is centrifuged removal somatic cells, and 0.45 μm of micro-filtrate membrane filtration of supernatant is analyzed with HPLC,
The yield of product (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters is 93.6%, ee 84.5%.
The preparation of embodiment 13 (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters
The resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM are taken, with the 100mM's of pH7.0
Sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution is resuspended.1g is added in the three neck round bottom flask of 50mLDry weightThe LbADH tranquillization of/L is thin
Born of the same parents and 0.5gDry weightThe GdhBM resting cells of/L in addition it is 20mL to state buffer solution and mend to total volume, then add substrate racemic 2- benzene first
The NADP of acyl aminomethyl -3- carbonyls methyl butyrate 500mg, cosubstrate glucose 1g and final concentration of 0.2mM+, it is subsequently placed in
It is stirred to react for 24 hours in 30 DEG C of constant temperature water baths, during which adjusting pH value of reaction system with 1M sodium hydrate aqueous solutions maintains 7.0.
After reaction, reaction solution is centrifuged removal somatic cells, and 0.45 μm of micro-filtrate membrane filtration of supernatant is analyzed with HPLC,
The yield of product (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters is 73.8%, ee 91.6%.
The preparation of embodiment 14 (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters
The resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM are taken, with the 100mM's of pH7.0
Sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution is resuspended.1g is added in the three neck round bottom flask of 50mLDry weightThe LbADH tranquillization of/L is thin
Born of the same parents and 2gDry weightThe GdhBM resting cells of/L in addition it is 20mL to state buffer solution and mend to total volume, then add substrate racemic 2- benzoyls
The NADP of aminomethyl -3- carbonyls methyl butyrate 500mg, cosubstrate glucose 1g and final concentration of 0.2mM+, it is subsequently placed in 30
It is stirred to react for 24 hours in DEG C constant temperature water bath, during which adjusting pH value of reaction system with 1M sodium hydrate aqueous solutions maintains 7.0.Instead
After answering, reaction solution is centrifuged removal somatic cells, and 0.45 μm of micro-filtrate membrane filtration of supernatant is analyzed with HPLC, is produced
The yield of object (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters is 83.5%, ee 87.3%.
Comparative example 1
Taking a certain amount of engineering bacteria EcoLkADH, (engineering bacteria contains clone from Lactobacillus kefiri DSM
20587 carbonyl reduction enzyme gene, specific construction method is with embodiment 1, resting cell 3), with the phosphoric acid of the 100mM of pH7.0
Sodium dihydrogen-disodium hydrogen phosphate buffer solution is resuspended.1g is added in the three neck round bottom flask of 50mLDry weightThe resting cell of/L LkADH,
Again plus substrate racemic 2- benzoyl aminomethyl -3- carbonyl methyl butyrate 500mg, cosubstrate isopropanol 1mL, in addition stating buffering
It is 20mL that liquid, which is mended to total volume, is eventually adding the NADP of final concentration of 0.2mM+, it is subsequently placed in 30 DEG C of constant temperature water baths and stirs
Reaction is for 24 hours.After reaction, reaction solution is centrifuged removal somatic cells, and 0.45 μm of micro-filtrate membrane filtration of supernatant is used
HPLC is analyzed, and the yield of product (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters is 31.6%, ee 95.7%.
Comparative example 2
Taking a certain amount of engineering bacteria EcoCR, (engineering bacteria contains clone from Candida magnoliae CGMCC
2.1919 carbonyl reduction enzyme gene, specific construction method with embodiment 1,3) and the resting cell of EcoGdhBM, with pH7.0's
The sodium dihydrogen phosphate of 100mM-disodium hydrogen phosphate buffer solution is resuspended.1g is added in the three neck round bottom flask of 50mLDry weight/L CR's
Resting cell and 1gDry weightThe resting cell of/L GdhBM in addition it is 20mL to state buffer solution and mend to total volume, then adds substrate racemic
The NADP of 2- benzoyl aminomethyl -3- carbonyls methyl butyrate 500mg, cosubstrate glucose 1g and final concentration of 0.2mM+, so
It is placed in 30 DEG C of constant temperature water baths and is stirred to react for 24 hours, during which adjusting pH value of reaction system with 1M sodium hydrate aqueous solutions maintains
7.0.After reaction, reaction solution is centrifuged removal somatic cells, and 0.45 μm of micro-filtrate membrane filtration of supernatant uses HPLC
Analysis, the yield of product (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters is 0.13%, ee 0.
