CN102732579A - Method for preparing (3S)-3-(tertbutyloxycarbonyl)amino-1-chlorin-4-phenyl-(2R)-butanol by microbial transformation - Google Patents
Method for preparing (3S)-3-(tertbutyloxycarbonyl)amino-1-chlorin-4-phenyl-(2R)-butanol by microbial transformation Download PDFInfo
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Abstract
The invention provides a method for preparing (3S)-3-(tertbutyloxycarbonyl)amino-1-chlorin-4-phenyl-(2R)-butanol by microbial transformation, comprising the following steps of: using (3S)-3-(tertbutyloxycarbonyl)amino-1-chlorin-4-phenyl-2-butanone as a substrate, using enzyme-containing somatic cells obtained by fermentation of saccharomyces cerevisiae (Saccharomycescerevisiae)CGMCCNo.2266 as a biocatalyst, and performing a conversion reaction to obtain the product. The strain produced by the invention is safe and nontoxic; and the microbial thallus is easy for large-scale culture and requires lower cost than a chemical catalyst and katalaze. In addition, the method provided by the invention requires mild reaction condition, is environmentally friendly, has high molar conversion rate, is easy to realize large-scale industrial production, and is a green technology for the industrial production of (3S)-3-(tertbutyloxycarbonyl)amino-1-chlorin-4-phenyl-(2R)-butanol.
Description
Technical field
The present invention relates to the microbial conversion process field, be specifically related to the method for a kind of biocatalysis preparation (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols.
Background technology
(3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols, English name: ((3S)-3-(tert-Butoxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanol), CAS number: 162536-40-5, molecular formula C
15H
22NClO
3, molecular weight 299.79.(3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols is the key intermediate of preparation anti-AIDS drug Reyataz R.Reyataz R is a novel azepine peptide proteinoid enzyme inhibitors (PI), is that the X-ray diffraction studies according to enzyme-azepine peptide complex designs, and has the symmetric chemical structure of C-2.It is the highly selective of HIV-1 proteolytic enzyme and suppressor factor efficiently; Cracking through blocking virus gap and gap-pol precursor polyprotein; Thereby suppress the generation of virus structural protein, reversed transcriptive enzyme, intergrase and proteolytic enzyme, the cell that HIV-1 is infected discharges noninfective jejune virion.These article have the inhibition activity stronger than other PI to many strains HIV-1 virus, and medium effective concentration in in-vitro culture medium (EC50) is 2.6~5.3 nmol/L, and 90% effective concentration (EC90) is 9~15 nmol/L.
Resolution of racemates (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-butanols can obtain (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols, but splitting the efficient maximum has only 50%, and production efficiency is not high.With (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone is that substrate adopts carbonyl in chemical method and the microbial method asymmetric reduction substrate all can obtain (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols.And the chemical method asymmetric reduction need prepare the chirality chemical catalyst, costs an arm and a leg, and the preparation process is loaded down with trivial details, and environmental pollution is bigger usually.
Summary of the invention
The object of the invention provides the method for a kind of microbial transformation preparation (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols, and this method reaction conditions is gentle, and is easy and simple to handle, environmental friendliness, and product yield is high, is easy to suitability for industrialized production.
The technical scheme that the present invention adopts is:
The method of a kind of microbial transformation preparation (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols, said method are to be substrate with (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone, with yeast saccharomyces cerevisiae (
Saccharomyces cerevisiae) the enzyme somatic cells that contains that obtains of CGMCC No. 2266 fermentation is biological catalyst, carry out conversion reaction and make said (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols.
Said yeast saccharomyces cerevisiae CGMCC No.2266; Formerly protect as new bacterial strain in the granted patent 200810059686; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms preservation center; Be positioned at Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, preserving number CGMCC No.2266, preservation date on November 26th, 2007.
The colony characteristics of said yeast saccharomyces cerevisiae CGMCC No.2266: on nutrient agar, demonstrate oyster white, glossy, smooth, neat in edge, moistening, smooth surface, the uniform colonial morphology of quality.
Further; The method of microbial transformation of the present invention preparation (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols is: the phosphate buffered saline buffer with pH 5.0 ~ 8.0 is a reaction solvent, with (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone be substrate, with yeast saccharomyces cerevisiae (
Saccharomyces cerevisiae) the enzyme somatic cells that contains that obtains of CGMCC No. 2266 fermentation is biological catalyst; In 25 ~ 45 ℃ of following conversion reactions 12 ~ 72 hours; After reaction finished, conversion fluid obtained said (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols through separation and purification.
Described (3S)-3-(tertbutyloxycarbonyl) amino-initial final concentration of 1-chloro-4-phenyl-2-butanone in phosphate buffered saline buffer is 0.1 ~ 1 mmol/L.
