CN102296094B - Method for prepared (2S,3S)-2,3-butanediol and (3S)-acetoin from glucose - Google Patents
Method for prepared (2S,3S)-2,3-butanediol and (3S)-acetoin from glucose Download PDFInfo
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- CN102296094B CN102296094B CN 201110247505 CN201110247505A CN102296094B CN 102296094 B CN102296094 B CN 102296094B CN 201110247505 CN201110247505 CN 201110247505 CN 201110247505 A CN201110247505 A CN 201110247505A CN 102296094 B CN102296094 B CN 102296094B
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Abstract
The invention discloses a method for prepared (2S,3S)-2,3-butanediol and (3S)-acetoin from glucose. The method comprises the following steps that glucose is converted into a mixture of (2S,3S)-2,3-butanediol and meso-2,3-butanediol by a Klebsiella pneumoma CICC 10011 resting cell biocatalyst; the mixture of (2S,3S)-2,3-butanediol and meso-2,3-butanediol is split by a bacillus subtilis ATCC 23857 resting cell biocatalyst; and (2S,3S)-2,3-butanediol and (3S)-acetoin are prepared simultaneously. The concentration of (2S,3S)-2,3-butanediol prepared by the method reaches 2.5g/L (wherein the optical purity is 96.9%). The concentration of (3S)-acetoin prepared by the method reaches 29.3g/L (wherein e.e. is 96.2%). Therefore, the method has great industrial application prospects.
Description
Technical field
The invention provides a kind of method by glucose preparation (2S, 3S)-2,3-butanediol and (3S)-acetoin, belong to technical field of biochemical industry.
Background technology
2,3-butanediol comprises 3 isomer (2R, 3R)-2,3-butanediol, (2S, 3S)-2,3-butanediol and meso-2,3-butyleneglycol.Acetoin comprises two enantiomers (3R)-acetoin and (3S)-acetoin.The various isomer of 2,3-butanediol and acetoin are the important source material in asymmetric synthesis, are all the medicine intermediates that has application potential.
The method of production chirality 2,3-butanediol and acetoin comprises chemical method and biological process at present.Wherein the chemical method reaction conditions is complicated, and restive, cost is very high, and the optical purity of product is very low.And that biological process has a reaction type is various, and atopic is strong, and temperature, pressure condition are gentle, environmental friendliness, the advantage such as energy consumption is low.Can produce various 2 with biological process, the isomer of 3-butyleneglycol and acetoin, for example with meso-2, the 3-butyleneglycol is substrate, take engineering bacteria intestinal bacteria (Escherichia coli) BL21 (DE3) (pETDuet-ydjL) as catalyzer, at pH 8.0, under 37 ℃, reaction is 12 hours, can obtain (the 3S)-acetoin (36.7 grams per liter) of high density, but this substrate is expensive, is not suitable for actual production.
Also have, that has reported utilizes biological process production (2S, 3S)-2,3-butanediol as follows with the document of (3S)-acetoin:
1 produces (2S, 3S)-2,3-butanediol, concentration 3.7 grams per liters, and enantiomeric excess per-cent (enantiomeric excess, e.e.) 95%, substrate is acetoin.its method contains (2S for building, 3S)-2, engineering bacteria e. coli jm109/the PMLBD119 of 3-butanediol dehydrogenation enzyme, transform acetoin preparation (2S take this project bacterium as biological catalyst, 3S)-2, 3-butyleneglycol (Ui, S., Takusagawa, Y., Ohtsuki, T., Mimura, A., Ohkuma, M., Kudo, T., 2001.Stereochemical applications of the expression of the L-2, 3-butanediol dehydrogenase gene in Escherichia coli.Lett.Appl.Microbiol.32, 93-98.).
2 produce (3S)-acetoin, concentration 3.2 grams per liters, and optical purity is unknown, and substrate is 2,3-butanediol.Its method is to contain (2S, 3S)-2, the bacterial strain Brevibacterium saccharolyticum of 3-butanediol dehydrogenation enzyme (Brevibacterium saccharolyticum) C-1012 transforms 2,3-butyleneglycol preparation (3S)-acetoin (Ui, S., Masuda, H., Muraki, H., 1984.Laboratory-scale production of acetoin isomers (D (-) and L (+)) by bacterial fermentation.J.Ferment.Technol.62,151-156.).
Yet the substrate 2,3-butanediol that uses in aforesaid method and the cost of acetoin are all higher, are not suitable for actual production, have restricted the application of the method.And result for retrieval is known, take the glucose of cheapness as substrate, produces simultaneously the method for (2S, 3S)-2,3-butanediol and (3S)-acetoin with two-step reaction, has no at present report.
