CN102703524A - Method for preparing (S)-(+)-1, 3-butanediol through microbial transformation - Google Patents
Method for preparing (S)-(+)-1, 3-butanediol through microbial transformation Download PDFInfo
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Abstract
The invention provides a method for preparing (S)-(+)-1, 3-butanediol through microbial transformation. The method comprises the following steps of: performing transformation reaction by taking 4-hydroxyl-2-butanone as a substrate and cells containing enzyme thalli obtained by fermentation of saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No. 2266 as a biocatalyst in potassium phosphate buffer solution with pH of 5.0-8.0 at 25-45 DEG C for 8-108 hours; and separating and purifying the transformation solution after completion of the reaction to obtain the (S)-(+)-1, 3-butanediol. The enantiomer excess of the (S)-(+)-1, 3-butanediol produced by using the method provided by the invention is greater than 99.9 percent, the molar transformation rate of the substrate can reach 100 percent, the product purity reaches 99.8 percent, and immobilized cell particles can be repeatedly used for 16 times.
Description
(1) technical field
The present invention relates to a kind of method of microbial transformation preparation (S)-(+)-1,3 butylene glycol.
(2) background technology
(S)-(+)-and 1,3 butylene glycol ((S)-(+)-1,3-Butanediol), CAS accession number: 24621-61-2, molecular formula C
4H
10O
2, molecular weight 90.12.(S)-(+)-1,3 butylene glycol is a kind of important chiral drug midbody, is that chiral source can be synthesized a large amount of chipal compounds with it.It is the chiral raw material of the important intermediate cyanalcohol of synthetic agricultural insecticide pyrethroid.Pyrethroid is the one type of broad spectrum pesticide that can prevent and treat various pests, and its insecticidal toxicity improves 10 ~ 100 times than older generation sterilant like organochlorine, organophosphorus, amino formate.The chemical structure of pyrethroid is complicated, and optically active isomer or cis-trans steric isomer are arranged, and the reactions step of production technique is more, to raw materials quality with control strict.In addition, (S)-(+)-1,3 butylene glycol can be introduced directly into synthesis of natural product in the target molecule; Can be used as chiral monomer is applied in the reaction of lewis acid mediated ethylidene ether and nucleophilic reagent; Can be applied to the synthetic of optically active phosphine oxide; Can also be as the ofhypoglycemic medicine of biologically active substance as the diabetics.
1991, people such as Larcheveque M. adopted L or D-Threonine to make pure R or S-1 through nitrous acid deamination bromination, esterification, palladium catalyzed hydrogenation and lithium aluminium hydride reduction four-step reaction, and 3-butyleneglycol enantiomer, total recovery are 64%.This process reaction step is more, and yield is not high.Adopt chemical reduction method asymmetric reduction 4-hydroxyl-2-butanone also can prepare (S)-(+)-1,3 butylene glycol, but need expensive chemical catalyst Raney's nickel as chiral catalyst, cost is higher.Calendar year 2001, people such as Isidoro I. adopt lypase resolution of racemates 1,3 butylene glycol to prepare R or S-1, the 3-butyleneglycol, but splitting the efficient maximum has only 50%, and the lypase price is comparatively expensive, so lypase method for splitting cost is higher, and production efficiency is lower.
(3) summary of the invention
The object of the invention provides a kind of method of utilizing yeast saccharomyces cerevisiae bio-transformation 4-hydroxyl-2-butanone to synthesize (S)-(+)-1,3 butylene glycol.
The technical scheme that the present invention adopts is:
A kind of microbial transformation prepares (S)-(+)-1; The method of 3-butyleneglycol, said method comprises: with 4-hydroxyl-2-butanone is substrate, the enzyme somatic cells that contains that obtains with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.2266 fermentation is a biological catalyst; In the potassium phosphate buffer of pH 5.0 ~ 8.0; Under 25 ~ 45 ℃, carry out conversion reaction 8 ~ 108 hours, reaction finishes the back conversion fluid and obtains described (S)-(+)-1,3 butylene glycol through separation and purification.
