CN104846025A - Method for preparing (2S, 3R)-2-benzoyl aminomethyl-3-hydroxy methyl butyrate - Google Patents
Method for preparing (2S, 3R)-2-benzoyl aminomethyl-3-hydroxy methyl butyrate Download PDFInfo
- Publication number
- CN104846025A CN104846025A CN201510149171.5A CN201510149171A CN104846025A CN 104846025 A CN104846025 A CN 104846025A CN 201510149171 A CN201510149171 A CN 201510149171A CN 104846025 A CN104846025 A CN 104846025A
- Authority
- CN
- China
- Prior art keywords
- resting cell
- benzoyl aminomethyl
- gene
- dry weight
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing (2S, 3R)-2-benzoyl aminomethyl-3-hydroxy methyl butyrate. The method comprises the following steps: preparing carbonyl reductase gene-containing engineering bacterium and glucose dehydrogenase gene-containing engineering bacterium; respectively preparing the resting cell suspensions of two engineering bacteria; mixing two resting cell suspensions of two engineering bacteria, mixing a substrate racemic 2-benzoyl aminomethyl-3-carbonyl methyl butyrate, a hydrogen donor and a cofactor, and performing an asymmetric reduction reaction to prepare the (2S, 3R)-2-benzoyl aminomethyl-3-hydroxy methyl butyrate; wherein a base sequence of the carbonyl reductase gene is shown as a SEQ ID NO.1, and the base sequence of the glucose dehydrogenase gene is shown as a SEQ ID NO.2. The method is capable of catalyzing the substrate racemic 2-benzoyl aminomethyl-3-carbonyl methyl butyrate to react for generating (2S, 3R)-2-benzoyl aminomethyl-3-hydroxy methyl butyrate, and can increase the conversion rate and purity of the products.
Description
Technical field
The present invention relates to biological pharmacy technical field, particularly relate to the method that one prepares (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters.
Background technology
(2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is that one has optically active beta-hydroxy Ester, is also the one of chiral alcohol, and its chemical structural formula is such as formula shown in (II).(2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is synthesis (3R, 4R)-3-[(R)-1-tert-butyl dimethyl silica ethyl]-4-AA is (referred to as 4-AA, chemical structural formula is such as formula (III) Suo Shi) key starting material, 4-AA is a kind of important medical fine chemicals, be mainly used in the microbiotic synthesizing all kinds of training south class, as imipenum, biapenem, meropenem and Faropenem etc.These pharmaceutical uses are extensive, all have broad spectrum high-effect anti-microbial effect, be thus subject to people and greatly pay attention to Grain-negative and positive bacteria, aerophil, anerobe etc.At present, in the synthesis technique of 4-AA, be comparatively superior with the synthetic route that racemic 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate is raw material, therefore, how set up by reaction highly selective the key point that chiral centre is reaction process.
In current existing report, effect is preferably and uses chiral catalyst (R)-BINAP-Ru, but this method needs to use noble metal ruthenium as catalyzer, and need to carry out under high-temperature and high-pressure conditions, require higher to reactor, therefore this route limits the large-scale industrial production of 4-AA.In addition, also some are had to utilize biological catalysis to carry out the report of asymmetric reaction, as (United States Patent (USP)s such as Peter Schneider, US 4927507) once reported a kind of method utilizing bread yeast reductase 12-benzoyl aminomethyl-3-carbonyl butyric ester, the product configuration obtained is (2S, 3S)-2-benzoyl aminomethyl-3-hydroxybutyrate ester and (2R, 3S)-2-benzoyl aminomethyl-3-hydroxybutyrate ester, thus need to carry out configuration reversal through chemical process again, complex operation, the rate of recovery is low.
Codexis company of the U.S. has screened a kind of carbonyl reductase AdhR, after a series of improvement having been carried out to it on molecular biology method, utilize Virahol to realize regenerating coenzyme as cosubstrate, make the product of the direct catalysis preparation of AdhR (2S, 3R) configuration.But profit is difficult to make the vigor of coenzyme consumption and regeneration match in this way, thus affects the catalytic effect of AdhR; Reaction is easily subject to the restriction of two substrates and product thermodynamic(al)equilibrium, the thermodynamic condition of more difficult acquisition optimal reaction; Excessive cosubstrate also can produce restraining effect to enzyme and reduce catalytic efficiency, and the complicacy adding membership increase product separation purifying of cosubstrate, increase cost.
Summary of the invention
The invention provides one and prepare (2S, 3R) the method for-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters, the method the reaction of catalytic substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate can generate (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters, improves transformation efficiency and the purity of product.