Summary of the invention
For the deficiencies in the prior art, the problem to be solved in the present invention be to provide a kind of can be take the glucose of cheapness as raw material production (2S, 3S)-2,3-butanediol and the method for (3S)-acetoin.
Technical conceive of the present invention is:
resting cell transforming glucose with Klebsiella pneumonia (Klebsiellapneumonia) CICC 10011 (available from Chinese industrial microbial strains preservation administrative center) is produced (2S, 3S)-2, 3-butyleneglycol and meso-2, the mixture of 3-butyleneglycol, then with the substrate of this mixture as subtilis (Bacillus subtilis) ATCC 23857 (available from the biological product preservation of USS center), utilize subtilis ATCC 23857 with meso-2, the 3-butyleneglycol is converted into (3S)-acetoin, and (2S, 3S)-2, the 3-butyleneglycol still is present in conversion fluid, thereby realize producing simultaneously (2S, 3S)-2, 3-butyleneglycol and (3S)-acetoin.
Whole process can be represented by the formula:
Concrete steps by glucose preparation (2S, 3S)-2,3-butanediol and (3S)-acetoin method of the present invention are:
1, the preparation biological catalyst, that is: final concentration be 70～100 gram dry cell weights/liter Klebsiella pneumonia CICC 10011 resting cells
(1) slant culture: Klebsiella pneumonia CICC 10011 is scoring on culture medium slant, cultivated 10～18 hours for 37 ℃.
(2) seed culture: under aseptic condition, with the ring bacterium mud of one on aseptic inoculation ring picking step (1) inclined-plane, be inoculated in 50 milliliters of liquid substratum, cultivated 10～14 hours for 37 ℃.
(3) fermentor cultivation: under aseptic condition, get the inoculum size of step (2) gained nutrient solution take volume ratio as 5% and be inoculated in 5 liters of liquid nutrient mediums, cultivated 12～20 hours for 37 ℃.
(4) collect thalline: with the culture 8 that obtains in step (3), 000 rev/min centrifugal 10 minutes, and wash thalline twice with the phosphoric acid buffer of pH 7.0, then cell is resuspended in the phosphoric acid buffer of pH 7.0, the final concentration that makes cell be 70～100 gram dry cell weights/liter, be biological catalyst, standby.
Wherein, described in above-mentioned steps (1), the solid culture based formulas is: peptone 1.0 grams per liters; Extractum carnis 1.0 grams per liters; Yeast powder 0.5 grams per liter; Glucose 0.5 grams per liter; Sodium acetate 0.3 grams per liter; Triammonium citrate 0.2 grams per liter; Tween 800.1 grams per liters; K
2HPO
43H
2O 0.2 grams per liter; CaCO
30.5 grams per liter; MgSO
40.02 grams per liter; MnSO
40.05 grams per liter; Agar 1.5 grams per liters, transferring pH is 6.8～7.2,115 ℃ of sterilizations 20 minutes.
Described in above-mentioned steps (2)～(3), the liquid culture based formulas is: glucose 80.0 grams per liters; Secondary ammonium phosphate 6.0 grams per liters; KCl 1.8 grams per liters; MgSO
47H
2O 0.6 grams per liter; EDTA 0.51 grams per liter; FeSO
47H
2O 0.0225 grams per liter; ZnSO
47H
2O 0.0075 grams per liter; MnSO
47H
2O 0.0038 grams per liter; Citric acid 0.21 grams per liter; Trisodium Citrate 0.294 grams per liter is transferred pH to 7.0, sterilizes 20 minutes for 115 ℃.
2, utilize Klebsiella pneumonia CICC 10011 resting cell biological catalyst preparation (2S, 3S)-2,3-butanediol and the meso-2 of step 1 preparation, the mixture of 3-butyleneglycol
Wherein: take cell concentration as 14～56 gram dry cell weights/liter biological catalyst be Klebsiella pneumonia CICC 10011 resting cells, at 37 ± 2 ℃, transforming starting point concentration under pH 5.0～9.0 conditions is the glucose of 80～160 grams per liters, react after 3～10 hours, contained (2S, 3S)-2,3-butanediol and meso-2, the conversion fluid of 3-mixture of butanediols; With the gained conversion fluid with 8,000 ± 500 rev/mins centrifugal 10～15 minutes, remove the biological catalyst add, collect supernatant liquor and carry out underpressure distillation, after the complete evaporate to dryness of moisture content, cut is (2S, 3S)-2,3-butanediol and meso-2, the mixture of 3-butyleneglycol;
Can measure (2S, 3S) in supernatant liquor-2,3-butanediol and meso-2 with gas-chromatography, the concentration of 3-butyleneglycol.