Bacterial strain uses therefor yeast saccharomyces cerevisiae CGMCC No.2266 of the present invention screens near the soil the brew-house of the West Lake, Hangzhou to obtain; In CN101230319A, disclose; Test discovery through the contriver, this bacterial strain has the ability of good conversion 4-hydroxyl-2-butanone production (S)-(+)-1,3 butylene glycol.This bacterial strain colony characteristics: on nutrient agar, demonstrate oyster white, glossy, smooth, neat in edge, moistening, smooth surface, the uniform colonial morphology of quality.
The initial final concentration of substrate 4-hydroxyl-2-butanone in potassium phosphate buffer is 2 ~ 100mmol/L.
The said enzyme somatic cells consumption that contains is counted 1 ~ 50g/g substrate with dried cell weight.The said enzyme somatic cells that contains can be to participate in reaction with the free cell form, also can after immobilization, participate in reaction with the immobilized cell form.Immobilized cell can make by entrapping method; Concrete preparation process can be according to carrying out as follows: will mix mutually with the sodium alginate soln of equal-volume 1 ~ 5% (w/w) through the fermented liquid that Wine brewing yeast strain CGMCC No.2266 fermentation culture obtains; Cell concentration is 3.0 ~ 10.0g/L in the described fermented liquid; Stirring obtains mixed solution, mixed solution is dropwise splashed in the calcium chloride solution that mass concentration is 2.5 ~ 4.0% (w/w) continuously stirring; The sodium-alginate mixed solution that contains thalline solidify to form the gel beads immobilization particle; The immobilization particle suspension-s that obtains is positioned under 35 ~ 38 ℃ of conditions solidifies, the immobilization particle that obtains washs with SPSS, obtains immobilized cell particle.The immobilized cell particle that obtains is scattered in the immobilized cell particle that carries out in the fermention medium after multiplication culture 16 ~ 72h obtains multiplication culture, with the immobilized cell particle behind the multiplication culture as the used biological catalyst of the present invention.
The diameter of described immobilized cell particle can be controlled through the needle sizes of syringe, is recommended as 1 ~ 5mm, most preferably 2mm.
In the embedding treatment process, the concentration of sodium-alginate is recommended as 1 ~ 5% (w/w), and most preferably 2%.
The present invention adopts immobilized cell particle as catalyzer, and the bio-transformation mechanism that 4-hydroxyl-2-butanone is reduced to (S)-(+)-1,3 butylene glycol is following:
The said enzyme somatic cells that contains prepares according to following method: yeast saccharomyces cerevisiae CGMCC No.2266 is seeded in the fermention medium; Shaking speed is 150 ~ 200r/min; Cultivate 18 ~ 30h down,, promptly get the said enzyme somatic cells that contains for 26 ~ 35 ℃ the centrifugal removal supernatant of fermented liquid; Said fermention medium final concentration consists of: glucose 26 ~ 32g/L, yeast powder 2 ~ 4g/L, ammonium sulfate 3 ~ 6g/L, anhydrous MgSO
40.2 ~ 0.4g/L, K
2HPO
43H
20 0.5 ~ 1.5g/L, KH
2PO
40.6 ~ 1.5g/L, pH7.0, solvent are water.
Said separation purification method is following: after reaction finished, conversion fluid filtered, and got the filtrate decompression rotary evaporation except that anhydrating; The sample dissolution that obtains adds dried over sodium sulfate in the acetone of 5 times of sample volumes, remove by filter sodium sulfate; Distillation is removed acetone and is promptly got product (S)-(+)-1,3 butylene glycol.