Further, can also be added with final concentration in the reaction system of the present invention is that 5 ~ 20% ethanol is as cosubstrate.Contain a large amount of ethanol dehydrogenases in the microorganism cells; Alcohol dehydrogenase enzymatic conversion ethanol generates acetaldehyde; Simultaneously with hydrogen pass to Reduced nicotinamide-adenine dinucleotide (NAD) generate Reduced nicotinamide-adenine dinucleotide go back ortho states (the reduced coenzyme I, NADH), alcoholic acid adds the in-situ regeneration that helps realizing coenzyme NAD H; For reaction process provides a large amount of hydrogen donors, thereby improve the transformation efficiency of reaction.Simultaneously, alcoholic acid adds the permeability that can suitably strengthen cell, impels more substrate to get in the microorganism cells and carries out bio-transformation, improves transformation efficiency.
The described enzyme somatic cells consumption that contains is counted 1 ~ 20 g/g substrate with dried cell weight, and the amount of substrate described here i.e. the quality of (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone.
The said enzyme somatic cells dry weight that contains is meant the fermented liquid spinning, and abandoning supernatant is dried 48 hours to constant weight, the weight of gained stem cell with the gained wet cell at 120 ℃.Quantitatively the required said enzyme somatic cells fermented liquid that contains of dried cell weight with method for determination of amount is: get spinning in the part fermented liquid; Abandoning supernatant; Wet cell is dried 48 hours to constant weight at 120 ℃; Measure the gained dried cell weight, the unit of calculating contains stem cell ratio in the enzyme somatic cells fermented liquid, calculates the required enzyme somatic cells fermented liquid consumption that contains of quantitative dried cell weight with this ratio again.
The described enzyme somatic cells that contains prepares according to following method: yeast saccharomyces cerevisiae CGMCC No. 2266 is seeded in the fermention medium, and shaking speed is 150 ~ 200 r/min, cultivates 18 ~ 30 h down for 26 ~ 35 ℃, and fermented liquid is centrifugal, makes to contain the enzyme somatic cells.
Further; (3S)-and the preparation method of 3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-pure article of butanols is following: after reaction finishes,, discard bacterial sediment with centrifugal 20 minutes of conversion fluid 4000 r/min; With supernatant with equal-volume ETHYLE ACETATE continuous extraction 3 times; The combined ethyl acetate extraction liquid adds SODIUM SULPHATE ANHYDROUS 99PCT and removes moisture, suction filtration in acetic acid ethyl acetate extract; Getting filtrating distillation and removing ETHYLE ACETATE, promptly getting said (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols.
(3S)-3-of the present invention (tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanol production method is recommended to carry out according to following steps:
(1) slant culture: yeast saccharomyces cerevisiae CGMCC No. 2266 is inoculated into slant medium, cultivates for 26 ~ 35 ℃ and got the thalline inclined-plane in 4 ~ 6 days; Described slant medium final concentration consists of: wort 5 ~ 15 g/L, and yeast powder 2 ~ 4 g/L, peptone 4 ~ 6 g/L, glucose 7 ~ 12 g/L, agar 15 ~ 25 g/L, natural pH value, solvent is a water;
(2) seed culture: get a transfering loop thalline from the thalline inclined-plane and be transferred to seed culture medium, shaking speed is 150 ~ 200 r/min, cultivates 18 ~ 26 h for 26 ~ 35 ℃ and gets seed liquor; Described seed culture medium final concentration consists of: glucose 26 ~ 32 g/L, yeast powder 2 ~ 4 g/L, ammonium sulfate 3 ~ 6 g/L, anhydrous MgSO
40. 2 ~ 0. 4 g/L, K
2HPO
43H
2O 0. 5 ~ 1.5 g/L, KH
2PO
40. 6 ~ 1.5 g/L, using the pH value of NaOH or HCl solution adjustment liquid nutrient medium is 7.0, and solvent is a water;
(3) fermentation culture: get seed liquor, be inoculated in the fermention medium with the inoculum size of volume(tric)fraction 10 ~ 20%, shaking speed is 150 ~ 200 r/min, cultivates 18 ~ 30 h, the fermented liquid spinning is obtained the said enzyme somatic cells that contains for 26 ~ 35 ℃; Said fermention medium final concentration consists of: glucose 26 ~ 32 g/L, yeast powder 2 ~ 4 g/L, ammonium sulfate 3 ~ 6 g/L, anhydrous MgSO
40. 2 ~ 0. 4 g/L, K
2HPO
43H
2O 0. 5 ~ 1.5 g/L, KH
2PO
40.6 ~ 1.5 g/L, using the pH value of NaOH or HCl solution adjustment liquid nutrient medium is 7.0, and solvent is a water;
(4) bio-transformation: in the phosphate buffered saline buffer of pH 5.0 ~ 8.0; Add final concentration and be 5 ~ 20% ethanol as cosubstrate; Adding final concentration is (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone of 0.1 ~ 1 mmol/L; And be equivalent to the dried cell weight quality for 1 ~ 20 times of (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone contain the enzyme somatic cells, 25 ~ 45 ℃ of following conversion reactions 12 ~ 72 hours, conversion fluid;
(5) to finish the method that the back obtains (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-pure article of butanols following in reaction: after reaction finishes; With conversion fluid centrifugal 20 minutes in 4000 r/min; Discard bacterial sediment, with supernatant with equal-volume ETHYLE ACETATE continuous extraction 3 times, combined ethyl acetate extraction liquid; In acetic acid ethyl acetate extract, add SODIUM SULPHATE ANHYDROUS 99PCT and remove moisture; Suction filtration being got filtrating distillation and removing ETHYLE ACETATE, promptly gets said (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols.