One prepares the method for (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters, comprising:
(1) engineering bacteria of preparation containing carbonyl reductase gene and the engineering bacteria containing glucose dehydrogenase gene;
(2) the resting cell suspension of two kinds of engineering bacterias is prepared respectively;
(3) by after two kinds of resting cell suspension mixing, mix with substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate, hydrogen donor and cofactor again, carry out asymmetric reduction reaction, obtained (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters;
The base sequence of described carbonyl reductase gene is as shown in SEQ ID NO.1, and the base sequence of described glucose dehydrogenase gene is as shown in SEQ ID NO.2.
Described carbonyl reductase gene (referred to as LbADH gene), clones from wild mushroom Lactobacillus brevis, and the aminoacid sequence of the protein of carbonyl reductase genes encoding is as shown in SEQ ID NO.7; Described glucose dehydrogenase gene (referred to as GdhBM gene), clones from Bacillus Megaterium, and the aminoacid sequence of the protein of glucose dehydrogenase gene coding is as shown in SEQ ID NO.8.
Wherein, described engineering bacteria contains expression vector pET-30a (+), and host cell is e. coli bl21 (DE3).
Described carbonyl reductase gene and glucose dehydrogenase gene are expressed respectively in e. coli bl21 (DE3), obtain carbonyl reductase and Hexose phosphate dehydrogenase.
Described carbonyl reductase and Hexose phosphate dehydrogenase are all using the resting cell form of genetic engineering bacterium as the catalyzer in asymmetric reduction reaction.The preparation method of resting cell suspension, comprise: described engineering bacteria is inoculated into and receives in the substratum of mycin containing card, after shaking table activation, when enlarged culturing to OD600 value reaches 0.8 ~ 1.2, add inductor, continue to cultivate, centrifugal collecting cell, resuspended with damping fluid, the resting cell suspension described in acquisition.
As preferably, described inductor is IPTG, and concentration is 0.5 ~ 0.8mM.As preferably, adding the culture condition after inductor is: culture temperature is 16 ~ 25 DEG C, and incubation time is 10 ~ 20h.Described damping fluid is SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic, and as preferably, the concentration of described damping fluid is 100 ~ 150mM; MgCl also containing 1 ~ 10mM in described damping fluid
2.
The reaction formula of asymmetric reduction reaction of the present invention, as follows:
In whole reaction process, on the one hand, carbonyl reductase LbADH catalysis racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate asymmetric reduction generates the pure (2S of stereoisomerism, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters, simultaneous reduced form cofactor NADPH is converted into oxidized form cofactor NADP
+process, on the other hand, glucose oxidase is gluconolactone by Hexose phosphate dehydrogenase, has regenerated reduced form cofactor NADPH simultaneously, forms the loop line of a cofactor consumption and regeneration, promotes the carrying out of main reaction.
In asymmetric reduction reaction, described hydrogen donor is glucose, and described cofactor is NADP
+/ NADPH.
In reaction process, the concentration of carbonyl reductase and Hexose phosphate dehydrogenase, has impact to the yield of final product.As preferably, in the mixed solution of two kinds of resting cell suspensions, the resting cell containing carbonyl reductase is 4: 1 ~ 1: 2 with the mass ratio of the resting cell containing Hexose phosphate dehydrogenase.More preferably, be 1: 1 containing the resting cell of carbonyl reductase with the mass ratio of resting cell containing Hexose phosphate dehydrogenase.
Particularly, in asymmetric reduction reaction, described concentration of substrate is 2.5 ~ 500g/L, and the concentration containing the resting cell of carbonyl reductase is 0.1g
dry weight/ L ~ 25g
dry weight/ L, the concentration containing the resting cell of Hexose phosphate dehydrogenase is 0.1g
dry weight/ L ~ 25g
dry weight/ L, the concentration of hydrogen donor is 5 ~ 750g/L, and the concentration of cofactor is 0 ~ 0.5mM.
As preferably, the temperature of described asymmetric reduction reaction is 25 ~ 40 DEG C, and reaction buffer pH is 6.0 ~ 8.0.More preferably, temperature of reaction is 30 ~ 37 DEG C, and reaction buffer pH is 6.5 ~ 7.5.
In whole reaction process, after question response to HPLC detection substrate exhausts completely, with isopyknic organic solvent extraction 2 ~ 4 times, merge extraction phase, anhydrous sodium sulfate drying, underpressure distillation removing organic solvent, obtains target product.