3, prepare again biological catalyst: final concentration be 20～50 gram dry cell weights/liter subtilis ATCC 23857 resting cells
(1) slant culture: subtilis ATCC 23857 is scoring to contains on the culture medium slant that mass volume ratio is 1.5% agar, cultivated 10～18 hours for 37 ℃.
(2) seed culture: under aseptic condition, with the ring bacterium mud of one on aseptic inoculation ring picking step (1) inclined-plane, be inoculated in 50 milliliters of liquid substratum, cultivated 10～14 hours for 37 ℃.
(3) fermentor cultivation: under aseptic condition, get the inoculum size of step (2) gained nutrient solution take volume ratio as 5% and be inoculated in 5 liters of liquid nutrient mediums, cultivated 12～20 hours for 37 ℃.
(4) collect thalline: with the culture 8 that obtains in step (3), 000 rev/min centrifugal 10 minutes, and wash thalline twice with the phosphoric acid buffer of pH 7.0, then cell is resuspended in the phosphoric acid buffer of pH 7.0, the final concentration that makes cell be 20～50 gram dry cell weights/liter, be biological catalyst, standby.
Wherein, described in above-mentioned steps (1), the solid culture based formulas is: peptone 10.0 grams per liters; Extractum carnis 8.0 grams per liters; Yeast powder 4.0 grams per liters; Glucose 20.0 grams per liters; Sodium acetate 5.0 grams per liters; Triammonium citrate 2.0 grams per liters; K
2HPO
43H
2The O2.0 grams per liter; MgSO
47H
2O 0.2 grams per liter; MnSO
40.2 grams per liter; Agar 15 grams per liters are transferred pH to 6.2, sterilize 20 minutes for 115 ℃.
Described in above-mentioned steps (2)～(3), the liquid culture based formulas is: glucose 70 grams per liters; Peptone 10 grams per liters; Yeast powder 30 grams per liters are transferred pH to 7.0, sterilize 20 minutes for 115 ℃.
4, utilize (the 2S that makes in the subtilis ATCC 23857 resting cell biological catalyst splitting step (2) of step (3) preparation, 3S)-2,3-butyleneglycol and meso-2, the mixture of 3-butyleneglycol, prepare simultaneously (2S, 3S)-2,3-butanediol and (3S)-acetoin
Wherein: take cell concentration as 13～20 gram dry cell weights/liter biological catalyst be subtilis ATCC 23857 resting cells, at 23～45 ℃, under pH 5.0～8.0 conditions with step (2) in (2S, the 3S)-2,3-butanediol and the meso-2 that make, the mixture of 3-butyleneglycol carries out the transformation experiment of 6～24 hours, split (2S, 3S)-2,3-butanediol and meso-2, the 3-butyleneglycol is contained the conversion fluid of (3S)-acetoin; With the gained conversion fluid with 8,000 ± 500 rev/mins centrifugal 10～15 minutes, remove the biological catalyst add, collect supernatant liquor and carry out underpressure distillation, distillate is the aqueous solution of (3S)-acetoin, cut is (2S, 3S)-2,3-butanediol; Then, distillate and ethyl acetate are pressed equal-volume hybrid extraction (3S)-acetoin, repeat 6～8 times with method, merge the ethyl acetate after each time extracts, carry out underpressure distillation, obtain sterling (3S)-acetoin; Cut and ethyl acetate are pressed equal-volume hybrid extraction (2S, 3S)-2,3-butanediol, repeat 6～8 times with method, merge the ethyl acetate after each time extracts, carry out underpressure distillation, obtain sterling (2S, 3S)-2,3-butanediol.
In above-mentioned method by glucose preparation (2S, 3S)-2,3-butanediol and (3S)-acetoin:
The described biological catalyst of step (2) be preferred 28～42 gram dry cell weights of the cell concentration of Klebsiella pneumonia CICC 10011 resting cells/liter.
The starting point concentration of the described glucose of step (2) is preferably 100～120 grams per liters.
The described pH of step (2) preferred 6.0～7.0; Preferred 4～8 hours of reaction times.
The described biological catalyst of step (4) be preferred 15～18 gram dry cell weights of the cell concentration of subtilis ATCC 23857 resting cells/liter.
Step (4) described (2S, 3S)-2,3-butanediol and meso-2, in the mixture of 3-butyleneglycol, meso-2,3-butyleneglycol concentration is 8.5～40 grams per liters, the concentration of (2S, 3S)-2,3-butanediol is 0.66～3.1 grams per liter.
Step (4) described (2S, 3S)-2,3-butanediol and meso-2, in the mixture of 3-butyleneglycol, meso-2,3-butyleneglycol concentration most preferably is 17～32 grams per liters, and the concentration of (2S, 3S)-2,3-butanediol most preferably is 1.3～2.5 grams per liters.