(3S)-3-of the present invention (tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-the superfluous value (ee%) of enantiomorph of butanols and confirming of substrate conversion efficiency: reaction is carried out in the process; Every interval 1 ~ 11 h 5 mL that take a sample; Centrifugal 20 minutes of sample 4000 r/min; Discard deposition, supernatant with 5 mL ethyl acetate extractions, is drawn acetic acid ethyl acetate extract 5 μ L and squeezed into liquid chromatography analysis.The liquid chromatography model is an Agilent 1200; Chromatographic column is chiral column CHIRACEL OD-H (4.6 mm * 250 mm; 5 um), the damping fluid composition is normal hexane-tertiary butyl dimethyl cellosolve-trifluoroacetic acid (800:200:2), and moving phase is prepared according to volume ratio 99:1 by above-mentioned damping fluid and ethanol; Flow rate of mobile phase is 0.5 mL/min, and sample size is 5 ul.Under this analysis condition can with substrate (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone, (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols with (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2S)-butanols separates fully, thereby the enantiomorph surplus value of the molar yield that further calculates substrate and product (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols.
(3S)-3-(tertbutyloxycarbonyl) amino that makes-1-chloro-4-phenyl-(2R)-the available liquid chromatographmass spectrometer detection of the pure article of butanols, confirm degree of purity of production and molecular weight.
Employing microorganism cells asymmetric reduction (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl among the present invention-2-butanone preparation (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols; Can obtain the product of the superfluous value of high antimer, the ethanol of interpolation 5 ~ 20% helps improving the molar yield of substrate as cosubstrate.
The present invention adopts microbe transformation method preparation (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols; Compare with chemical synthesis, enzyme catalysis method and to have the following advantages: (1) produces the bacterial strain safety non-toxic; Microbial cells is easy to large scale culturing; Can obtain a large amount of biological catalysts, more with low cost than chemical catalyst and katalaze(enzyme); (2) reaction conditions is gentle, environmental friendliness, and molar yield is high, is easy to realize large-scale industrial production, is the friendly process of suitability for industrialized production (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols.
(1) embodiment
Below in conjunction with specific embodiment the present invention is described further, but scope of the present invention is not limited in this.
The slant medium preparation: wort 10 g/L, yeast powder 3 g/L, peptone 5 g/L, glucose 10 g/L, agar 20 g/L, natural pH value, solvent is a water; 121 ℃ of sterilization 20 min, sterilization postcooling bevel.
Seed and fermention medium preparation: glucose 30 g/L, yeast powder 3 g/L, ammonium sulfate 5 g/L, anhydrous MgSO
40.25 g/L, K
2HPO
43H
2O 1 g/L, KH
2PO
41 g/L, solvent are water, and the pH value of using NaOH or HCl solution adjustment liquid nutrient medium is 7.0,121 ℃ of sterilization 20 min.
Embodiment 1
Yeast saccharomyces cerevisiae CGMCC No. 2266 bacterial classification inoculations to slant medium, are cultivated for 30 ℃ and made the thalline inclined-plane in 6 days.Get a transfering loop thalline from the thalline inclined-plane with inoculating needle and be seeded in the 250 mL triangular flasks that contain 100 mL liquid nutrient mediums, under 30 ℃, the condition of 180 r/min, cultivate 24 h and obtain seed liquor.10mL seed liquor (the inoculum size consumption is a liquid nutrient medium volume 10%) is inoculated in the 250 mL triangular flasks that contain 100 mL liquid nutrient mediums, under 30 ℃, the condition of 180 r/min, cultivates 24 h and obtain fermented liquid, fermented liquid is centrifugal, must contain the enzyme somatic cells.
Through measuring, the dry weight that contains the enzyme somatic cells in every liter of fermented liquid of present embodiment is 50 grams.