Compared with prior art, the present invention has following beneficial effect:
(1) engineering bacteria containing carbonyl reductase LbADH built and the engineering bacteria containing Hexose phosphate dehydrogenase GdhBM are applied to catalytic substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate and generate (2S by the present invention, in the reaction of 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters, production for (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters provide a kind of newly prepare approach;
(2) the present invention adopts carbonyl reductase LbADH and Hexose phosphate dehydrogenase GdhBM to combine, the vigor of coenzyme consumption and regeneration in reaction process is matched, obtain best preparation (2S, 3R) the method for-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters, improves transformation efficiency and the purity of product.
Accompanying drawing explanation
Fig. 1 is the electrophorogram of gene Lbadh of the present invention;
M: nucleic acid Marker, 1,2: gene Lbadh sample.
Fig. 2 is the electrophorogram of gene GdhBM of the present invention;
M: nucleic acid Marker, 1,2: gene GdhBM sample.
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 electrophorogram of genetic engineering bacterium EcoLbADH abduction delivering;
M: the broken born of the same parents' supernatant of protein Marker, 1:pET-30a (+) empty plasmid contrast, 2: genetic engineering bacterium EcoLbADH induction thalline breaks born of the same parents' supernatant, 3: genetic engineering bacterium EcoLbADH induces thalline to break born of the same parents' precipitation.
Fig. 6 is the protein s DS-PAGE electrophorogram of genetic engineering bacterium EcoGdhBM abduction delivering;
M: protein Marker, 1: genetic engineering bacterium EcoGdhBM induction thalline breaks born of the same parents' supernatant, 2: genetic engineering bacterium EcoGdhBM induces thalline to break born of the same parents' precipitation.
Fig. 7 is that the HPLC of 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate standard substance analyzes collection of illustrative plates.
Fig. 8 is that the HPLC of (2S, 3R)-2-benzoyl aminomethyl-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 aminomethyl-3-hydroxybutyrate methyl esters surveys ee value.
Figure 10 is 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate
1h NMR spectrogram.
Figure 11 is reaction product (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters
1h NMR spectrogram.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
The structure of embodiment 1 plasmid pET30-LbADH
Clone Lbadh gene with primers F _ LbADH/R_LbADH, obtain the Lbadh gene (as shown in SEQ ID NO.1) that length is 759bp.Nucleic acid electrophoresis checking gene size, as Fig. 1.
The sequence of primers F _ LbADH is: 5 ' CGC
gGATCCaTGTCTAACCGTTTGGATG-3 ';
The sequence of primer R_LbADH is: 5 ' CCG
cTCGAGcTATTGAGCAGTGTAGCCAC-3 '.
BamHI and XhoI double digestion Lbadh gene, reclaim enzyme cut after gene band, BamHI and XhoI double digestion pET-30a (+) plasmid, reclaim enzyme cut after plasmid band, Lbadh gene after enzyme is cut and enzyme cut after pET-30a (+) plasmid, connect with ligase enzyme, transformed clone host Escherichia coli DH5 α.Carry out bacterium colony PCR with primers F _ LbADH/R_LbADH, checking transforms recon, then extracts recombinant plasmid, checks order.The recombinant plasmid that sequencing result is errorless, is recombinant plasmid pET30-LbADH, and as shown in Figure 3 ,-20 DEG C save backup plasmid map.
The structure of embodiment 2 plasmid pET30-GdhBM
Clone GdhBM gene with primers F _ GdhBM/R_GdhBM, obtain the GdhBM gene (as shown in SEQ ID NO.2) that length is 786bp.Nucleic acid electrophoresis checking gene size, as Fig. 2.
The sequence of primers F _ GdhBM is: 5 '-GGA
aGATCTGaTGTATAAAGATTTAGAAGGA-3 ';
The sequence of primer R_GdhBM is: 5 ' CCG
cTCGAGtTATCCGCGTCCTGCTTGGAA-3 '.
GlII and XhoI double digestion GdhBM gene, reclaim enzyme cut after gene band, BglII and XhoI double digestion pET-30a (+) plasmid, reclaim enzyme cut after plasmid band, GdhBM gene after enzyme is cut and enzyme cut after pET-30a (+) plasmid, connect with ligase enzyme, transformed clone host Escherichia coli DH5 α.Carry out bacterium colony PCR with primers F _ GdhBM/R_GdhBM, checking transforms recon, then extracts recombinant plasmid, checks order.The recombinant plasmid that sequencing result is errorless, is recombinant plasmid pET30-GdhBM, and as shown in Figure 4 ,-20 DEG C save backup plasmid map.