This patent adopts immobilized brewing yeast asymmetric reduction 4-hydroxyl-2-butanone to obtain (S)-(+)-1,3 butylene glycol of high-optical-purity.This yeast saccharomyces cerevisiae low price is easy to realize in the short period of time large scale culturing.The substrate conversion efficiency of this technological process 4-hydroxyl-2-butanone is high.The application of immobilized cell in the bio-conversion process process helps realizing the recycling of microorganism cells, and reaction conditions is gentle, environmental friendliness; With low cost, be easy to realize the in-situ regeneration of coenzyme be easy to suitability for industrialized production; It is the green synthesis process of (S)-(+)-1,3 butylene glycol.
The present invention can obtain fermented liquid as follows:
(1) slant culture: yeast saccharomyces cerevisiae CGMCC No.2266 thalline is inoculated into slant medium, cultivates the thalline that obtained slant culture in 4 ~ 6 days for 26 ~ 35 ℃; Described slant medium is formed: wort 5 ~ 15g/L, and yeast powder 2 ~ 4g/L, peptone 4 ~ 6g/L, glucose 7 ~ 12g/L, agar 15 ~ 25g/L, natural pH value, solvent is a water; 121 ℃ of sterilization 20min, sterilization postcooling bevel;
(2) seed culture: get a ring thalline from slant medium and be transferred to seed culture medium, 26 ~ 35 ℃, shaking speed is 150 ~ 200r/min, cultivates 18 ~ 26h and gets seed liquor; Described seed culture medium is formed: glucose 26 ~ 32g/L, yeast powder 2 ~ 4g/L, ammonium sulfate 3 ~ 6g/L, anhydrous MgSO
40.2 ~ 0.4g/L, K
2HPO
43H
2O 0.5 ~ 1.5g/L, KH
2PO
40.6 ~ 1.5g/L, natural pH value, solvent is a water; Sterilized 20 minutes for 121 ℃, the sterilization postcooling promptly gets seed culture medium;
(3) fermentation culture: get seed liquor, the inoculum size with 10 ~ 20% is inoculated in the fermention medium, and culture temperature is 26 ~ 35 ℃, and shaking speed is 150 ~ 200r/min, cultivates 18 ~ 30h and obtains fermented liquid; The composition of described fermention medium is with the seed substratum.
The concrete recommendation obtains biological catalyst according to following steps: the resulting fermented liquid in front is mixed with the sodium alginate soln of equal-volume 2% mutually; Described fermented liquid cell concentration is 4.30g/L; Stirring obtains mixed solution, and it is in 3.5% the calcium chloride solution that mixed solution is dropwise splashed into mass concentration, continuously stirring; The sodium-alginate mixed solution that contains thalline solidify to form the gel beads immobilization particle; The immobilization suspension-s that obtains is positioned under 37 ℃ of conditions solidifies, the immobilization particle that obtains washs with SPSS, obtains immobilized cell particle; The immobilized cell particle that obtains is scattered in and carries out multiplication culture 72h in the fermention medium, obtains the immobilized cell particle behind the multiplication culture, is biological catalyst with the immobilized cell particle behind the multiplication culture.
Concrete recommend described being applied as: with the immobilized cell particle behind the multiplication culture as biological catalyst; With 4-hydroxyl-2-butanone is substrate; In potassium phosphate buffer; Conversion reaction 84h under 30 ℃, 180r/min condition, conversion fluid obtains product (S)-(+)-1,3 butylene glycol through separation and purification; Described separation and purification can be adopted following steps: conversion fluid is removed by filter immobilized cell particle; The filtrate decompression rotary evaporation removed anhydrate; The sample dissolution that obtains adds sodium sulfate and removes minor amount of water in the acetone of 5 times of sample volumes, removes by filter sodium sulfate; Distillation is removed acetone and can be obtained product (S)-(+)-1,3 butylene glycol.In potassium phosphate buffer; The starting point concentration of substrate 4-hydroxyl-2-butanone is 2 ~ 100mmol/L, and it is the dry cell weight that the fermented liquid of 4.3g/L is processed the immobilized cell particle behind the immobilized cell particle multiplication culture that multiplication culture obtained after 24 hours in fermention medium with the 0.33L cell concentration that the dry cell weight that contains in every L phosphoric acid buffer is equivalent to entrapping method.