In ten parts of triangular flasks that contain 100 mL pH7.0 phosphate buffered saline buffers, add above-mentioned gained fermented liquid 200 mL respectively; The dry weight that wherein contains the enzyme somatic cells is 10 g; Add (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone respectively; Make the final concentration of (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone be respectively 0.1 mmol/L, 0.2 mmol/L, 0.3 mmol/L, 0.4 mmol/L, 0.5 mmol/L, 0.6 mmol/L, 0.7 mmol/L, 0.8 mmol/L, 0.9 mmol/L, 1.0 mmol/L; All place 30 ℃, reaction 36 h in the shaking table of 180 r/min.After reaction finishes with conversion fluid centrifugal 20 minutes in 4000 r/min; Discard bacterial sediment; With equal-volume ETHYLE ACETATE continuous extraction 3 times, the combined ethyl acetate extraction liquid adds SODIUM SULPHATE ANHYDROUS 99PCT and removes small amount of moisture in acetic acid ethyl acetate extract with supernatant; Suction filtration; ETHYLE ACETATE is removed in filtrating distillation, promptly get said (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols, the substrate starting point concentration to molar yield and (3S)-influence of the corresponding body surplus value (ee%) of 3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols sees table 1.
Table 1 substrate starting point concentration is to the influence of transformation efficiency and (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-superfluous value of butanols enantiomorph
Concentration of substrate (mmol/L) | Transformation efficiency (%) | (3S)-the superfluous value of enantiomorph (ee%) of 3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols |
0.1 | 100 | 100 |
0.2 | 92.2 | 100 |
0.3 | 80.5 | 100 |
0.4 | 77.3 | 95.6 |
0.5 | 68.8 | 83.2 |
0.6 | 52.1 | 70.3 |
0.7 | 43.2 | 68.2 |
0.8 | 35.9 | 62.1 |
0.9 | 28.2 | 58.5 |
1.0 | 19.5 | 51.2 |
Table 1 can be found out: along with the raising transformation efficiency of concentration of substrate reduces gradually.Transformation efficiency is 100% when concentration of substrate is 0.1 mmol/L.Concentration of substrate has bigger influence to the superfluous value of the enantiomorph of product (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols, (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-and the enantiomorph surplus value of butanols reduces along with the raising of concentration of substrate.When concentration of substrate was lower than 0.3mmol/L, the superfluous value of the enantiomorph of product (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols was 100%.
Embodiment 2
Yeast saccharomyces cerevisiae CGMCC No. 2266 bacterial classification inoculations to slant medium, are cultivated for 30 ℃ and got the thalline inclined-plane in 6 days.Get a transfering loop thalline from the thalline inclined-plane with inoculating needle and be seeded in the 250 mL triangular flasks that contain 100 mL liquid nutrient mediums, under 30 ℃, the condition of 180 r/min, cultivate 24 h and obtain seed liquor.10 mL seed liquor (the inoculum size consumption is a liquid nutrient medium volume 10%) are inoculated in the 250 mL triangular flasks that contain 100 mL liquid nutrient mediums; Under 30 ℃, the condition of 180 r/min, cultivate 24 h and obtain fermented liquid; Fermented liquid is centrifugal; Must contain the enzyme somatic cells, the dry weight that contains the enzyme somatic cells in every liter of fermented liquid is 50 grams.
In seven parts of triangular flasks that contain 100 mL pH7.0 phosphate buffered saline buffers, add above-mentioned gained fermented liquid 200 mL respectively; The dry weight that wherein contains the enzyme somatic cells is 10 g; Adding (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone respectively, to make (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone final concentration be 0.3 mmol/L; Add the volume percent (v/v) that cosubstrate ethanol makes the ethanol volumetric usage account for reaction system separately and be respectively 0%, 5%, 10%, 15%, 20%, 25% and 30%; Place 30 ℃, reaction 36 h in the shaking table of 180 r/min, after reaction finishes with conversion fluid centrifugal 20 minutes in 4000 r/min; Discard bacterial sediment; With equal-volume ETHYLE ACETATE continuous extraction 3 times, the combined ethyl acetate extraction liquid adds SODIUM SULPHATE ANHYDROUS 99PCT and removes small amount of moisture in acetic acid ethyl acetate extract with supernatant; Suction filtration; ETHYLE ACETATE is removed in the distillation of will filtrating, and promptly get said (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols, the cosubstrate alcohol concn to molar yield and (3S)-influence of the corresponding body surplus value (ee%) of 3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols sees table 2.
Table 2: add ethanol to transformation efficiency and (3S)-influence of 3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-superfluous value of butanols enantiomorph
Ethanol (%) | Transformation efficiency (%) | (3S)-the superfluous value of enantiomorph (ee%) of 3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols |
0 | 80.5 | 100 |
5 | 92.8 | 100 |
10 | 100 | 100 |
15 | 98.9 | 100 |
20 | 96.2 | 100 |
25 | 90.5 | 100 |
30 | 82.7 | 100 |
Table 2 can be found out: an amount of alcoholic acid adds and helps improving the substrate molar yield, and the ethanol addition is 10% ~ 15% o'clock, and effect is better, and alcohol concn more is higher than the bio-transformation ability that can reduce mikrobe on the contrary, and transformation efficiency can reduce gradually.(3S)-the superfluous value of enantiomorph of 3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols do not change with the change of ethanol addition, all remains on 100%.
Embodiment 3
Yeast saccharomyces cerevisiae CGMCC No. 2266 bacterial classification inoculations to slant medium, are cultivated for 30 ℃ and got the thalline inclined-plane in 6 days.Get a transfering loop thalline from the thalline inclined-plane with inoculating needle and be seeded in the 250 mL triangular flasks that contain 100 mL liquid nutrient mediums, under 30 ℃, the condition of 180 r/min, cultivate 24 h and obtain seed liquor.Seed liquor is inoculated in the 250 mL triangular flasks that contain 100 mL liquid nutrient mediums with 10% inoculum size; Under 30 ℃, the condition of 180 r/min, cultivate 24 h and obtain fermented liquid; Fermented liquid is centrifugal, must contain the enzyme somatic cells, and the dry weight that contains the enzyme somatic cells in every liter of fermented liquid is 50 grams.
In five parts of triangular flasks that contain 100 mL pH7.0 phosphate buffered saline buffers, add above-mentioned gained fermented liquid 200 mL respectively; The dry weight that wherein contains the enzyme somatic cells is 10 g; Add (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone separately and make (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone final concentration 0.3 mmol/L; Add cosubstrate ethanol; Making the ethanol final concentration is 10%, in the shaking table of 180 r/min respectively at 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃ following conversion reaction 36 h.After reaction finishes with conversion fluid centrifugal 20 minutes in 4000 r/min; Discard bacterial sediment; With equal-volume ETHYLE ACETATE continuous extraction 3 times, the combined ethyl acetate extraction liquid adds SODIUM SULPHATE ANHYDROUS 99PCT and removes small amount of moisture in acetic acid ethyl acetate extract with supernatant; Suction filtration; ETHYLE ACETATE is removed in filtrating distillation, promptly get said (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols, temperature of reaction to the substrate molar yield and (3S)-influence of the corresponding body surplus value (ee%) of 3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols sees table 3.
Table 3 temperature of reaction is to the influence of transformation efficiency and (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-superfluous value of butanols enantiomorph
Invert point (℃) | Transformation efficiency (%) | (3S)-the superfluous value of enantiomorph (ee%) of 3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols |
25 | 89.7 | 100 |
30 | 100 | 100 |
35 | 86.2 | 100 |
40 | 72.5 | 100 |
45 | 63.1 | 100 |
Table 3 can be found out: temperature of reaction has considerable influence to transformation efficiency.The too high part inactivation that can cause enzyme of temperature of reaction, thereby transformation efficiency reduces.Preferable invert point is about 30 ℃, (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-and the superfluous value of enantiomorph of butanols do not change with the change of invert point, all remains on 100%.
Embodiment 4:
Yeast saccharomyces cerevisiae CGMCC No. 2266 bacterial classification inoculations to slant medium, are cultivated for 30 ℃ and got the thalline inclined-plane in 6 days.Get a transfering loop thalline from the thalline inclined-plane with inoculating needle and be seeded in the 250 mL triangular flasks that contain 100 mL liquid nutrient mediums, under 30 ℃, the condition of 180 r/min, cultivate 24 h and obtain seed liquor.Seed liquor is inoculated in the 250 mL triangular flasks that contain 100 mL liquid nutrient mediums with 10% inoculum size; Under 30 ℃, the condition of 180 r/min, cultivate 24 h and obtain fermented liquid; Fermented liquid is centrifugal, must contain the enzyme somatic cells, and the dry weight that contains the enzyme somatic cells in every liter of fermented liquid is 50 grams.
In five parts of triangular flasks that contain 100 mL pH7.0 phosphate buffered saline buffers, add above-mentioned gained fermented liquid 200 mL respectively; The dry weight that wherein contains the enzyme somatic cells is 10 g; Add (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone; Making (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone final concentration is 0.3 mmol/L, adds cosubstrate ethanol, and making the ethanol final concentration is 10%; Place 30 ℃, react 12 h, 24 h, 36 h, 48 h, 72 h in the shaking table of 180 r/min respectively.After reaction finishes with conversion fluid centrifugal 20 minutes in 4000 r/min; Discard bacterial sediment; With equal-volume ETHYLE ACETATE continuous extraction 3 times, the combined ethyl acetate extraction liquid adds SODIUM SULPHATE ANHYDROUS 99PCT and removes small amount of moisture in acetic acid ethyl acetate extract with supernatant; Suction filtration; Filtrating is heated up in a steamer and is removed ETHYLE ACETATE, promptly get said (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols, the reaction times to the substrate molar yield and (3S)-influence of the corresponding body surplus value (ee%) of 3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols sees table 4.
Table 4: the reaction times is to the influence of transformation efficiency and (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-superfluous value of butanols enantiomorph
Transformation time (h) | Transformation efficiency (%) | (3S)-the superfluous value of enantiomorph (ee%) of 3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols |
12 | 35.0 | 100 |
24 | 80.2 | 100 |
36 | 100 | 100 |
48 | 100 | 100 |
72 | 100 | 100 |
Table 4 can be found out: along with the prolongation transformation efficiency of transformation time improves gradually.Transformation efficiency can reach 100% basically after reacting 36 hours, and therefore best transformation time is 36 hours.(3S)-the superfluous value of enantiomorph of 3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols do not change with the change of transformation time, all remains on 100%.
Embodiment 5:
Yeast saccharomyces cerevisiae CGMCC No. 2266 bacterial classification inoculations to slant medium, are cultivated for 30 ℃ and got the thalline inclined-plane in 6 days.Get a ring thalline from the thalline inclined-plane with inoculating needle and be seeded in the 250 mL triangular flasks that contain 100 mL liquid nutrient mediums, under 30 ℃, the condition of 180 r/min, cultivate 24 h and obtain seed liquor.Seed liquor is inoculated in the 250 mL triangular flasks that contain 100 mL liquid nutrient mediums with 10% inoculum size; Under 30 ℃, the condition of 180 r/min, cultivate 24 h and obtain fermented liquid; Fermented liquid is centrifugal, must contain the enzyme somatic cells, and the dry weight that contains the enzyme somatic cells in every liter of fermented liquid is 50 grams.
In five parts of triangular flasks that contain 100 mLpH7.0 phosphate buffered saline buffers, add 40 milliliters of above-mentioned gained fermented liquids, 100 milliliters, 200 milliliters, 300 milliliters and 400 milliliters respectively, the dry weight that wherein contains the enzyme somatic cells is respectively 2 g, 5 g, 10 g, 15 g and 20 g.In above-mentioned five triangular flasks, add (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone respectively; Making (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone final concentration is 0.3 mmol/L; Adding cosubstrate ethanol more separately, to make the ethanol final concentration be 10%; Place 30 ℃, reaction 36 h in the shaking table of 180 r/min.After reaction finishes with conversion fluid centrifugal 20 minutes in 4000 r/min; Discard bacterial sediment; With equal-volume ETHYLE ACETATE continuous extraction 3 times, the combined ethyl acetate extraction liquid adds SODIUM SULPHATE ANHYDROUS 99PCT and removes small amount of moisture in acetic acid ethyl acetate extract with supernatant; Suction filtration; ETHYLE ACETATE is removed in filtrating distillation, promptly get said (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols, the somatic cells amount to the substrate molar yield and (3S)-influence of the corresponding body surplus value (ee%) of 3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols sees table 5.
Table 5 biomass is to the influence of transformation efficiency and (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-superfluous value of butanols enantiomorph
Table 5 biomass is to the influence of transformation efficiency and (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-superfluous value of butanols enantiomorph
Biomass (dry weight) (g) | Transformation efficiency (%) | (3S)-the superfluous value of enantiomorph (ee%) of 3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols |
2 | 32.1 | 100 |
5 | 52.6 | 100 |
10 | 100 | 100 |
15 | 100 | 100 |
20 | 100 | 100 |
Table 5 can be found out: along with the increase transformation efficiency of thalline consumption increases gradually.The increase of thalline consumption has not only improved the consumption of enzyme, has improved the consumption of coenzyme NAD H simultaneously, therefore helps improving reaction conversion ratio.(3S)-the superfluous value of enantiomorph of 3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols do not change with the change of thalline add-on, all remains on 100%.
Claims (9)
1. the method for microbial transformation preparation (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols is characterized in that, said method is to be substrate with (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone, with yeast saccharomyces cerevisiae (
Saccharomyces cerevisiae) the enzyme somatic cells that contains that obtains of CGMCC No. 2266 fermentation is biological catalyst, carry out conversion reaction and make said (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols.
2. preparation method as claimed in claim 1 is characterized in that, described method is: the phosphate buffered saline buffer with pH 5.0 ~ 8.0 is a reaction solvent, with (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone be substrate, with yeast saccharomyces cerevisiae (
Saccharomyces cerevisiae) the enzyme somatic cells that contains that obtains of CGMCC No. 2266 fermentation is biological catalyst; In 25 ~ 45 ℃ of following conversion reactions 12 ~ 72 hours; After reaction finished, conversion fluid obtained said (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols through separation and purification.
3. preparation method as claimed in claim 1 is characterized in that, the initial final concentration of said substrate in reaction system is 0.1 ~ 1 mmol/L.
4. preparation method as claimed in claim 1 is characterized in that, can also be added with final concentration in the described reaction system and be 5 ~ 20% ethanol.
5. preparation method as claimed in claim 1 is characterized in that, the described enzyme somatic cells consumption that contains is counted 1 ~ 20 g/g substrate with dried cell weight.
6. preparation method as claimed in claim 1; It is characterized in that; The described enzyme somatic cells that contains prepares according to following method: yeast saccharomyces cerevisiae CGMCC No. 2266 is seeded in the fermention medium, and shaking speed is 150 ~ 200 r/min, cultivates 18 ~ 30 h down for 26 ~ 35 ℃; Fermented liquid is centrifugal, make and contain the enzyme somatic cells.
7. preparation method as claimed in claim 2 is characterized in that, described (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-the butanols separation purification method is following: after reaction finishes; With centrifugal 20 minutes of conversion fluid 4000 r/min; Discard bacterial sediment, with supernatant with equal-volume ETHYLE ACETATE continuous extraction 3 times, combined ethyl acetate extraction liquid; In acetic acid ethyl acetate extract, add SODIUM SULPHATE ANHYDROUS 99PCT and remove moisture; Suction filtration being got filtrating distillation and removing ETHYLE ACETATE, promptly gets said (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols.
8. preparation method as claimed in claim 1 is characterized in that, described method is carried out according to following steps:
(1) slant culture: yeast saccharomyces cerevisiae CGMCC No. 2266 is inoculated into slant medium, cultivates for 26 ~ 35 ℃ and got the thalline inclined-plane in 4 ~ 6 days;
(2) seed culture: get a transfering loop thalline from the thalline inclined-plane and be transferred to seed culture medium, shaking speed is 150 ~ 200 r/min, cultivates 18 ~ 26 h for 26 ~ 35 ℃ and gets seed liquor;
(3) fermentation culture: get seed liquor, be inoculated in the fermention medium with the inoculum size of volume(tric)fraction 10 ~ 20%, shaking speed is 150 ~ 200 r/min, cultivates 18 ~ 30 h, the fermented liquid spinning is obtained the said enzyme somatic cells that contains for 26 ~ 35 ℃;
(4) bio-transformation: in the phosphate buffered saline buffer of pH 5.0 ~ 8.0; Add final concentration and be 5 ~ 20% ethanol as cosubstrate; The adding final concentration is that (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone of 0.1 ~ 1 mmol/L is a substrate; And be equivalent to the dried cell weight quality for 1 ~ 20 times of (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone contain the enzyme somatic cells, 25 ~ 45 ℃ of following conversion reactions 12 ~ 72 hours, reaction finishes to make conversion fluid;
(5) separation and purification: after conversion reaction finishes,, discard bacterial sediment with conversion fluid centrifugal 20 minutes in 4000 r/min; With supernatant with equal-volume ETHYLE ACETATE continuous extraction 3 times; The combined ethyl acetate extraction liquid adds SODIUM SULPHATE ANHYDROUS 99PCT and removes moisture, suction filtration in acetic acid ethyl acetate extract; ETHYLE ACETATE is removed in filtrating distillation, promptly gets said (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols.