The structure of embodiment 3 genetic engineering bacterium and abduction delivering
With plasmid pET30-LbADH and pET30-GdhBM built in example 1 and 2, transform expressive host Escherichia coli BL21 (DE3) respectively.Bacterium colony PCR is respectively, the recon that checking transforms with primers F _ LbADH/R_LbADH and F_GdhBM/R_GdhBM.Verify that errorless genetic engineering bacterium is EcoLbADH and EcoGdhBM.EcoLbADH and EcoGdhBM is inoculated into respectively and receives in 3 ~ 5mL liquid LB test-tube culture medium of chloramphenicol resistance containing card, at 35 DEG C, shaking table activates 12 hours, being transferred to by 1% switching amount by the culture obtained after activation receives in the liquid LB Shake flask medium of chloramphenicol resistance containing card, in fermention medium, isothermal vibration cultivates 3h, culture condition is 37 DEG C, 200rpm.Treat that cell concentration grows to OD
600when=0.8, add 0.5mM IPTG (final concentration), 16 DEG C of induction 16h, 10,000g centrifugal 5min collecting cells, after washing 1 time, abandon supernatant, obtain resting cell with pH7.0 sodium phosphate buffer, be placed in-80 DEG C frozen.Protein expression situation SDS-pAGE detects, as illustrated in Figures 5 and 6.
The structure of embodiment 4 genetic engineering bacterium and abduction delivering
With plasmid pET30-LbADH and pET30-GdhBM built in example 1 and 2, transform expressive host Escherichia coli BL21 (DE3) respectively.Bacterium colony PCR is respectively, the recon that checking transforms with primers F _ LbADH/R_LbADH and F_GdhBM/R_GdhBM.Verify that errorless genetic engineering bacterium is EcoLbADH and EcoGdhBM.EcoLbADH and EcoGdhBM is inoculated into respectively and receives in 3 ~ 5mL liquid LB test-tube culture medium of chloramphenicol resistance containing card, at 40 DEG C, shaking table activates 8 hours, being transferred to by 1% switching amount by the culture obtained after activation receives in the liquid LB Shake flask medium of chloramphenicol resistance containing card, in fermention medium, isothermal vibration cultivates 3h, culture condition is 35 DEG C, 200rpm.Treat that cell concentration grows to OD
600when=1.2, add 0.8mM IPTG (final concentration), 25 DEG C of induction 10h, 10,000g centrifugal 5min collecting cells, after washing 1 time, abandon supernatant, obtain resting cell with pH7.0 sodium phosphate buffer, be placed in-80 DEG C frozen.
Embodiment 5 reaction process monitoring method
HPLC detection method is adopted to measure the conversion situation of 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate to (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters.Sample preparation: get different time points reaction solution 50 μ L, adds acetonitrile 150 ~ 950 μ L, in the centrifugal 5min of 12,000g after mixing, gets supernatant 0.45 μm of filtering with microporous membrane and treats sample detection.Chromatographic condition is: chromatographic column: Pursuit C18 (150*4.6mm); Moving phase: 10mM ammonium acetate solution: acetonitrile=55: 45 (v/v); Flow velocity: 0.5mL/min; Sample size: 5 μ L; Determined wavelength: 254nm.HPLC collection of illustrative plates as shown in Figure 7.The retention time of 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate is 9.811min, and the H spectrum of its Structural Identification as shown in Figure 10; The retention time of (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is 7.438min, and the H spectrum of its Structural Identification as shown in figure 11.
Embodiment 6 product ee values determination method
The ee pH-value determination pH of target product (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters adopts the method for chirality HPLC to measure.Sample preparation: get different time points reaction solution 100 μ L, extraction into ethyl acetate three times, combining extraction liquid, uses saturated NaHCO
3respectively wash once with saturated NaCl, anhydrous sodium sulfate drying dewaters, and filter, revolve steaming and desolventize, residue is dissolved in 2mL chromatographic grade dehydrated alcohol, treats that HPLC detects.
Chiral HPLC condition is as follows: chromatographic column: Chiralpak ID (4.6 × 250mm); Moving phase: normal hexane: Virahol: trifluoroacetic acid=80: 20: 0.1; Flow velocity: 0.5ml/min; Sample size: 5 μ L; Determined wavelength: 254nm.The HPLC collection of illustrative plates of four kinds of anomeric product as shown in Figure 9.The retention time of (2S, 3R) configuration is 38.34min, (2R, 3S) retention time of configuration is 49.31min, and the retention time of (2R, 3R) configuration is 54.14min, the retention time of (2S, 3S) configuration is 57.97min.
The preparation of embodiment 7 (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters
Get the resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM, resuspended with the SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid of the 100mM of pH6.0.0.1g is added in three mouthfuls of round-bottomed flasks of 50mL
dry weightthe LbADH resting cell of/L and 0.1g
dry weightthe GdhBM resting cell of/L, adding above-mentioned damping fluid benefit is 20mL to cumulative volume, then adds substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate 50mg, and cosubstrate glucose 100mg and final concentration are the NADP of 0.2mM
+, be then placed in 37 DEG C of constant temperature water bath stirring reaction 24h, period regulates pH value of reaction system to maintain 6.0 with 1M aqueous sodium hydroxide solution.After reaction terminates, reaction solution removes somatic cells through centrifugation, supernatant liquor 0.45 μm of micro-filtrate membrane filtration, and analyze with HPLC, the yield of product (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is 90.3%, ee is 86.9%.