The pure article of (S)-(+)-1,3 butylene glycol that obtain detect with gas chromatograph-mass spectrometer confirms degree of purity of production and molecular weight.
Confirming of the superfluous value of molar yield and product (S)-(+)-1,3 butylene glycol enantiomorph (ee%):
Adopt derivatization method to detect, derivatization method is following, after bioconversion reaction finishes; Conversion fluid is removed by filter immobilized cell particle, the liquid sample rotary evaporation is removed anhydrate, after dewatering sample is dissolved in acetone; Add sodium sulfate and remove micro-moisture, remove by filter sodium sulfate, acetone is removed in volatilization; Add and sample isopyknic 3-gifblaar poison acid anhydride and pyridine, behind 45 ℃ of insulation 2h, the samples using Agilent 7820 gas chromatograph analyzing and testing behind the derivatize.Chromatographic column is a chiral column, and model is Chiral CYCLODEX-B (0.25mm * 30m * 0.25 μ m); Carrier gas is a nitrogen, and flow rate of carrier gas is 1ml/min.Chiral chromatographic column can detect (R)-(+)-1,3 butylene glycol with (S)-(+)-content of two kinds of enantiomorphs of 1,3 butylene glycol, further calculate the molar yield of reaction with (S)-(+)-the superfluous value of enantiomorph of 1,3 butylene glycol.
The immobilized cell particle that filtration obtains is reusable in the reaction of microbial transformation preparation (S)-(+)-1,3 butylene glycol.
Effect intentionally of the present invention is embodied in the following aspects:
1, microbe transformation method preparation (S)-(+)-1,3 butylene glycol of the present invention's employing is compared with chemical synthesis and had the following advantages: 1. production operation is easy, and biological transformation ratio is higher.2. produce used bacterial strain safety non-toxic.3. environmental friendliness, reaction conditions is gentle, can transform smoothly under the normal temperature and pressure.4. do not receive seasonal effect, be easy to realize large-scale industrial production.5. biological catalyst is more with low cost than chemical catalyst.
2, adopting immobilized cell in phosphate buffered saline buffer, to carry out bio-transformation among the present invention compares with traditional bio-transformation and have following advantage: 1. immobilized cell helps realizing the separation and Extraction of product as biological catalyst.2. immobilized cell helps realizing the recycling of catalyzer as biological catalyst, practices thrift cost.3. be easy to realize large-scale industrial production.
The superfluous value of (S)-(+)-1,3 butylene glycol enantiomorph that utilizes the inventive method to produce is greater than 99.9%, and the substrate molar yield can reach 100%, and product purity reaches 99.8%, and immobilized cell particle can reuse 16 times.
(4) embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1:
Slant culture: CGMCC No.2266 bacterial classification inoculation is being contained wort 10g/L, yeast powder 3g/L, peptone 5g/L, glucose 10g/L, agar 20g/L cultivates in the substratum of natural pH value.Cultivated 4～6 days for 30 ℃.
Seed culture and fermentation: seed and fermention medium all adopt liquid nutrient medium, and composition is glucose 30g/L, yeast powder 3g/L, ammonium sulfate 5g/L, anhydrous MgSO
40.25g/L, K
2HPO
43H
2O 1g/L, KH
2PO
41g/L.From slant medium, get a ring thalline with inoculating needle and be seeded in the 250ml triangular flask that contains the 100ml liquid nutrient medium, under 30 ℃, the condition of 180r/min, cultivate 24h and obtain seed.The inoculum size of seed liquor with 10% (v/v) is inoculated in the 250ml triangular flask that contains the 100ml liquid nutrient medium, under 30 ℃, the condition of 180r/min, cultivates 24h and obtain fermented liquid, be used for the immobilization of thalline with the fermented liquid that obtains.
With dry cell weight is that 100ml fermented liquid and the equal-volume concentration of 430mg is that 2% sodium alginate soln is mixed and made into mixed solution, and mixed solution is packed in the syringe, splashes into 3.5%CaCl
2Form immobilization particle in the solution; The immobilization particle diameter that forms is 2mm; Solidify 30min in 37 ℃; The immobilization particle that obtains washs with SPSS, and calcium ion that flush away is excessive and captured cell not continue multiplication culture 72h in the liquid medium within the immobilized cell particle that obtains.
6 parts of good immobilized cell particles of multiplication culture are joined 6 bottles of 100ml pH values respectively be respectively 5.8,6.2, in 6.8,7.2,7.6 and 8.0 the potassium phosphate buffer (0.1mol/L).Begin bioconversion reaction after in five bottles, adding substrate 5.5mmol/L respectively.Behind the reaction 84h; Adopt filtering method to carry out solid-liquid separation, the liquid concentrating under reduced pressure that obtains removes and anhydrates, and adds the acetone of 5 times of volumes; Add sodium sulfate and remove minor amount of water; Remove by filter sodium sulfate, will contain the enantiomorph superfluous value of the acetone soln of product with the molar yield and product (S)-(+)-1,3 butylene glycol of gas chromatographic analysis substrate.The result shows that in the buffered soln of pH5.8 ~ 8.0, immobilization CGMCC No.2266 has the better conversion ability to 4-hydroxyl-2-butanone, and the best pH value of potassium phosphate buffer is 6.8.In the damping fluid of pH6.8,4-hydroxyl-2-butanone of 5.5mmol/L can be converted into (S)-(+)-1,3 butylene glycol in 100% ground.Immobilization CGMCC No.2266 transforms superfluous the value all greater than 99.9% of enantiomorph of (S)-(+)-1,3 butylene glycol of 4-hydroxyl-2-butanone acquisition in the buffer solution of potassium phosphate of pH5.8 ~ 8.0.
Table 1: immobilization CGMCC No.2266 transforms 4-hydroxyl-2-butanone in different pH potassium phosphate buffers
| pH | 5.8 | 6.2 | 6.8 | 7.2 | 7.6 | 8.0 |
| Molar yield (%) | 90.7 | 95.2 | 100 | 99.6 | 98.2 | 96.5 |
Embodiment 2:
CGMCC No.2266 is cultivated acquisition thalline fermented liquid according to the method in the instance 1.With 4 bottles of dry cell weights fermented liquid that is 430mg is that 2% sodium alginate soln is mixed and made into mixed solution with equal-volume concentration respectively, and mixed solution is respectively charged in the syringe, splashes into 3.5%CaCl
2Form immobilization particle in the solution; The immobilization particle diameter that forms is 2mm; Solidify 30min in 37 ℃; The immobilization particle that obtains washs with SPSS, and calcium ion that flush away is excessive and captured cell not continue multiplication culture 0h, 24h, 48h and 72h in the liquid medium within respectively with 4 bottles of immobilized cell particles that obtain.
4 kinds of good immobilized cell particles of multiplication culture are added respectively in the 100ml pH6.8 potassium phosphate buffer (0.1mol/L) that contains 5.5mmol 4-hydroxyl-2-butanone; Carry out bio-transformation 84h under 30 ℃, the condition of 180r/min, transform finish after, adopt filtering method to carry out solid-liquid separation; The liquid concentrating under reduced pressure that obtains removes and anhydrates; The acetone that adds 5 times of volumes adds sodium sulfate and removes minor amount of water, removes by filter sodium sulfate; To contain the enantiomorph superfluous value of the acetone soln of product with the molar yield and product (S)-(+)-1,3 butylene glycol of gas chromatographic analysis substrate.The result is as shown in table 2.The result shows that the raising of molar yield is grown and more helped to the multiplication culture time of immobilized cell particle, and the immobilized cell particle that the multiplication culture time reaches 72h is used to transform 4-hydroxyl-2-butanone molar yield and can reaches 100%.The time of immobilized cell multiplication culture is long more, and cell concentration is many more, helps improving the transformation efficiency of biotransformation more.The immobilized cell particle that obtains with above-mentioned 4 kinds of different multiplication culture times is that the superfluous value of enantiomorph of (S)-(+)-1,3 butylene glycol of biological Preparation of Catalyst is all greater than 99.9%.
Table 2: the immobilized cell particle of different multiplication culture times transforms 4-hydroxyl-2-butanone
| The multiplication culture time (h) | 0 | 24 | 48 | 72 |
| Molar yield (%) | 0 | 65.2 | 82.1 | 100 |
Embodiment 3:
CGMCC No.2266 is cultivated acquisition thalline fermented liquid according to the method in the instance 1.With dry cell weight is that 100ml fermented liquid and the equal-volume concentration of 430mg is that 2% sodium alginate soln is mixed and made into mixed solution, and mixed solution is packed in the syringe, splashes into 3.5%CaCl
2Form immobilization particle in the solution; The immobilization particle diameter that forms is 2mm; Solidify 30min in 37 ℃; The immobilization particle that obtains washs with SPSS, and calcium ion that flush away is excessive and captured cell not continue multiplication culture 72h in the liquid medium within the immobilized cell particle that obtains.
The immobilized cell particle that multiplication culture is good joins 5 bottles of 4-hydroxyl-2-butanone starting point concentrations and is respectively in the 100ml pH6.8 potassium phosphate buffer (0.1mol/L) of 5.5mmol/L, 11.0mmol/L, 16.5mmol/L, 22.0mmol/L and 27.5mmol/L; Carry out bio-transformation 84h under 30 ℃, the condition of 180r/min; After transforming end; Adopt filtering method to carry out solid-liquid separation, the liquid concentrating under reduced pressure that obtains removes and anhydrates, and adds the acetone of 5 times of volumes; Add sodium sulfate and remove minor amount of water; Remove by filter sodium sulfate, will contain the enantiomorph superfluous value of the acetone soln of product with the molar yield and product (S)-(+)-1,3 butylene glycol of gas chromatographic analysis substrate.The result is as shown in table 3.The result shows that the substrate starting point concentration has considerable influence to the molar yield of immobilized cell bio-transformation 4-hydroxyl-2-butanone.The molar yield of reaction reduces along with the raising of concentration of substrate.The amount that substrate is described increases there is restraining effect in bio-transformation.When the substrate starting point concentration was 5.5mmol/L, the molar yield of reaction can reach 100%.In the biotransformation under above-mentioned 5 kinds of different substrate starting point concentration conditions, the superfluous value of the enantiomorph of product (S)-(+)-1,3 butylene glycol is all greater than 99.9%.
Table 3: immobilization CGMCCNo.2266 transforms the 4-hydroxyl-2-butanone of different starting point concentrations
Embodiment 4:
CGMCC No.2266 is cultivated acquisition thalline fermented liquid according to the method in the instance 1.With 4 bottles of dry cell weights 100ml fermented liquid that is 430mg is that the sodium alginate soln of 2% (w/w) is mixed and made into mixed solution with equal-volume concentration respectively, and mixed solution is respectively charged in the different syringe of needle sizes, splashes into 3.5%CaCl
2Form immobilization particle in the solution; The immobilization particle diameter that forms is respectively 2mm, 3mm, 4mm and 5mm; Solidify 30min in 37 ℃; The immobilization particle that obtains washs with SPSS, and calcium ion that flush away is excessive and captured cell not continue multiplication culture 72h in the liquid medium within the immobilized cell particle that obtains.