Preferred 30～37 ℃ of the temperature of the described transformation experiment of step (4), pH is preferred 6.0～7.5, preferred 10～18 hours of reaction times.
The condition optimization of step (2) or (4) described underpressure distillation is: 50 ± 2 ℃ of temperature, pressure 0.015～0.098 MPa.
Analytical procedure used in the present invention is as follows:
Measure optical density(OD) with 2100 visible spectrophotometers in wavelength 620 nanometers.
With SBA-50B type bio-sensing analysis-e/or determining glucose concn.
Chiral product (2R, 3R)-2,3-butanediol, (2S, 3S)-2,3-butanediol, meso-2, the 3-butyleneglycol, (3R)-acetoin all uses model to detect as the gas chromatograph of Agilent 1790 with (3S)-acetoin.Concrete grammar is:
After sample centrifugal (12,000 rev/mins, 4 minutes), after supernatant liquor contains the ethyl acetate extraction of interior mark primary isoamyl alcohol with equal-volume, get 1 microlitre organic phase sample introduction.Sampler temperature and detector temperature are 280 ℃.The heating schedule of column oven is: kept 3 minutes for 40 ℃, then be warming up to 80 ℃ with the speed of 1.5 ℃/minute, then be warming up to 85 ℃ with the speed of 0.5 ℃/minute, and then be warming up to 200 ℃ with the speed of 75 ℃/minute, kept 3 minutes at 200 ℃ at last.
Advantage of the present invention and characteristics are:
1. realized (2S, 3S)-2,3-butanediol and (3S)-acetoin time preparation.
2. with the high value of raw materials of glucose preparation chipal compounds (2S, the 3S)-2,3-butanediol and (3S)-acetoin of cheapness, greatly reduce production cost.
3. (the 2S of the inventive method preparation, 3S)-2, the 3-butyleneglycol is high with (3S)-acetoin purity, (the 2S of experiment confirm the inventive method preparation, 3S)-2,3-butyleneglycol concentration can reach 3.3 grams per liters (optical purity 96.9%), and (3S)-acetoin concentration can reach 29.3 grams per liters (e.e.96.2%), has good application prospect.
4. the resting cell of the inventive method use original strain is as biological catalyst, and requirement for environmental conditions is low and simple to operate, is easy to carry out preparation of industrialization.
5. the follow-up rectifying separation of the inventive method is easy to operate, and the extraction expense is cheap.
Description of drawings
Fig. 1 prepares the GC collection of illustrative plates of gained (3S)-acetoin with the present invention.
Fig. 2 prepares the GC collection of illustrative plates of gained (2S, 3S)-2,3-butanediol with the present invention.
Embodiment
Embodiment 1: the preparation of Klebsiella pneumonia CICC 10011 resting cell biological catalysts
(1) slant culture: Klebsiella pneumonia CICC 10011 (available from Chinese industrial microbial strains preservation administrative center) is scoring to contains on the culture medium slant that mass volume ratio is 1.5% agar, cultivated 14 hours for 37 ℃.
(2) seed culture: under aseptic condition, with the ring bacterium mud of one on aseptic inoculation ring picking step (1) inclined-plane, be inoculated in 50 milliliters of liquid substratum, cultivated 12 hours for 37 ℃.
(3) fermentor cultivation: under aseptic condition, get the inoculum size of step (2) gained nutrient solution take volume ratio as 5% and be inoculated in 5 liters of liquid nutrient mediums, cultivated 16 hours for 37 ℃.
(4) collect thalline: with the culture 8 that obtains in step (3), 000 rev/min centrifugal 10 minutes, and wash thalline twice with the phosphoric acid buffer of pH 7.0, then cell is resuspended in the phosphoric acid buffer of pH 7.0, the final concentration that makes cell be 70 gram dry cell weights/liter, be biological catalyst, standby.
Wherein, described in above-mentioned steps (1), the solid culture based formulas is: peptone 1.0 grams per liters; Extractum carnis 1.0 grams per liters; Yeast powder 0.5 grams per liter; Glucose 0.5 grams per liter; Sodium acetate 0.3 grams per liter; Triammonium citrate 0.2 grams per liter; Tween 800.1 grams per liters; K
2HPO
43H
2O 0.2 grams per liter; CaCO
30.5 grams per liter; MgSO
40.02 grams per liter; MnSO
40.05 grams per liter; Agar 1.5 grams per liters, transferring pH is 6.8～7.2,115 ℃ of sterilizations 20 minutes.