The preparation of embodiment 8 (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters
Get the resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM, resuspended with the SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid of the 100mM of pH7.0.1g is added in three mouthfuls of round-bottomed flasks of 50mL
dry weightthe LbADH resting cell of/L and 1g
dry weightthe GdhBM resting cell of/L, adding above-mentioned damping fluid benefit is 20mL to cumulative volume, then adds substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate 500mg, and cosubstrate glucose 1g and final concentration are the NADP of 0.2mM
+, be then placed in 30 DEG C of constant temperature water bath stirring reaction 24h, period regulates pH value of reaction system to maintain 7.0 with 1M aqueous sodium hydroxide solution.After reaction terminates, reaction solution removes somatic cells through centrifugation, supernatant liquor 0.45 μm of micro-filtrate membrane filtration, and analyze with HPLC, the yield of product (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is 80.5%, ee is 89.6%.
The preparation of embodiment 9 (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters
Get the resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM, resuspended with the SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid of the 100mM of pH8.0.5g is added in three mouthfuls of round-bottomed flasks of 50mL
dry weightthe LbADH resting cell of/L and 5g
dry weightthe GdhBM resting cell of/L, adding above-mentioned damping fluid benefit is 20mL to cumulative volume, then adds substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate 2g, and cosubstrate glucose 4g and final concentration are the NADP of 0.2mM
+, be then placed in 40 DEG C of constant temperature water bath stirring reaction 12h, period regulates pH value of reaction system to maintain 8.0 with 1M aqueous sodium hydroxide solution.After reaction terminates, reaction solution removes somatic cells through centrifugation, supernatant liquor 0.45 μm of micro-filtrate membrane filtration, and analyze with HPLC, the yield of product (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is 73.5%, ee is 91.4%.
The preparation of embodiment 10 (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters
Get the resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM, resuspended with the SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid of the 100mM of pH7.0.10g is added in three mouthfuls of round-bottomed flasks of 50mL
dry weightthe LbADH resting cell of/L and 10g
dry weightthe GdhBM resting cell of/L, adding above-mentioned damping fluid benefit is 20mL to cumulative volume, then adds substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate 5g, and cosubstrate glucose 10g and final concentration are the NADP of 0.5mM
+, be then placed in 25 DEG C of constant temperature water bath stirring reaction 24h, period regulates pH value of reaction system to maintain 7.0 with 1M aqueous sodium hydroxide solution.After reaction terminates, reaction solution removes somatic cells through centrifugation, supernatant liquor 0.45 μm of micro-filtrate membrane filtration, and analyze with HPLC, the yield of product (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is 75.2%, ee is 90.2%.
The preparation of embodiment 11 (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters
Get the resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM, resuspended with the SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid of the 100mM of pH7.0.20g is added in three mouthfuls of round-bottomed flasks of 50mL
dry weightthe LbADH resting cell of/L and 20g
dry weightthe GDHBM resting cell of/L, adding above-mentioned damping fluid benefit is 20mL to cumulative volume, then adds substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate 10g, and cosubstrate glucose 15g and final concentration are the NADP of 0.5mM
+, be then placed in 30 DEG C of constant temperature water bath stirring reaction 24h, period regulates pH value of reaction system to maintain 7.0 with 1M aqueous sodium hydroxide solution.After reaction terminates, reaction solution removes somatic cells through centrifugation, supernatant liquor 0.45 μm of micro-filtrate membrane filtration, and analyze with HPLC, the yield of product (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is 83.2%, ee is 87.6%.
The preparation of embodiment 12 (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters
Get the resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM, resuspended with the SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid of the 100mM of pH7.0.25g is added in three mouthfuls of round-bottomed flasks of 50mL
dry weightthe LbADH resting cell of/L and 25g
dry weightthe GdhBM resting cell of/L, adding above-mentioned damping fluid benefit is 20mL to cumulative volume, then adds substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate 10g, and cosubstrate glucose 15g and final concentration are the NADP of 0.5mM
+, be then placed in 37 DEG C of constant temperature water bath stirring reaction 24h, period regulates pH value of reaction system to maintain 7.0 with 1M aqueous sodium hydroxide solution.After reaction terminates, reaction solution removes somatic cells through centrifugation, supernatant liquor 0.45 μm of micro-filtrate membrane filtration, and analyze with HPLC, the yield of product (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is 93.6%, ee is 84.5%.