4 kinds of good immobilized cell particles of multiplication culture are joined respectively in the 100ml pH6.8 potassium phosphate buffer (0.1mol/L) that contains 5.5mmol 4-hydroxyl-2-butanone; Carry out bio-transformation 108h under 30 ℃, the condition of 180r/min, every in the conversion process at a distance from the 12h sampling, adopt filtering method to carry out solid-liquid separation; The liquid concentrating under reduced pressure that obtains removes and anhydrates; The acetone that adds 5 times of volumes adds sodium sulfate and removes minor amount of water, removes by filter sodium sulfate; To contain the enantiomorph superfluous value of the acetone soln of product with the molar yield and product (S)-(+)-1,3 butylene glycol of gas chromatographic analysis substrate.The result is as shown in table 4.The result shows; The difference of immobilized cell particle diameter is influential to the molar yield of bio-transformation; When optimal fixation cell granulations diameter is 2mm; The molar yield of bio-transformation reaches 100%, with the immobilization particle of above-mentioned four kinds of diameters as the superfluous value of the enantiomorph of (S)-(+)-1,3 butylene glycol of Preparation of Catalyst all greater than 99.9%.
Table 4: the immobilized cell particle of different diameter transforms the molar yield (%) of 4-hydroxyl-2-butanone
Embodiment 5:
CGMCC No.2266 is cultivated acquisition thalline fermented liquid according to the method in the instance 1.With 5 bottles of dry cell weights 100ml fermented liquid that is 430mg is that the sodium alginate soln of 1%, 2%, 3%, 4% and 5% (w/w) is mixed and made into mixed solution with equal-volume concentration respectively, and mixed solution is respectively charged into syringe, splashes into 3.5%CaCl
2Form immobilization particle in the solution; The particle diameter size is 2mm, and in 37 ℃ of curing 30min, the immobilization particle that obtains washs with SPSS; Calcium ion that flush away is excessive and captured cell not continue multiplication culture 72h in the liquid medium within the immobilized cell particle that obtains.
5 kinds of good immobilized cell particles of multiplication culture are joined respectively in the 100ml pH6.8 potassium phosphate buffer (0.1mol/L) that contains 5.5mmol 4-hydroxyl-2-butanone; Carry out bio-transformation 108h under 30 ℃, the condition of 180r/min, every in the conversion process at a distance from the 12h sampling, adopt filtering method to carry out solid-liquid separation; The liquid concentrating under reduced pressure that obtains removes and anhydrates; The acetone that adds 5 times of volumes adds sodium sulfate and removes minor amount of water, removes by filter sodium sulfate; To contain the enantiomorph superfluous value of the acetone soln of product with the molar yield and product (S)-(+)-1,3 butylene glycol of gas chromatographic analysis substrate.The result shows; In the immobilization process; The concentration of sodium-alginate is influential to the bio-transformation ability of immobilized cell particle, and when the concentration of sodium-alginate was optimum concn 2%, the molar yield of bio-transformation can reach 100%; The superfluous value of the enantiomorph of resulting (S)-(+) of the immobilization CGMCC No.2266 particle conversion of substrate-1,3 butylene glycol that under the sodium alginate to embed condition of 5 kinds of different concns, obtains is all greater than 99.9%.
Table 5: the sodium alginate soln immobilization CGMCC No.2266 of different concns transforms the molar yield (%) of 4-hydroxyl-2-butanone
Embodiment 6:
CGMCC No.2266 is cultivated acquisition thalline fermented liquid according to the method in the instance 1.With dry cell weight is that fermented liquid and the equal-volume concentration of 430mg is that 2% sodium alginate soln is mixed and made into mixed solution, and mixed solution is packed in the syringe, splashes into 3.5%CaCl
2Form immobilization particle in the solution; The immobilization particle diameter that forms is 2mm; Solidify 30min in 37 ℃; The immobilization particle that obtains washs with SPSS, and calcium ion that flush away is excessive and captured cell not continue multiplication culture 72h in the liquid medium within the immobilized cell particle that obtains.