Described in above-mentioned steps (2)～(3), the liquid culture based formulas is: glucose 80.0 grams per liters; Secondary ammonium phosphate 6.0 grams per liters; KCl 1.8 grams per liters; MgSO
47H
2O 0.6 grams per liter; EDTA 0.51 grams per liter; FeSO
47H
2O 0.0225 grams per liter; ZnSO
47H
2O 0.0075 grams per liter; MnSO
47H
2O 0.0038 grams per liter; Citric acid 0.21 grams per liter; Trisodium Citrate 0.294 grams per liter is transferred pH to 7.0, sterilizes 20 minutes for 115 ℃.
Embodiment 2: utilize biological catalyst preparation (2S, the 3S)-2,3-butanediol and the meso-2 that obtain in embodiment 1, the mixture of 3-butyleneglycol
Transformation experiment: the biological catalyst that makes gained be the cell concn of Klebsiella pneumonia CICC 10011 be 28 gram dry cell weights/liter, initial glucose concentration is 100 grams per liters, at 37 ℃, reacts under pH 6.0 conditions, reaction medium is distilled water.8 hours termination reactions are contained (2S, 3S)-2,3-butanediol and meso-2 the conversion fluid of 3-mixture of butanediols.With the gained conversion fluid with 8,000 rev/mins centrifugal 15 minutes, remove the biological catalyst add, in the gas Chromatographic Determination supernatant liquor (2S, 3S)-2,3-butanediol concentration as 3.2 grams per liters, meso-2,3-butyleneglycol concentration is 40.7 grams per liters.
Collect supernatant liquor at 50 ℃, 0.098 MPa is carried out underpressure distillation, and cut is heavy molten with a small amount of water, obtains 2 of high density, 3-BD solution, and wherein (2S, 3S)-2,3-BD concentration is 64 grams per liters, meso-2,3-BD concentration is 815 grams per liters, and is standby.
Embodiment 3: utilize biological catalyst preparation (2S, the 3S)-2,3-butanediol and the meso-2 that obtain in embodiment 1, the mixture of 3-butyleneglycol
Transformation experiment: the biological catalyst that makes gained be the cell concn of Klebsiella pneumonia CICC 10011 be 36 gram dry cell weights/liter, initial glucose concentration is 110 grams per liters, at 37 ℃, reacts under pH 7.0 conditions.6 hours termination reactions are contained (2S, 3S)-2,3-butanediol and meso-2 the conversion fluid of 3-mixture of butanediols.With the gained conversion fluid with 8,000 rev/mins centrifugal 15 minutes, remove the biological catalyst add, in the gas Chromatographic Determination supernatant liquor (2S, 3S)-2,3-butanediol concentration as 3.0 grams per liters, meso-2,3-butyleneglycol concentration is 38.8 grams per liters.
Collect supernatant liquor at 50 ℃, 0.060 MPa is carried out underpressure distillation, and cut is heavy molten with a small amount of water, obtains 2 of high density, 3-BD solution, and wherein (2S, 3S)-2,3-BD concentration is 62 grams per liters, meso-2,3-BD concentration is 794 grams per liters, and is standby.
Embodiment 4: utilize biological catalyst preparation (2S, the 3S)-2,3-butanediol and the meso-2 that obtain in embodiment 1, the mixture of 3-butyleneglycol
Transformation experiment: the biological catalyst that makes gained be the cell concn of Klebsiella pneumonia CICC 10011 be 42.0 gram dry cell weights/liter, initial glucose concentration is 120 grams per liters, at 37 ℃, reacts under pH 6.5 conditions, reaction medium is distilled water.4 hours termination reactions are contained (2S, 3S)-2,3-butanediol and meso-2 the conversion fluid of 3-mixture of butanediols.With the gained conversion fluid with 8,000 rev/mins centrifugal 15 minutes, remove the biological catalyst add, in the gas Chromatographic Determination supernatant liquor (2S, 3S)-2,3-butanediol concentration as 2.8 grams per liters, meso-2,3-butyleneglycol concentration is 35.5 grams per liters.
Collect supernatant liquor at 50 ℃, 0.015 MPa is carried out underpressure distillation, and cut is heavy molten with a small amount of water, obtains 2 of high density, 3-BD solution, and wherein (2S, 3S)-2,3-BD concentration is 61 grams per liters, meso-2,3-BD concentration is 779 grams per liters, and is standby.
Embodiment 5: the preparation of subtilis ATCC 23857 resting cell biological catalysts
(1) slant culture: subtilis ATCC 23857 (available from the biological product preservation of USS center) is scoring to and contains on the culture medium slant that mass volume ratio is 1.5% agar, cultivated 14 hours for 37 ℃.
(2) seed culture: under aseptic condition, with the ring bacterium mud of one on aseptic inoculation ring picking step (1) inclined-plane, be inoculated in 50 milliliters of liquid substratum, cultivated 12 hours for 37 ℃.