The preparation of embodiment 13 (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters
Get the resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM, resuspended with the SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid of the 100mM of pH7.0.1g is added in three mouthfuls of round-bottomed flasks of 50mL
dry weightthe LbADH resting cell of/L and 0.5g
dry weightthe GdhBM resting cell of/L, adding above-mentioned damping fluid benefit is 20mL to cumulative volume, then adds substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate 500mg, and cosubstrate glucose 1g and final concentration are the NADP of 0.2mM
+, be then placed in 30 DEG C of constant temperature water bath stirring reaction 24h, period regulates pH value of reaction system to maintain 7.0 with 1M aqueous sodium hydroxide solution.After reaction terminates, reaction solution removes somatic cells through centrifugation, supernatant liquor 0.45 μm of micro-filtrate membrane filtration, and analyze with HPLC, the yield of product (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is 73.8%, ee is 91.6%.
The preparation of embodiment 14 (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters
Get the resting cell of a certain amount of embodiment 3 engineering bacteria EcoLbADH and EcoGdhBM, resuspended with the SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid of the 100mM of pH7.0.1g is added in three mouthfuls of round-bottomed flasks of 50mL
dry weightthe LbADH resting cell of/L and 2g
dry weightthe GdhBM resting cell of/L, adding above-mentioned damping fluid benefit is 20mL to cumulative volume, then adds substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate 500mg, and cosubstrate glucose 1g and final concentration are the NADP of 0.2mM
+, be then placed in 30 DEG C of constant temperature water bath stirring reaction 24h, period regulates pH value of reaction system to maintain 7.0 with 1M aqueous sodium hydroxide solution.After reaction terminates, reaction solution removes somatic cells through centrifugation, supernatant liquor 0.45 μm of micro-filtrate membrane filtration, and analyze with HPLC, the yield of product (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is 83.5%, ee is 87.3%.
Comparative example 1
(this project bacterium contains the carbonyl reductase gene of clone from Lactobacillus kefiri DSM 20587 to get a certain amount of engineering bacteria EcoLkADH, concrete construction process is with embodiment 1,3) resting cell, resuspended with the SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid of the 100mM of pH7.0.1g is added in three mouthfuls of round-bottomed flasks of 50mL
dry weightthe resting cell of/L LkADH, then add substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate 500mg, cosubstrate Virahol 1mL, adding above-mentioned damping fluid benefit is 20mL to cumulative volume, finally adds the NADP that final concentration is 0.2mM
+, be then placed in 30 DEG C of constant temperature water bath stirring reaction 24h.After reaction terminates, reaction solution removes somatic cells through centrifugation, supernatant liquor 0.45 μm of micro-filtrate membrane filtration, and analyze with HPLC, the yield of product (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is 31.6%, ee is 95.7%.
Comparative example 2
(this project bacterium contains the carbonyl reductase gene of clone from Candida magnoliae CGMCC 2.1919 to get a certain amount of engineering bacteria EcoCR, concrete construction process is with embodiment 1,3) and the resting cell of EcoGdhBM, resuspended with the SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid of the 100mM of pH7.0.1g is added in three mouthfuls of round-bottomed flasks of 50mL
dry weight/the resting cell of L CR and 1g
dry weightthe resting cell of/L GdhBM, adding above-mentioned damping fluid benefit is 20mL to cumulative volume, then adds substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate 500mg, and cosubstrate glucose 1g and final concentration are the NADP of 0.2mM
+, be then placed in 30 DEG C of constant temperature water bath stirring reaction 24h, period regulates pH value of reaction system to maintain 7.0 with 1M aqueous sodium hydroxide solution.After reaction terminates, reaction solution removes somatic cells through centrifugation, supernatant liquor 0.45 μm of micro-filtrate membrane filtration, and analyze with HPLC, the yield of product (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is 0.13%, ee is 0.
Comparative example 3
(this project bacterium contains the carbonyl reductase gene of clone from Acinetobacter baylyi EU273886.1 to get a certain amount of engineering bacteria EcoDkR, concrete construction process is with embodiment 1,3) and the resting cell of EGdhBM, resuspended with the SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid of the 100mM of pH7.0.1g is added in three mouthfuls of round-bottomed flasks of 50mL
dry weightthe DkR resting cell of/L and 1g
dry weightthe GdhBM resting cell of/L, adding above-mentioned damping fluid benefit is 20mL to cumulative volume, then adds substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate 500mg, and cosubstrate glucose 1g and final concentration are the NADP of 0.2mM
+, be then placed in 30 DEG C of constant temperature water bath stirring reaction 24h, period regulates pH value of reaction system to maintain 7.0 with 1M aqueous sodium hydroxide solution.After reaction terminates, reaction solution removes somatic cells through centrifugation, supernatant liquor 0.45 μm of micro-filtrate membrane filtration, and analyze with HPLC, the yield of product (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is 0.31%, ee is 0.