9. method as claimed in claim 2 is characterized in that said method carries out as follows:
(1) slant culture: yeast saccharomyces cerevisiae CGMCC No. 2266 is inoculated into slant medium, cultivates for 26 ~ 35 ℃ and got the thalline inclined-plane in 4 ~ 6 days; Described slant medium final concentration consists of: wort 5 ~ 15 g/L, and yeast powder 2 ~ 4 g/L, peptone 4 ~ 6 g/L, glucose 7 ~ 12 g/L, agar 15 ~ 25 g/L, natural pH value, solvent is a water;
(2) seed culture: get a transfering loop thalline from the thalline inclined-plane and be transferred to seed culture medium, 26 ~ 35 ℃, shaking speed is 150 ~ 200 r/min, cultivates 18 ~ 26 h and gets seed liquor; Described seed culture medium final concentration consists of: glucose 26 ~ 32 g/L, yeast powder 2 ~ 4 g/L, ammonium sulfate 3 ~ 6 g/L, anhydrous MgSO
40.2 ~ 0.4 g/L, K
2HPO
43H
2O 0.5 ~ 1.5 g/L, KH
2PO
40.6 ~ 1.5 g/L, using the pH value of NaOH or HCl solution adjustment liquid nutrient medium is 7.0, and solvent is a water;
(3) fermentation culture: get seed liquor; With volume(tric)fraction is that 10 ~ 20% inoculum size is inoculated in the fermention medium; Culture temperature is 26 ~ 35 ℃; Shaking speed is 150 ~ 200 r/min, cultivates the fermented liquid that 18 ~ 30 h obtain containing the enzyme somatic cells, and spinning obtains the said enzyme somatic cells that contains; Said fermention medium final concentration consists of: glucose 26 ~ 32 g/L, yeast powder 2 ~ 4 g/L, ammonium sulfate 3 ~ 6 g/L, anhydrous MgSO
40.2 ~ 0.4 g/L, K
2HPO
43H
2O 0.5 ~ 1.5 g/L, KH
2PO
40.6 ~ 1.5 g/L, using the pH value of NaOH or HCl solution adjustment liquid nutrient medium is 7.0, and solvent is a water;
(4) bio-transformation: in the phosphate buffered saline buffer of pH 5.0 ~ 8.0; Adding final concentration is (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone of 0.1 ~ 1 mmol/L; Add final concentration and be 5 ~ 20% ethanol as cosubstrate; And be equivalent to the dried cell weight quality for 1 ~ 20 times of (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-2-butanone contain the enzyme somatic cells, in 25 ~ 45 ℃ of following conversion reactions 12 ~ 72 hours conversion fluid;
(5) separation and purification: after reaction finishes,, discard bacterial sediment with conversion fluid centrifugal 20 minutes in 4000 r/min; With supernatant with equal-volume ETHYLE ACETATE continuous extraction 3 times; The combined ethyl acetate extraction liquid adds SODIUM SULPHATE ANHYDROUS 99PCT and removes moisture, suction filtration in acetic acid ethyl acetate extract; Getting filtrating distillation and removing ETHYLE ACETATE, promptly getting said (3S)-3-(tertbutyloxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanols.
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CN104911224A (en) * | 2015-06-26 | 2015-09-16 | 南京工业大学 | Method for catalytic synthesis of atazanavir intermediate |
CN109897872A (en) * | 2017-12-11 | 2019-06-18 | 湖州颐辉生物科技有限公司 | Enzyme process prepares (2S, 3S)-N- tertbutyloxycarbonyl -3- amino -1- chlorine-2-hydroxyl -4- phenyl butane |
CN111235123A (en) * | 2020-03-27 | 2020-06-05 | 长兴制药股份有限公司 | Carbonyl reductase with high-concentration tolerance of alcoholic solution and application thereof |
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CN101230319A (en) * | 2008-02-04 | 2008-07-30 | 浙江工业大学 | Saccharomyces cerevisiae CGMCC No.2266 and its application in preparation of (S)-(-)- beta-hydroxyphenyl propionic acid ethyl |
CN101824438A (en) * | 2010-02-09 | 2010-09-08 | 浙江工业大学 | Method for preparing (S)-3-hydroxy butyric acid ethyl ester through ethyl acetoacetate microbial conversion |
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CN1993464A (en) * | 2004-08-06 | 2007-07-04 | 株式会社钟化 | Novel carbonyl reductase, gene thereof and method of using the same |
CN101230319A (en) * | 2008-02-04 | 2008-07-30 | 浙江工业大学 | Saccharomyces cerevisiae CGMCC No.2266 and its application in preparation of (S)-(-)- beta-hydroxyphenyl propionic acid ethyl |
CN101824438A (en) * | 2010-02-09 | 2010-09-08 | 浙江工业大学 | Method for preparing (S)-3-hydroxy butyric acid ethyl ester through ethyl acetoacetate microbial conversion |
Cited By (6)
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CN104911224A (en) * | 2015-06-26 | 2015-09-16 | 南京工业大学 | Method for catalytic synthesis of atazanavir intermediate |
CN104911224B (en) * | 2015-06-26 | 2018-12-25 | 南京工业大学 | Method for catalytic synthesis of atazanavir intermediate |
CN109897872A (en) * | 2017-12-11 | 2019-06-18 | 湖州颐辉生物科技有限公司 | Enzyme process prepares (2S, 3S)-N- tertbutyloxycarbonyl -3- amino -1- chlorine-2-hydroxyl -4- phenyl butane |
CN109897872B (en) * | 2017-12-11 | 2023-12-22 | 湖州颐盛生物科技有限公司 | Enzymatic preparation of (2S, 3S) -N-t-butoxycarbonyl-3-amino-1-chloro-2-hydroxy-4-phenylbutane |
CN111235123A (en) * | 2020-03-27 | 2020-06-05 | 长兴制药股份有限公司 | Carbonyl reductase with high-concentration tolerance of alcoholic solution and application thereof |
CN111235123B (en) * | 2020-03-27 | 2020-10-27 | 长兴制药股份有限公司 | Carbonyl reductase with high-concentration tolerance of alcoholic solution and application thereof |
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