(3) fermentor cultivation: under aseptic condition, get the inoculum size of step (2) gained nutrient solution take volume ratio as 5% and be inoculated in 5 liters of liquid nutrient mediums, cultivated 16 hours for 37 ℃.
(4) collect thalline: with the culture 8 that obtains in step (3), 000 rev/min centrifugal 10 minutes, and wash thalline twice with the phosphoric acid buffer of pH 7.0, then cell is resuspended in the phosphoric acid buffer of pH 7.0, the final concentration that makes cell be 50 gram dry cell weights/liter, be biological catalyst, standby.
Wherein, described in above-mentioned steps (1), the solid culture based formulas is: peptone 10.0 grams per liters; Extractum carnis 8.0 grams per liters; Yeast powder 4.0 grams per liters; Glucose 20.0 grams per liters; Sodium acetate 5.0 grams per liters; Triammonium citrate 2.0 grams per liters; K
2HPO
43H
2The O2.0 grams per liter; MgSO
47H
2O 0.2 grams per liter; MnSO
40.2 grams per liter; Agar 15 grams per liters are transferred pH to 6.2, sterilize 20 minutes for 115 ℃.
Described in above-mentioned steps (2)～(3), the liquid culture based formulas is: glucose 70.0 grams per liters; Peptone 10.0 grams per liters; Yeast powder 30.0 grams per liters are transferred pH to 7.0, sterilize 20 minutes for 115 ℃.
Embodiment 6: utilize the biological catalyst that obtains in embodiment 5 to split (2S, 3S)-2,3-butanediol and the meso-2 that obtains in embodiment 2, the mixture of 3-butyleneglycol, preparation (2S, 3S)-2,3-butanediol and (3S)-acetoin simultaneously
Transformation experiment: the biological catalyst that makes gained be the cell concn of subtilis ATCC 23857 be 15 gram dry cell weights/liter, adding the cut that obtains in embodiment 2 is (2S, 3S)-2,3-butanediol and meso-2, the mixture of 3-butyleneglycol, make initial meso-2,3-butyleneglycol concentration is 32 grams per liters, (2S, 3S)-2,3-butyleneglycol concentration is 2.5 grams per liters, at 30 ℃, reacts under pH 7.0 conditions.18 hours termination reactions are contained the conversion fluid of (3S)-acetoin.
With the gained conversion fluid with 8,000 rev/mins centrifugal 15 minutes, remove the biological catalyst add, collect supernatant liquor at 50 ℃, 0.098 MPa is carried out underpressure distillation, and distillate i.e. the aqueous solution of (3S)-acetoin, cut i.e. (2S, 3S)-2,3-butanediol.Distillate and ethyl acetate are pressed equal-volume hybrid extraction (3S)-acetoin, repeat 6 times with method, merge the ethyl acetate after each time extracts, carry out underpressure distillation, obtain sterling (3S)-acetoin.Cut and ethyl acetate are pressed equal-volume hybrid extraction (2S, 3S)-2,3-butanediol, repeat 6 times with method, merge the ethyl acetate after each time extracts, carry out underpressure distillation, obtain sterling (2S, 3S)-2,3-butanediol.
After weighing molten (3S)-acetoin sterling with the isopyknic distilled water of reaction solution, wherein (3S)-acetoin concentration is as 29.3 grams per liters take gas Chromatographic Determination, and corresponding e.e. is 96.2%.The GC collection of illustrative plates of this sample is seen Fig. 1, and retention time is that the peak of 32.7 minutes is the peak of (3S)-acetoin.With with heavy molten (2S, the 3S)-2,3-butanediol sterling of the isopyknic distilled water of reaction solution after, (2S, 3S)-2,3-butanediol concentration is as 2.5 grams per liters in the heavy solution of gas Chromatographic Determination, corresponding optical purity is 96.9%.The GC collection of illustrative plates of this sample is seen Fig. 2, and retention time is that the peak of 43.3 minutes is the peak of (2S, 3S)-2,3-butanediol.
Embodiment 7: utilize the biological catalyst that obtains in embodiment 5 to split (2S, 3S)-2,3-butanediol and the meso-2 that obtains in embodiment 2, the mixture of 3-butyleneglycol, preparation (2S, 3S)-2,3-butanediol and (3S)-acetoin simultaneously
Transformation experiment: the biological catalyst that makes gained be the cell concn of subtilis ATCC 23857 be 16.8 gram dry cell weights/liter, adding the cut that obtains in embodiment 2 is (2S, 3S)-2,3-butanediol and meso-2, the mixture of 3-butyleneglycol, initial meso-2,3-butyleneglycol concentration is 17 grams per liters, (2S, 3S)-2,3-butyleneglycol concentration is 1.3 grams per liters, at 37 ℃, reacts under pH 7.0 conditions.10 hours termination reactions are contained the conversion fluid of (3S)-acetoin.