Comparative example 3
Taking a certain amount of engineering bacteria EcoDkR, (engineering bacteria contains clone from Acinetobacter baylyi
The carbonyl reduction enzyme gene of EU273886.1, specific construction method use pH7.0 with embodiment 1,3) with the resting cell of EGdhBM
The sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution of 100mM be resuspended.1g is added in the three neck round bottom flask of 50mLDry weight/ L's
DkR resting cells and 1gDry weightThe GdhBM resting cells of/L in addition it is 20mL to state buffer solution and mend to total volume, then adds and disappear outside substrate
Revolve the NADP of 2- benzoyl aminomethyl -3- carbonyls methyl butyrate 500mg, cosubstrate glucose 1g and final concentration of 0.2mM+,
It is subsequently placed in 30 DEG C of constant temperature water baths and is stirred to react for 24 hours, during which adjusting pH value of reaction system with 1M sodium hydrate aqueous solutions ties up
It holds 7.0.After reaction, reaction solution is centrifuged removal somatic cells, and 0.45 μm of micro-filtrate membrane filtration of supernatant is used
HPLC is analyzed, and the yield of product (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters is 0.31%, ee 0.
Comparative example 4
Taking a certain amount of engineering bacteria EcoXRADH, (engineering bacteria contains clone from Rubrobacter xylanophilus
The carbonyl reduction enzyme gene of DSM9941, specific construction method with embodiment 1,3) and the resting cell of GdhBM, with pH7.0's
The sodium dihydrogen phosphate of 100mM-disodium hydrogen phosphate buffer solution is resuspended.1g is added in the three neck round bottom flask of 50mLDry weight/ L's
XRADH resting cells and 1gDry weightThe GdhBM resting cells of/L in addition it is 20mL to state buffer solution and mend to total volume, then add outside substrate
The NADP of racemization 2- benzoyl aminomethyl -3- carbonyls methyl butyrate 500mg, cosubstrate glucose 1g and final concentration of 0.2mM+, it is subsequently placed in 30 DEG C of constant temperature water baths and is stirred to react for 24 hours, during which adjusts pH value of reaction system with 1M sodium hydrate aqueous solutions
Maintain 7.0.After reaction, reaction solution is centrifuged removal somatic cells, and 0.45 μm of micro-filtrate membrane filtration of supernatant is used
HPLC is analyzed, and the yield of product (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters is 0, ee 0.
Comparative example 5
Taking a certain amount of engineering bacteria EcoYDL326, (engineering bacteria contains clone from Saccharomyces cerevisiae
Carbonyl reduction enzyme gene, specific construction method with embodiment 1,3) and the resting cell of engineering bacteria EcoGdhBM, with pH7.0's
The sodium dihydrogen phosphate of 100mM-disodium hydrogen phosphate buffer solution is resuspended.1g is added in the three neck round bottom flask of 50mLDry weight/ L's
YDL326 resting cells and 1gDry weightThe GdhBM resting cells of/L in addition it is 20mL to state buffer solution and mend to total volume, then add outside substrate
The NADP of racemization 2- benzoyl aminomethyl -3- carbonyls methyl butyrate 500mg, cosubstrate glucose 1g and final concentration of 0.2mM+, it is subsequently placed in 30 DEG C of constant temperature water baths and is stirred to react for 24 hours, during which adjusts pH value of reaction system with 1M sodium hydrate aqueous solutions
Maintain 7.0.After reaction, reaction solution is centrifuged removal somatic cells, and 0.45 μm of micro-filtrate membrane filtration of supernatant is used
HPLC is analyzed, and the yield of product (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters is 1.72%, ee 0.
Claims (1)
- A kind of 1. method for preparing (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters, which is characterized in that including：(1) engineering bacteria of the enzyme gene containing carbonyl reduction and the engineering bacteria containing glucose dehydrogenase gene are prepared；The engineering bacteria Containing expression vector pET-30a (+), host cell is e. coli bl21 (DE3)；(2) the resting cell suspension of two kinds of engineering bacterias is prepared respectively；The preparation method of resting cell suspension, including：It will be described Engineering bacteria be inoculated into received containing card mycin culture medium in, shaking table activation after, expand culture to OD600 values reach 0.8~1.2 when, Derivant is added in, continues to cultivate, cell is collected by centrifugation, be resuspended with buffer solution, obtains the resting cell suspension；Described lures Agent is led as IPTG, the concentration of derivant is 0.5~0.8mM；In the mixed liquor of two kinds of resting cell suspensions, containing carbonyl reductase The mass ratio of resting cell and the resting cell containing glucose dehydrogenase is 4：1~1：2；(3) after two kinds of resting cell suspensions are mixed, then with substrate racemic 2- benzoyl aminomethyl -3- carbonyls methyl butyrate, Hydrogen donor and co-factor mixing, carry out asymmetric reduction reaction, (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate first are made Ester；The base sequence of the carbonyl reduction enzyme gene is as shown in SEQ ID NO.1, the base of the glucose dehydrogenase gene Sequence is as shown in SEQ ID NO.2；The hydrogen donor is glucose, and the co-factor is NADP+/NADPH；In asymmetric reduction reaction, the concentration of substrate is 2.5~500g/L, and the concentration of the resting cell containing carbonyl reductase is 0.1gDry weight/ L~25gDry weight/ L, the concentration of the resting cell containing glucose dehydrogenase is 0.1gDry weight/ L~25gDry weight/ L, hydrogen donor Concentration is 5~750g/L, and the concentration of co-factor is 0~0.5mM；The temperature of the asymmetric reduction reaction is 25~40 DEG C, instead It is 6.0~8.0 to answer pH of buffer.
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| CN1940079A (en) * | 2006-09-08 | 2007-04-04 | 鲁南制药集团股份有限公司 | Synthesis of (2S,3S)-2-benzoyl aminometh-3-hydroxy-butyrate ester series compound by asymmetric yeast cell |
| CN101855342A (en) * | 2007-09-13 | 2010-10-06 | 科德克希思公司 | The Ketoreductase polypeptides that is used for reduction of acetophenones |
| CN101883846A (en) * | 2007-10-01 | 2010-11-10 | 科德克希思公司 | Ketoreductase polypeptides for the production of azetidinone |
| CN104388373A (en) * | 2014-12-10 | 2015-03-04 | 江南大学 | Construction of escherichia coli system with coexpression of carbonyl reductase Sys1 and glucose dehydrogenase Sygdh |
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| CN1940079A (en) * | 2006-09-08 | 2007-04-04 | 鲁南制药集团股份有限公司 | Synthesis of (2S,3S)-2-benzoyl aminometh-3-hydroxy-butyrate ester series compound by asymmetric yeast cell |
| CN101855342A (en) * | 2007-09-13 | 2010-10-06 | 科德克希思公司 | The Ketoreductase polypeptides that is used for reduction of acetophenones |
| CN101883846A (en) * | 2007-10-01 | 2010-11-10 | 科德克希思公司 | Ketoreductase polypeptides for the production of azetidinone |
| CN104388373A (en) * | 2014-12-10 | 2015-03-04 | 江南大学 | Construction of escherichia coli system with coexpression of carbonyl reductase Sys1 and glucose dehydrogenase Sygdh |
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