Comparative example 4
(this project bacterium contains the carbonyl reductase gene of clone from Rubrobacter xylanophilus DSM9941 to get a certain amount of engineering bacteria EcoXRADH, concrete construction process is with embodiment 1,3) and the resting cell of GdhBM, resuspended with the SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid of the 100mM of pH7.0.1g is added in three mouthfuls of round-bottomed flasks of 50mL
dry weightthe XRADH resting cell of/L and 1g
dry weightthe GdhBM resting cell of/L, adding above-mentioned damping fluid benefit is 20mL to cumulative volume, then adds substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate 500mg, and cosubstrate glucose 1g and final concentration are the NADP of 0.2mM
+, be then placed in 30 DEG C of constant temperature water bath stirring reaction 24h, period regulates pH value of reaction system to maintain 7.0 with 1M aqueous sodium hydroxide solution.After reaction terminates, reaction solution removes somatic cells through centrifugation, supernatant liquor 0.45 μm of micro-filtrate membrane filtration, and analyze with HPLC, the yield of product (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is 0, ee is 0.
Comparative example 5
(this project bacterium contains the carbonyl reductase gene of clone from Saccharomyces cerevisiae to get a certain amount of engineering bacteria EcoYDL326, concrete construction process is with embodiment 1,3) and the resting cell of engineering bacteria EcoGdhBM, resuspended with the SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid of the 100mM of pH7.0.1g is added in three mouthfuls of round-bottomed flasks of 50mL
dry weightthe YDL326 resting cell of/L and 1g
dry weightthe GdhBM resting cell of/L, adding above-mentioned damping fluid benefit is 20mL to cumulative volume, then adds substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate 500mg, and cosubstrate glucose 1g and final concentration are the NADP of 0.2mM
+, be then placed in 30 DEG C of constant temperature water bath stirring reaction 24h, period regulates pH value of reaction system to maintain 7.0 with 1M aqueous sodium hydroxide solution.After reaction terminates, reaction solution removes somatic cells through centrifugation, supernatant liquor 0.45 μm of micro-filtrate membrane filtration, and analyze with HPLC, the yield of product (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters is 1.72%, ee is 0.
Claims (8)
1. prepare a method for (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters, it is characterized in that, comprising:
(1) engineering bacteria of preparation containing carbonyl reductase gene and the engineering bacteria containing glucose dehydrogenase gene;
(2) the resting cell suspension of two kinds of engineering bacterias is prepared respectively;
(3) by after two kinds of resting cell suspension mixing, mix with substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate, hydrogen donor and cofactor again, carry out asymmetric reduction reaction, obtained (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters;
The base sequence of described carbonyl reductase gene is as shown in SEQ ID NO.1, and the base sequence of described glucose dehydrogenase gene is as shown in SEQ ID NO.2.
2. the method for claim 1, is characterized in that, described engineering bacteria contains expression vector pET-30a (+), and host cell is e. coli bl21 (DE3).
3. the method for claim 1, is characterized in that, described hydrogen donor is glucose, and described cofactor is NADP
+/ NADPH.
4. the method for claim 1, is characterized in that, in the mixed solution of two kinds of resting cell suspensions, the resting cell containing carbonyl reductase is 4: 1 ~ 1: 2 with the mass ratio of the resting cell containing Hexose phosphate dehydrogenase.
5. the method for claim 1, is characterized in that, in asymmetric reduction reaction, described concentration of substrate is 2.5 ~ 500g/L, and the concentration containing the resting cell of carbonyl reductase is 0.1g
dry weight/ L ~ 25g
dry weight/ L, the concentration containing the resting cell of Hexose phosphate dehydrogenase is 0.1g
dry weight/ L ~ 25g
dry weight/ L, the concentration of hydrogen donor is 5 ~ 750g/L, and the concentration of cofactor is 0 ~ 0.5mM.
6. the method for claim 1, is characterized in that, the temperature of described asymmetric reduction reaction is 25 ~ 40 DEG C, and reaction buffer pH is 6.0 ~ 8.0.
7. the method for claim 1, it is characterized in that, the preparation method of resting cell suspension, comprising: be inoculated into by described engineering bacteria and receive in the substratum of mycin containing card, after shaking table activation, when enlarged culturing to OD600 value reaches 0.8 ~ 1.2, add inductor, continue to cultivate, centrifugal collecting cell, resuspended with damping fluid, the resting cell suspension described in acquisition.