The immobilized cell particle that multiplication culture is good joins in the 100ml pH6.8 potassium phosphate buffer (0.1mol/L) that contains 5.5mmol 4-hydroxyl-2-butanone, carries out bio-transformation 84h under 30 ℃, the condition of 180r/min, transforms to finish after-filtration and go out immobilized cell particle; Immobilized cell particle is suspended in the 100ml pH6.8 potassium phosphate buffer (0.1mol/L) again; Carry out bio-transformation 84h under 30 ℃, the condition of 180r/min, so reuse immobilized cell particle 16 times, after each reaction finishes; Adopt filtering method to carry out solid-liquid separation; The liquid concentrating under reduced pressure that obtains removes and anhydrates, and adds the acetone of 5 times of volumes, adds sodium sulfate and removes minor amount of water; Remove by filter sodium sulfate; To contain the enantiomorph superfluous value of the acetone soln of product with the molar yield and product (S)-(+)-1,3 butylene glycol of gas chromatographic analysis substrate, the result is as shown in table 6.The result shows; Immobilized cell particle can be re-used in the biosynthesizing of (S)-(+)-1,3 butylene glycol well, product (S)-(+)-1; The superfluous value of the enantiomorph of 3-butyleneglycol all greater than 99.9%, the 16 time molar yield be the first time molar yield 15.2%.
Table 6: immobilization CGMCC No.2266 transforms the recycling of 4-hydroxyl-2-butanone
| The recycling number of times | Molar yield (%) | The recycling number of times | Molar yield (%) |
| 1 | 100 | 9 | 48.0 |
| 2 | 95.2 | 10 | 41.0 |
| 3 | 88.9 | 11 | 35.2 |
| 4 | 85.7 | 12 | 30.9 |
| 5 | 76.8 | 13 | 26.7 |
| 6 | 69.5 | 14 | 23.2 |
| 7 | 62.3 | 15 | 18.9 |
| 8 | 55.2 | 16 | 15.2 |
Claims (5)
1. a microbial transformation prepares (S)-(+)-1; The method of 3-butyleneglycol, said method comprises: with 4-hydroxyl-2-butanone is substrate, the enzyme somatic cells that contains that obtains with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.2266 fermentation is a biological catalyst; In the potassium phosphate buffer of pH 5.0 ~ 8.0; Under 25 ~ 45 ℃, carry out conversion reaction 8 ~ 108 hours, reaction finishes the back conversion fluid and obtains described (S)-(+)-1,3 butylene glycol through separation and purification.
2. the method for claim 1 is characterized in that the initial final concentration of substrate 4-hydroxyl-2-butanone in potassium phosphate buffer is 2 ~ 100mmol/L.
3. the method for claim 1 is characterized in that the said enzyme somatic cells consumption that contains counts 1 ~ 50g/g substrate with dried cell weight.
4. the method for claim 1; It is characterized in that the said enzyme somatic cells that contains prepares according to following method: yeast saccharomyces cerevisiae CGMCC No.2266 is seeded in the fermention medium; Shaking speed is 150 ~ 200r/min; Cultivate 18 ~ 30h down,, promptly get the said enzyme somatic cells that contains for 26 ~ 35 ℃ the centrifugal removal supernatant of fermented liquid; Said fermention medium final concentration consists of: glucose 26 ~ 32g/L, yeast powder 2 ~ 4g/L, ammonium sulfate 3 ~ 6g/L, anhydrous MgSO
40.2 ~ 0.4g/L, K
2HPO
43H
200.5 ~ 1.5g/L, KH
2PO
40.6 ~ 1.5g/L, pH7.0, solvent are water.
5. the method for claim 1 is characterized in that said separation purification method is following: after reaction finished, conversion fluid filtered; Get the filtrate decompression rotary evaporation except that anhydrating; The sample dissolution that obtains adds dried over sodium sulfate in the acetone of 5 times of sample volumes, remove by filter sodium sulfate; Distillation is removed acetone and is promptly got product (S)-(+)-1,3 butylene glycol.
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| CN201210179794.3A CN102703524B (en) | 2012-05-31 | 2012-05-31 | Method for preparing (S)-(+)-1, 3-butanediol through microbial transformation |
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| CN102703524B CN102703524B (en) | 2014-06-11 |
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