With the gained conversion fluid with 8,000 rev/mins centrifugal 15 minutes, remove the biological catalyst add, collect supernatant liquor at 50 ℃, 0.015 MPa is carried out underpressure distillation, and distillate i.e. the aqueous solution of (3S)-acetoin, cut i.e. (2S, 3S)-2,3-butanediol.Distillate and ethyl acetate are pressed equal-volume hybrid extraction (3S)-acetoin, repeat 7 times with method, merge the ethyl acetate after each time extracts, carry out underpressure distillation, obtain sterling (3S)-acetoin.Cut and ethyl acetate are pressed equal-volume hybrid extraction (2S, 3S)-2,3-butanediol, repeat 7 times with method, merge the ethyl acetate after each time extracts, carry out underpressure distillation, obtain sterling (2S, 3S)-2,3-butanediol.
After weighing molten (3S)-acetoin sterling with the isopyknic distilled water of reaction solution, wherein (3S)-acetoin concentration is as 14.6 grams per liters take gas Chromatographic Determination, and corresponding e.e. is 96.1%.With with heavy molten (2S, the 3S)-2,3-butanediol sterling of the isopyknic distilled water of reaction solution after, (2S, 3S)-2,3-butanediol concentration is as 1.3 grams per liters in the heavy solution of gas Chromatographic Determination, corresponding optical purity is 97.2%.
Embodiment 8: utilize the biological catalyst that obtains in embodiment 5 to split (2S, 3S)-2,3-butanediol and the meso-2 that obtains in embodiment 2, the mixture of 3-butyleneglycol, preparation (2S, 3S)-2,3-butanediol and (3S)-acetoin simultaneously
Transformation experiment: the biological catalyst that makes gained be the cell concn of subtilis ATCC 23857 be 18 gram dry cell weights/liter, adding the cut that obtains in embodiment 2 is (2S, 3S)-2,3-butanediol and meso-2, the mixture of 3-butyleneglycol, initial meso-2,3-butyleneglycol concentration is 24 grams per liters, (2S, 3S)-2,3-butyleneglycol concentration is 1.9 grams per liters, at 37 ℃, reacts under pH 6.0 conditions.14 hours termination reactions are contained the conversion fluid of (3S)-acetoin.
With the gained conversion fluid with 8,000 rev/mins centrifugal 15 minutes, remove the biological catalyst add, collect supernatant liquor at 50 ℃, 0.050 MPa is carried out underpressure distillation, and distillate i.e. the aqueous solution of (3S)-acetoin, cut i.e. (2S, 3S)-2,3-butanediol.Distillate and ethyl acetate are pressed equal-volume hybrid extraction (3S)-acetoin, repeat 8 times with method, merge the ethyl acetate after each time extracts, carry out underpressure distillation, obtain sterling (3S)-acetoin.Cut and ethyl acetate are pressed equal-volume hybrid extraction (2S, 3S)-2,3-butanediol, repeat 8 times with method, merge the ethyl acetate after each time extracts, carry out underpressure distillation, obtain sterling (2S, 3S)-2,3-butanediol.
After weighing molten (3S)-acetoin sterling with the isopyknic distilled water of reaction solution, wherein (3S)-acetoin concentration is as 20.9 grams per liters take gas Chromatographic Determination, and corresponding e.e. is 96.4%.With with heavy molten (2S, the 3S)-2,3-butanediol sterling of the isopyknic distilled water of reaction solution after, (2S, 3S)-2,3-butanediol concentration is as 1.9 grams per liters in the heavy solution of gas Chromatographic Determination, corresponding optical purity is 96.7%.
Claims (9)
1. one kind by the method for glucose preparation (2S, 3S)-2,3-butanediol with (3S)-acetoin, and step is as follows:
(1), the preparation biological catalyst: final concentration be 70～100 gram dry cell weights/liter Klebsiella pneumonia (
Klebsiella pneumonia) CICC 10011 resting cells;
(2), utilize the Klebsiella pneumonia CICC 10011 resting cell biological catalysts of step (1) preparation to prepare (2S, 3S)-2,3-butanediol and meso-2, the mixture of 3-butyleneglycol;
Wherein: take cell concentration as 14～56 gram dry cell weights/liter biological catalyst be Klebsiella pneumonia CICC 10011 resting cells, at 37 ± 2 ℃, transforming starting point concentration under pH 6.0～7.0 conditions is the glucose of 80～160 grams per liters, react after 3～10 hours, contained (2S, 3S)-2,3-butanediol and meso-2, the conversion fluid of 3-mixture of butanediols; With gained conversion fluid with 8,000 ± 500rpm centrifugal 10～15 minutes, remove the biological catalyst that adds, collect supernatant liquor and carry out underpressure distillation, after the complete evaporate to dryness of moisture content, cut is (2S, 3S)-2,3-butanediol and meso-2, the mixture of 3-butyleneglycol;
(3), prepare again biological catalyst: final concentration be 20～50 gram dry cell weights/liter subtilis (
Bacillus subtilis) ATCC 23857 resting cells;
(4), utilize (the 2S that makes in the subtilis ATCC 23857 resting cell biological catalyst splitting step (2) of step (3) preparation, 3S)-2,3-butyleneglycol and meso-2, the mixture of 3-butyleneglycol, prepare simultaneously (2S, 3S)-2,3-butanediol and (3S)-acetoin;
Wherein: take cell concentration as 13～20 gram dry cell weights/liter biological catalyst be subtilis ATCC 23857 resting cells, at 23～45 ℃, under pH 5.0～8.0 conditions with step (2) in (2S, the 3S)-2,3-butanediol and the meso-2 that make, the mixture of 3-butyleneglycol carries out the transformation experiment of 6～24 hours, split (2S, 3S)-2,3-butanediol and meso-2, the 3-butyleneglycol is contained the conversion fluid of (3S)-acetoin; With gained conversion fluid with 8,000 ± 500rpm centrifugal 10～15 minutes, remove the biological catalyst that adds, to collect supernatant liquor and carry out underpressure distillation, distillate is the aqueous solution of (3S)-acetoin, and cut is (2S, 3S)-2,3-butanediol; Then, distillate and ethyl acetate are pressed equal-volume hybrid extraction (3S)-acetoin, repeat 6～8 times with method, merge the ethyl acetate after each time extracts, carry out underpressure distillation, obtain sterling (3S)-acetoin; Cut and ethyl acetate are pressed equal-volume hybrid extraction (2S, 3S)-2,3-butanediol, repeat 6～8 times with method, merge the ethyl acetate after each time extracts, carry out underpressure distillation, obtain sterling (2S, 3S)-2,3-butanediol.
2. as claimed in claim 1 by glucose preparation (2S, 3S)-2, the method of 3-butyleneglycol and (3S)-acetoin is characterized in that: the described biological catalyst of step (2) be the cell concentration of Klebsiella pneumonia CICC 10011 resting cells be 28～42 gram dry cell weights/liter.
3. as claimed in claim 1 by the method for glucose preparation (2S, 3S)-2,3-butanediol with (3S)-acetoin, it is characterized in that: the starting point concentration of the described glucose of step (2) is 100～120 grams per liters.
4. as claimed in claim 1 by the method for glucose preparation (2S, 3S)-2,3-butanediol with (3S)-acetoin, it is characterized in that: the described reaction times of step (2) is 4～8 hours.
5. as claimed in claim 1 by glucose preparation (2S, 3S)-2, the method of 3-butyleneglycol and (3S)-acetoin is characterized in that: the described biological catalyst of step (4) be the cell concentration of subtilis ATCC 23857 resting cells be 15～18 gram dry cell weights/liter.
6. as claimed in claim 1 by glucose preparation (2S, the method of 3S)-2,3-butanediol and (3S)-acetoin is characterized in that: the described (2S of step (4), 3S)-2,3-butyleneglycol and meso-2, in the mixture of 3-butyleneglycol, meso-2,3-butyleneglycol concentration is 8.5～40 grams per liters, (2S, 3S)-2,3-butanediol concentration is 0.66～3.1 grams per liter.
7. as claimed in claim 6 by glucose preparation (2S, the method of 3S)-2,3-butanediol and (3S)-acetoin is characterized in that: the described (2S of step (4), 3S)-2,3-butyleneglycol and meso-2, in the mixture of 3-butyleneglycol, meso-2,3-butyleneglycol concentration is 17～32 grams per liters, (2S, 3S)-2,3-butanediol concentration is 1.3～2.5 grams per liters.
8. as claimed in claim 1 by glucose preparation (2S, the method of 3S)-2,3-butanediol and (3S)-acetoin, it is characterized in that: the temperature of the described transformation experiment of step (4) is 30～37 ℃, pH is 6.0～7.5, and the reaction times is 10～18 hours.
9. as claimed in claim 1 by glucose preparation (2S, 3S)-2, the method of 3-butyleneglycol and (3S)-acetoin, it is characterized in that: the condition of step (2) or (4) described underpressure distillation is: 50 ± 2 ℃ of temperature, pressure 0.015～0.098 MPa.
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