8. the method for claim 1, is characterized in that, described inductor is IPTG, and the concentration of inductor is 0.5 ~ 0.8mM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510149171.5A CN104846025B (en) | 2015-03-31 | 2015-03-31 | A kind of method for preparing (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510149171.5A CN104846025B (en) | 2015-03-31 | 2015-03-31 | A kind of method for preparing (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104846025A true CN104846025A (en) | 2015-08-19 |
| CN104846025B CN104846025B (en) | 2018-06-01 |
Family
ID=53846012
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510149171.5A Active CN104846025B (en) | 2015-03-31 | 2015-03-31 | A kind of method for preparing (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104846025B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106811491A (en) * | 2015-12-01 | 2017-06-09 | 上海朴颐化学科技有限公司 | The Preparation Method And Their Intermediate of optically active beta-hydroxy ester type compound |
| CN115433721A (en) * | 2022-06-24 | 2022-12-06 | 山东理工大学 | Carbonyl reductase mutant and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2015
- 2015-03-31 CN CN201510149171.5A patent/CN104846025B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106811491A (en) * | 2015-12-01 | 2017-06-09 | 上海朴颐化学科技有限公司 | The Preparation Method And Their Intermediate of optically active beta-hydroxy ester type compound |
| CN106811491B (en) * | 2015-12-01 | 2022-07-19 | 焦作健康元生物制品有限公司 | Preparation method of optically active beta-hydroxy ester compound and intermediate thereof |
| CN115433721A (en) * | 2022-06-24 | 2022-12-06 | 山东理工大学 | Carbonyl reductase mutant and application thereof |
| CN115433721B (en) * | 2022-06-24 | 2024-01-23 | 山东理工大学 | Carbonyl reductase mutant and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104846025B (en) | 2018-06-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106191170B (en) | A kind of method that enzyme process prepares atriphos | |
| CN105274070A (en) | Mutant of 7 beta-hydroxyl steroid dehydrogenase, application of mutant and synthesis method | |
| CN109134594A (en) | A kind of method that enzyme process prepares glutathione | |
| CN110423717A (en) | Multienzyme recombinant cell and multienzyme cascade the method for catalyzing and synthesizing D-pantoyl lactone | |
| CN109136309A (en) | A kind of production method for replacing ATP to carry out enzymatic reaction using adenosine | |
| CN110396505A (en) | Ketone group pantoic acid lactone reductase and its application | |
| CN106520715B (en) | 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 | |
| CN108690854A (en) | A method of producing L-glufosinate-ammonium using chemo-enzymatic process | |
| CN106995808A (en) | One kind restructuring transaminase and its application | |
| CN106047837A (en) | Serratia lipase mutant, recombinant expression transformant, enzyme preparation and application | |
| CN104630125B (en) | Engineering bacteria and its application in the dihydroxy hecanoic acid t-butyl ester of (3R, 5S) 6 chlorine 3,5 is prepared | |
| CN104846025A (en) | Method for preparing (2S, 3R)-2-benzoyl aminomethyl-3-hydroxy methyl butyrate | |
| CN106947772A (en) | A kind of carbonyl reduction enzyme mutant and its application in chiral alcohol preparation | |
| CN103695379A (en) | Recombinant ketoreductase and method for preparing oxiracetam intermediate compound by using same | |
| CN106701723A (en) | D-fructose-6-phosphate aldolase A mutant, recombinant expression vector, genetically engineered bacterium and application and reaction product thereof | |
| CN106399398A (en) | Bio-preparation method for (R)-3,5-bis(trifluoromethyl) phenyl ethanol | |
| CN103898178B (en) | Enzyme process prepares high chiral pure (S)-3-piperidine alcohols and the method for derivant thereof | |
| CN107828752B (en) | Saccharopolyase, preparation method and application in production of alpha-arbutin | |
| CN101285085B (en) | Process for synthesizing adenosine methilanin by intact cell catalysis | |
| CN104531627A (en) | Carbonyl reductase, engineering strain and application of carbonyl reductase | |
| CN114426977A (en) | Recombinant engineering bacterium and construction method and application thereof | |
| CN101880694B (en) | Method for preparing chiral 3R, 5S-dihydroxyl compound by nonaqueous phase | |
| CN104651290B (en) | Engineering bacteria and its application in Atorvastatin pharmaceutical intermediate is prepared | |
| CN102558261A (en) | Nucleotide analogue and synthesis and application thereof | |
| CN103409478B (en) | Method for synthesizing biotin intermediate lactone through chemical enzyme method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |