CN102250970A - Method for synthesizing 1,3-dioxyacetone by glycerol fermentation - Google Patents
Method for synthesizing 1,3-dioxyacetone by glycerol fermentation Download PDFInfo
- Publication number
- CN102250970A CN102250970A CN2011101376102A CN201110137610A CN102250970A CN 102250970 A CN102250970 A CN 102250970A CN 2011101376102 A CN2011101376102 A CN 2011101376102A CN 201110137610 A CN201110137610 A CN 201110137610A CN 102250970 A CN102250970 A CN 102250970A
- Authority
- CN
- China
- Prior art keywords
- otan
- fermentation
- dioxyacetone
- glycerol
- substratum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the synthesis of a 1,3-dioxyacetone. A method for synthesizing 1,3-dioxyacetone by glycerol fermentation comprises the following steps: performing the gene recombination and DNA rearrangement technology on bacillus licheniformis B-05571 separated and screened from acid soil, integrating a dehydrogenase gene into an Escherichia coli JM109, constructing industrial engineering bacteria of 1,3-dioxyacetone synthesized by glycerol dehydrogenization, immobilizing by using a supermolecule self-assembly template, then fermenting in a culture medium containing glycerol to synthesize 1,3-dioxyacetone. The product 1,3-dioxyacetone can be obtained by the steps of filtering fermentation liquor, extracting with an organic solvent and recrystallizing with a combined solvent. The synthesis process route is simple and convenient, the product is high in quality and low in cost, and the total mass yield is higher than 75%.
Description
Technical field: the invention belongs to biological technical field of organic synthesis, relate to 1, the biological methodology of organic synthesis of 3-otan.
Background technology
1, a kind of important chemical of 3-otan, biochemical raw material, medicine, agricultural chemicals synthetic intermediate and polyfunctional food additive, purposes is very extensive.The synthetic method of otan mainly contains noble metal catalytic oxidation and microbial method.The synthesis method of heavy metal catalyzed oxidation glycerine owing to can't overcome the fatal shortcoming of catalytic selectivity difference, causes glycerol conversion yield to be lower than 40%, and the productive rate of DHA is lower than 25%.Industrial process is to utilize the microorganism batch fermentation at present.This method is to arrive suitable period at thalli growth, and the desaturase that utilizes thalline to produce is that substrate carries out dehydrogenation reaction with glycerine, produces DHA.If be used to produce microbial host acetobacter (Acetobacter) and Gluconobacter (Gluconobacter) microorganism, especially weak oxidized acetic acid bacteria (Acetobacter Suboxy-dans) and the bacillus of oxidizing glucose (Gluoonobacter 0xydans) of DHA.Bacterial classification is cultivated earlier after recovery in advance, changes the fermentor tank enlarged culturing then, treat that bacterial classification reaches finite concentration after, be inoculated in the middle of the fermention medium that contains glycerine, after the fermentation of the oxygen consumption of 60-80h, again fermented liquid is once emitted, obtain DHA through separation and purification.Each batch fermentation process all experiences inoculation, growth and breeding, thalline is old and feeble and then finish fermentation, finally extracts product.The existing at present shunting glycerol adding method that adopts, when thalli growth arrives logarithmic phase, and after product D HA reaches certain level, emit portion of product, and add raw material and substratum, so just can reduce the time of the biomass that reaches production level, improve the utilization ratio of substrate glycerine, save cost.The characteristics of batch fermentation are that microorganism environment of living in is constantly to change, and living contaminants takes place, and can be easy to terminating operation.In fermentative production, the bacterial classification of use is in logarithmic phase, when they are inoculated into the fermentor tank fresh culture, the adjustment period of the appearance hardly, can obtain the vigorous thalline of raised growth at short notice like this, helps shortening the production cycle.But the subject matter of this method has produced high osmotic pressure when being excessive concentration in substratum of substrate glycerine and product D HA, makes fermentation cellular lysate, inactivation, thereby causes the productive rate of DHA to be difficult to improve.The present invention is the Bacillus licheniformis B-05571 that will separate from acid soil and screen, use gene recombination and DNA to reset technology, dehydrogenase gene is integrated into intestinal bacteria J M 109, make up glycerol dehydrogenase synthetic 1, the industrial engineering bacterium of 3-otan, and adopt the immobilization of supramolecule self-assembly template, fermentation synthesizes 1 in containing the substratum of glycerine then, the 3-otan.Obtain product 1,3-otan by filtering fermentating liquid, organic solvent extraction and combination solvent recrystallization etc.Synthesis route is simple and direct, not only energy-conservation but also environmental protection, and the quality product that is obtained is high and cost is low, and the total mass yield reaches more than 75%.
Summary of the invention
The object of the present invention is to provide a kind of synthetic by glycerol fermentation synthetic 1, the method of 3-otan, be the Bacillus licheniformis B-05571 that will separate and screen from acid soil, use gene recombination and DNA to reset technology, dehydrogenase gene is integrated into intestinal bacteria J M 109, make up glycerol dehydrogenase synthetic 1, the industrial engineering bacterium of 3-otan, and adopt the immobilization of supramolecule self-assembly template, fermentation synthesizes 1 in containing the substratum of glycerine then, the 3-otan.Obtain product 1,3-otan by filtering fermentating liquid, organic solvent extraction and combination solvent recrystallization etc.
The present invention realizes by following method:
The Bacillus licheniformis isolation medium: distilled water 1000mL, glycerine 5g, NaCl 5g, yeast powder 5g, glucose 20g, agar powder 15g, transfer pH7.0,0.1MPa sterilization 20min. is cooled to 35 ℃～45 ℃, adds Neil red (Nile Red) 2mL/L (0.30mg Nile Red is dissolved in the 100mL dimethyl sulfoxide (DMSO)), pour culture dish under the aseptic condition, the cooling back is standby.
The eutrophy substratum: distilled water 1000mL, yeast powder 10g, agar powder 10g, glycerine 3g, (NH
4)
2SO
45g transfers pH7.0, the 0.1MPa 10min. that sterilizes
The product fermention medium:
The preparation of phosphate buffered saline buffer: distilled water 1000mL, NaH
2PO
4.12H
2O 8.95g, KH
2PO
41.5g, the pH7.00.1MPa sterilization, 15min～20min, standby.
The genetic engineering bacterium enrichment medium is the LB substratum: fermention medium is for adding the LB substratum of 15.0% glycerine, to blast air as the genetic engineering bacterium oxygenant; Culture temperature is 35 ℃.
The extraction of Bacillus licheniformis B-05571 fermentation and DHA:
Glycerol fermentation: preceding cultivation is in the L-test tube, adds 5ml eutrophy substratum with aseptic technique, inserts single bacterium colony of Bacillus licheniformis B-05571 with aseptic toothpick.35 ℃, 120r/min are cultivated 15h.Preceding culture 0.5ml is seeded in the 500ml triangular flask that contains 100ml eutrophy substratum 30 ℃, 130r/min shaking culture 24h.4 ℃, the aseptic centrifugal 10min of 6000*g abandons supernatant, and with sterile phosphate damping fluid vibration mixing, then at 4 ℃, the aseptic centrifugal 12min of 6000*g abandons supernatant in centrifuge tube.Preceding culture 0.5ml is seeded in the 500ml triangular flask that contains 100ml eutrophy substratum 35 ℃, 130r/min shaking culture 24h.4 ℃, the aseptic centrifugal 10min of 6000*g abandons supernatant, and with sterile phosphate damping fluid vibration mixing, once more at 4 ℃, the aseptic centrifugal 12min of 6000*g abandons supernatant in centrifuge tube.Supramolecule self-assembly masterplate 3g is added wherein, under 20～25 ℃, carry out immobilization 3h.Contain in the 500ml triangular flask of 100m fermention medium 30 ℃, 130r/min shaking culture 72h 10 bottles of the aseptic accesses of thalline that will not contain the eutrophy substratum respectively.
The separation of tunning: after the fermentation results, centrifugation and recovery katalaze enzyme, fermentation mother liquor uses n-butyl acetate extraction, after organic the concentrating with the extraction gained, uses butylacetate and sherwood oil (1: 4 volume) recrystallization to obtain product.
Embodiment
Below in conjunction with specific embodiment, the invention will be further elaborated, but be not limited to these specific embodiments, and all embodiment are all by above-mentioned operation steps operation.
Embodiment 1
The Bacillus licheniformis isolation medium: distilled water 1000mL, glycerine 5g, NaCl 5g, yeast powder 5g, glucose 20g, agar powder 15g, transfer pH7.0,0.1MPa sterilization 20min. is cooled to 35 ℃～45 ℃, adds Neil red (Nile Red) 2mL/L (0.30mg Nile Red is dissolved in the 100mL dimethyl sulfoxide (DMSO)), pour culture dish under the aseptic condition, the cooling back is standby.
The eutrophy substratum: distilled water 1000mL, yeast powder 10g, agar powder 10g, glycerine 3g, (NH
4)
2SO
45g transfers pH7.0, the 0.1MPa 10min. that sterilizes
The product fermention medium:
The preparation of phosphate buffered saline buffer: distilled water 1000mL, NaH
2PO
4.12H
2O 8.95g, KH
2PO
41.5g, the pH7.00.1MPa sterilization, 15min～20min, standby.
The genetic engineering bacterium enrichment medium is the LB substratum: fermention medium is for adding the LB substratum of 15.0% glycerine, to blast air as the genetic engineering bacterium oxygenant; Culture temperature is 35 ℃.
The extraction of Bacillus licheniformis B-05571 fermentation and DHA:
Glycerol fermentation: preceding cultivation is in the L-test tube, adds 5ml eutrophy substratum with aseptic technique, inserts single bacterium colony of Bacillus licheniformis B-05571 with aseptic toothpick.35 ℃, 120r/min are cultivated 15h.Preceding culture 0.5ml is seeded in the 500ml triangular flask that contains 100ml eutrophy substratum 30 ℃, 130r/min shaking culture 24h.4 ℃, the aseptic centrifugal 10min of 6000*g abandons supernatant, and with sterile phosphate damping fluid vibration mixing, then at 4 ℃, the aseptic centrifugal 12min of 6000*g abandons supernatant in centrifuge tube.Preceding culture 0.5ml is seeded in the 500ml triangular flask that contains 100ml eutrophy substratum 35 ℃, 130r/min shaking culture 24h.4 ℃, the aseptic centrifugal 10min of 6000*g abandons supernatant, and with sterile phosphate damping fluid vibration mixing, once more at 4 ℃, the aseptic centrifugal 12min of 6000*g abandons supernatant in centrifuge tube.Supramolecule self-assembly masterplate 4g is added wherein, under 20～25 ℃, carry out immobilization 4h.10 bottles of the aseptic accesses of thalline that will not contain the eutrophy substratum respectively contain in the 500ml triangular flask of 200m fermention medium 30 ℃, 130r/min shaking culture 72h.
The separation of tunning: after the fermentation results, centrifugation and recovery katalaze enzyme, fermentation mother liquor uses n-butyl acetate extraction, after organic the concentrating with the extraction gained, use butylacetate and sherwood oil (1: 4 volume) recrystallization to obtain 1,3-otan 23.1g (78.9～80.0 ℃ of fusing points, yield 77.0%).
Embodiment 2
The composition and the culture condition of each substratum are the same.
Glycerol fermentation: preceding cultivation is in the L-test tube, adds 5ml eutrophy substratum with aseptic technique, inserts single bacterium colony of Bacillus licheniformis B-05571 with aseptic toothpick.33 ℃, 120r/min are cultivated 20h.Preceding culture 0.5ml is seeded in the 500ml triangular flask that contains 100ml eutrophy substratum 30 ℃, 120r/min shaking culture 30h.4 ℃, the aseptic centrifugal 10min of 6000*g abandons supernatant, and with sterile phosphate damping fluid vibration mixing, then at 4 ℃, the aseptic centrifugal 12min of 6000*g abandons supernatant in centrifuge tube.Preceding culture 0.5ml is seeded in the 500ml triangular flask that contains 100ml eutrophy substratum 35 ℃, 130r/min shaking culture 24h.4 ℃, the aseptic centrifugal 10min of 6000*g abandons supernatant, and with sterile phosphate damping fluid vibration mixing, once more at 4 ℃, the aseptic centrifugal 12min of 6000*g abandons supernatant in centrifuge tube.Supramolecule self-assembly masterplate 4g is added wherein, under 20～25 ℃, carry out immobilization 3h.5 bottles of the aseptic accesses of thalline that will not contain the eutrophy substratum respectively contain in the 1000ml triangular flask of 300m fermention medium 30 ℃, 130r/min shaking culture 96h.
The separation of tunning: after the fermentation results, centrifugation and recovery katalaze enzyme, fermentation mother liquor uses n-butyl acetate extraction, after organic the concentrating with the extraction gained, use butylacetate and sherwood oil (1: 4 volume) recrystallization to obtain 1,3-otan 36.33g (78.7～80.0 ℃ of fusing points, yield 80.7%).
Claims (5)
1. the present invention relates to 1, synthesizing of 3-otan, be the Bacillus licheniformis B-05571 that will separate and screen from acid soil, use gene recombination and DNA to reset technology, dehydrogenase gene is integrated into e. coli jm109, make up glycerol dehydrogenase synthetic 1, the industrial engineering bacterium of 3-otan, and adopt the immobilization of supramolecule self-assembly template, fermentation synthesizes 1 in containing the substratum of glycerine then, the 3-otan.Obtain product 1,3-otan by filtering fermentating liquid, organic solvent extraction and combination solvent recrystallization etc.
2. described according to claim 1, it is characterized in that glycerol dehydrogenase synthetic 1, the structure of the industrial engineering bacterium of 3-otan is the Bacillus licheniformis B-05571 that will separate from acid soil and screen, and uses gene recombination and DNA to reset technology, and dehydrogenase gene is integrated into e. coli jm109.
3. described according to claim 1, it is characterized in that adopting supramolecule self-assembly template to glycerol dehydrogenase synthetic 1, it is 15～35 nanometers that the industrial engineering bacteria immobilization of 3-otan, the supramolecule self-assembly template that is adopted require mean pore size, and in acidic aqueous solution Stability Analysis of Structures.
4. described according to claim 1, it is characterized in that and will synthesize 1 through immobilized glycerol dehydrogenase, the industrial engineering bacterium of 3-otan, fermentation synthetic 1 in containing the substratum of glycerine, 3-otan, the technology condition of fermentation are 30 ℃ of temperature, 130r/min shaking culture 72h.
5. described according to claim 1, it is characterized in that fermented liquid separates earlier katalaze enzyme after filtration and reuses, utilize n-butyl acetate extraction filter secondary fermentation liquid, after again extraction liquid being concentrated, utilize 1: 4 by volume combination solvent recrystallization of butylacetate and sherwood oil, obtain product 1, the 3-otan.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101376102A CN102250970A (en) | 2011-05-26 | 2011-05-26 | Method for synthesizing 1,3-dioxyacetone by glycerol fermentation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101376102A CN102250970A (en) | 2011-05-26 | 2011-05-26 | Method for synthesizing 1,3-dioxyacetone by glycerol fermentation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102250970A true CN102250970A (en) | 2011-11-23 |
Family
ID=44978538
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011101376102A Pending CN102250970A (en) | 2011-05-26 | 2011-05-26 | Method for synthesizing 1,3-dioxyacetone by glycerol fermentation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102250970A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102492732A (en) * | 2011-11-24 | 2012-06-13 | 厦门大学 | Preparation method of dihydroxyacetone by transforming glycerol |
| CN104845961A (en) * | 2015-04-16 | 2015-08-19 | 徐州奥格曼新材料科技有限公司 | Immobilization and application of 1,3-dihydroxy acetone producing recombinant genetic engineering bacteria |
| CN105695517A (en) * | 2016-04-06 | 2016-06-22 | 常州市阿曼特化工有限公司 | Synthesis method of dihydroxyacetone |
| CN108486092A (en) * | 2018-04-25 | 2018-09-04 | 安徽瑞赛生化科技有限公司 | Produce the immobilization and its application of Pfansteihl recombination engineering bacteria |
| CN109234217A (en) * | 2018-06-29 | 2019-01-18 | 安徽瑞赛生化科技有限公司 | Produce the immobilization and its application of 2,5- furandicarboxylic acid recombination engineering bacteria |
-
2011
- 2011-05-26 CN CN2011101376102A patent/CN102250970A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102492732A (en) * | 2011-11-24 | 2012-06-13 | 厦门大学 | Preparation method of dihydroxyacetone by transforming glycerol |
| CN102492732B (en) * | 2011-11-24 | 2014-09-03 | 厦门大学 | Preparation method of dihydroxyacetone by transforming glycerol |
| CN104845961A (en) * | 2015-04-16 | 2015-08-19 | 徐州奥格曼新材料科技有限公司 | Immobilization and application of 1,3-dihydroxy acetone producing recombinant genetic engineering bacteria |
| CN105695517A (en) * | 2016-04-06 | 2016-06-22 | 常州市阿曼特化工有限公司 | Synthesis method of dihydroxyacetone |
| CN108486092A (en) * | 2018-04-25 | 2018-09-04 | 安徽瑞赛生化科技有限公司 | Produce the immobilization and its application of Pfansteihl recombination engineering bacteria |
| CN109234217A (en) * | 2018-06-29 | 2019-01-18 | 安徽瑞赛生化科技有限公司 | Produce the immobilization and its application of 2,5- furandicarboxylic acid recombination engineering bacteria |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105838622B (en) | Aspergillus niger HC306 and conversion aurantiin prepare the application in naringenin | |
| CN104862348A (en) | Method for fermentation-membrane separation combined production of long-chain dicarboxylic acid | |
| CN101148646B (en) | Method for producing high-activity chitosanase preparation | |
| CN102250970A (en) | Method for synthesizing 1,3-dioxyacetone by glycerol fermentation | |
| CN103290071B (en) | Method for preparing 2-keto-L-gulonic acid | |
| CN101008019A (en) | Use of Bacillus subtilis (Ehrenberg)Cohn in preparing 3-hydroxy butanone | |
| CN100552035C (en) | A kind of trispore Bruce mould fermentative preparation lycopene method of utilizing | |
| CN103224960B (en) | Method for producing erythritol by continuously fermenting and extracting Torula.sp strain | |
| CN103773827A (en) | Method for improving output of alpha-arbutin | |
| CN105543108A (en) | Penicillium purpurescens QL-9204 and application for preparing phloretin during phlorizin conversion | |
| CN104845999A (en) | Construction and application of 1,3-dihydroxy acetone recombinant genetic engineering bacteria | |
| CN101565679B (en) | Rhodotorula cell and method for producing optical active alcohols by asymmetric conversion | |
| CN103710291B (en) | The method of one strain bacillus megaterium Z2013513 and production phenyl-lactic acid thereof | |
| CN104845961A (en) | Immobilization and application of 1,3-dihydroxy acetone producing recombinant genetic engineering bacteria | |
| CN105420303A (en) | Fermentation process for producing trans-4-hydroxyl-L-proline | |
| CN103571856A (en) | Preparation method of engineering bacteria for producing1,3-dihydroxy acetone | |
| CN110438015B (en) | Immature bitter orange endophytic fungus for producing hesperidinase and method for producing hesperidinase by fermenting immature bitter orange endophytic fungus | |
| CN104762360A (en) | High-content nicotinamide synthesis induced by new-feature nitrile hydratase | |
| CN109136313A (en) | Utilize the method for Michigan's Klebsiella synthesis 2'-deoxyadenosine | |
| CN105112392A (en) | Preparation method for cephalosporin C acylase | |
| CN108486173B (en) | Preparation method of alpha-ketoglutaric acid | |
| CN102382868B (en) | Method for producing dihydroxyacetone by using gluconobacter sp. | |
| CN101812437A (en) | Method for producing chymosin by adopting mildew solid-state fermentation | |
| CN104651243B (en) | Pichia membranaefaciens and its application in chiral synthesis (R) 1,3 butanediol | |
| CN104293851A (en) | Method for producing hydroxyethyl pyridine by alternaria alternata |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| DD01 | Delivery of document by public notice |
Addressee: Xuzhou Hengyuan Bioengineering Co.,Ltd. Document name: Notification of Passing Preliminary Examination of the Application for Invention |
|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| DD01 | Delivery of document by public notice |
Addressee: Xuzhou Hengyuan Bioengineering Co.,Ltd. Document name: the First Notification of an Office Action |
|
| DD01 | Delivery of document by public notice |
Addressee: Xuzhou Hengyuan Bioengineering Co.,Ltd. Document name: Notification that Application Deemed to be Withdrawn |
|
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20111123 |