CN104845961A - Immobilization and application of 1,3-dihydroxy acetone producing recombinant genetic engineering bacteria - Google Patents
Immobilization and application of 1,3-dihydroxy acetone producing recombinant genetic engineering bacteria Download PDFInfo
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Abstract
The invention discloses immobilization and an application of 1,3-dihydroxy acetone producing recombinant genetic engineering bacteria; a supramolecular template is used for immobilization of the engineering bacteria of an escherichia coli expressed glycerol dehydrogenase gene (gldA). Construction of the engineering bacteria comprises that with a klebsiella pneumoniae genome DNA as a template, PCR amplification is applied to obtain the gene (gldA) encoding glycerol dehydrogenase (GDH), the gene (gldA) is cloned onto an escherichia coli expression vector PET 28a, and a cloned vector PET-gldA is constructed and is expressed successfully in E.coli JM109. A method for expressing 1,3-dihydroxy acetone comprises that the recombinant genetic engineering bacteria immobilized by the supramolecular template are fermented in a glycerol-containing culture medium to obtain the 1,3-dihydroxy acetone. With the immobilized stain, the product expression quantity is high and is up to a maximum of 120 g/L, the repeated use is good, and the product expression quantity still can reach 107 g/L after repeated use for 15 times, an active role is provided in microbial fermentation preparation of the 1,3-dihydroxy acetone, and application prospects are wide.
Description
Technical field: the invention belongs to microbial fermentation technology, relates to the immobilization and application of producing 1,3-Dihydroxyacetone recombination engineering bacteria.
Background technology
A kind of important chemical industry of 1,3-Dihydroxyacetone, bio feedstocks, medicine, pesticide synthesis 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 cannot overcome the fatal defects of catalytic selectivity difference, causes glycerol conversion yield lower than the productive rate of 40%, DHA lower than 25%.Industrial process is utilize microorganism batch fermentation at present.This method is at thalli growth to suitable period, and the desaturase utilizing thalline to produce is that substrate carries out dehydrogenation reaction with glycerine, produces DHA.If for the production of microbial host acetobacter (Acetobacter) and Gluconobacter (Gluconobacter) microorganism of DHA, especially weak oxidized acetic acid bacteria (Acetobacter Suboxy-dans) and bacillus of oxidizing glucose (Gluoonobacter Oxydans).Bacterial classification first carries out preculture after recovery, then turns fermentor tank enlarged culturing, reaches after finite concentration until bacterial classification, is inoculated in the middle of the fermention medium containing glycerine, after the oxygen consumption fermentation of 60-80h, is more once released by fermented liquid, obtains DHA through separation and purification.Each batch fermentation process experiences inoculation, growth and breeding, thalline is old and feeble and then terminate fermentation, finally extracts product.Existing employing shunts glycerol adding method, when thalli growth is to logarithmic phase, and after product D HA reaches certain level at present, release portion of product, and add raw material and substratum, so just can reduce the time of the biomass reaching production level, improve the utilization ratio of substrate glycerol, cost-saving.The feature of batch fermentation is the environment residing for microorganism is constantly change, living contaminants occurs, can be easy to terminating operation.In fermentative production, the bacterial classification of use is in logarithmic phase, when they are inoculated into fermentor tank fresh culture, the adjustment period of appearance hardly, can obtain the vigorous thalline of raised growth at short notice like this, is conducive to shortening the production cycle.But the subject matter of this method creates high osmotic pressure when being substrate glycerol and product D HA excessive concentration in the medium, makes fermentation thalli cracking, inactivation, thus causes the productive rate of DHA to be difficult to improve.
Along with the development of immobilized cell technology, increasing investigator pays close attention to immobilization Bacillus coli cells.Immobilization Bacillus coli cells has following advantage: compared to free cell, and immobilized cell is in catalytic reaction process, and its plasmid is more stable, and goal gene product vigor is higher, and product is easy to separation and purification.Since Bacillus coli cells is adsorbed on resin achieves Bacillus coli cells immobilization by nineteen fifty-nine Hattori first, fixing intestinal bacteria viable cell or be called that the research work of fixed hyperplasia Bacillus coli cells launches in succession.
Supramolecule typically refers to and relies on molecular interaction to combine by two or more molecule, and composition is complicated, organized aggregate, and keeps certain integrity to make it have clear and definite microtexture and macroscopic property.People can, according to Supramolecular self assembly principle, use intermolecular interaction force as instrument, and the component or building block with specific structure and function is assembled into new super molecular compound according to certain mode.These new compounds not only can show the ins and outs not available for individual molecule, greatly can also increase kind and the number of compound.If people can be good at controlling Supramolecular self assembly process, just the compound with ad hoc structure and function can be obtained more simply, more reliably according to re-set target.MCM-41, Dealuminated USY, mesoporous carbon etc. are all the supramolecules that can be used as host-guest chemistry with regular pore canal properity excellence.
The present invention fixes recombination engineering bacteria with supermolecular module, recombination engineering bacteria is klebsiella spp (Klebsiella pneumoniae) genomic dna is template, pcr amplification is used to obtain the gene (gldA) of encoding glycerol desaturase (GDH), and be cloned on coli expression carrier PET 28a, build cloning vector PET-gldA, and in E.Coli JM109 successful expression.The glycerol dehydrogenase synthesis 1,3-Dihydroxyacetone immobilization engineering bacteria built, 1,3-Dihydroxyacetone is synthesized in fermentation in containing the substratum of glycerine.Product 1,3-Dihydroxyacetone is obtained by filtering fermentating liquid, organic solvent extraction and combination solvent recrystallization etc.Product Expression amount preferably can reach 120g/L, and after reusing 20 times, Product Expression amount still can reach 107g/L.
Summary of the invention
The object of the invention is to provide the process for fixation producing 1,3-Dihydroxyacetone recombination engineering bacteria.
Another object of the present invention is the method and the application that provide the immobilization recombination bacillus coli producing 1,3-Dihydroxyacetone.
The present invention is realized by following method:
The engineering bacteria of escherichia coli expression glycerol dehydrogenase gene (gldA) is fixed with supermolecular module.The structure of engineering bacteria is for template with klebsiella spp (Klebsiella pneumoniae) genomic dna, pcr amplification is used to obtain the gene (gldA) of encoding glycerol desaturase (GDH), and be cloned on coli expression carrier PET 28a, build cloning vector PET-gldA, and in E.Coli JM109 successful expression.Be with the fixing recombination engineering bacteria of supermolecular module in glycerinated substratum fermentation for 1,3-Dihydroxyacetone.
Plasmid and bacterial strain:
Cloning vector PET28a, intestinal bacteria E.Coli JM109 available from commercial company.
Restriction enzyme, Taq enzyme, T4 DNA ligase is all purchased from the precious biotech firm in Dalian.MCM-41, Dealuminated USY, mesoporous carbon is all from Southern Yangtze University.Other chemical reagent are domestic analytical pure chemical reagent.
Klebsiella spp isolation medium: distilled water 1000mL, glycerine 5g, NaCl 5g, yeast powder 5 g, glucose 20g, agar powder 15g, adjust pH7.0,0.1MPa sterilizing 20min. to be cooled to 35 DEG C ~ 45 DEG C, add Neil red (Nile Red) 2mL/L (0.30mg Nile Red is dissolved in 100mL dimethyl sulfoxide (DMSO)), culture dish is poured under aseptic condition, for subsequent use after cooling.
Enrich media: distilled water 1000mL, yeast powder 10 g, agar powder 10g, glycerine 3g, (NH
4)
2sO
45g, adjusts pH7.0,0.1MPa sterilizing 10min.
product fermention medium:
The preparation of phosphate buffered saline buffer: distilled water 1000mL, NaH
2pO
4.12H
2o 8.95g, KH
2pO
41.5g, pH7.0,0.1MPa sterilizing, 15min ~ 20min, for subsequent use.
Genetic engineering bacterium enrichment medium is LB substratum: fermention medium is the LB substratum of interpolation 15.0% glycerine, to blast air as genetic engineering bacterium oxygenant; Culture temperature is 35 DEG C.
The acquisition of klebsiella spp dehydrogenase gene and the structure of recombinant plasmid:
By carrying out sequential analysis to 1,3-Dihydroxyacetone synthetic enzyme gldA, devise conservative primer: primer-I:5`-GGTGGGATCCTACATGCGCACTTATTTGAG-3`primer-II:5 `-AATGCTCGAGCGAATTAACGCGCCAGCCAC-3`.Be that masterplate amplification obtains a large fragment with klebsiella spp (Klebsiella pneumoniae) genomic dna, therefrom analyze the open reading frame obtaining DHA synthase gene 1 133bp size, according to the amplimer of the sequences Design DHA synthase gene gldA obtained, introduce XbaI and EcoRI position respectively in upstream and downstream.The two ferment treatment of XbaI and EcoRI is carried out to PET28a and pcr amplification product, after recovery under the effect of T4DNA ligase enzyme 16 DEG C spend the night continuously, in order to transforming.
Pcr amplification: 97 DEG C of denaturation 10min, 94 DEG C of distortion 60s, 58 DEG C of annealing 30s, 72 DEG C extend 60-120s, 30 rear 72 DEG C of extension 10min of circulation.
GldA gene and vector plasmid PET28a are carried out XbaI and EcoRI double digestion simultaneously, and enzyme utilizes T4 ligase enzyme to be connected in plasmid PET28a by gldA gene after cutting, obtain recombinant plasmid PET-gldA.
The preparation of competent escherichia coli cell and conversion:
According to " Molecular Cloning: A Laboratory guide ", calcium legal system is for competent escherichia coli cell and transform, be connected to 1mlLB after conversion and cultivate concentrated 36 DEG C, 150r/min shaking culture 2h, be spread evenly across on the LB flat board containing penbritin and screen recombination bacillus coli.Screen the bacterial strain obtained and utilize bacterium colony PCR and digestion with restriction enzyme recombinant plasmid checking recon.
Supermolecular module is fixed:
The phosphoric acid buffer of appropriate supramolecule and 5mLPH7.0 is added in the Erlenmeyer flask of 25mL, and then adds the activator of 2mL and the colibacillus engineering solution of 2mL, drip the glutaraldehyde of 7.5% in the most backward solution until liquid quality fraction is 0.75%.Immobilized reactant, at 180r/min, carries out 2h under the condition of 15 degree, more after filtration, 50mL PH be 7.0 phosphoric acid liquid washing after being fixed colibacillus engineering.
The extraction of klebsiella spp fermentation and DHA:
Front cultivation is in L-test tube, adds 5ml enrich media with aseptic technique, with single bacterium colony of sterile toothpick access klebsiella spp (Klebsiella pneumoniae).35 DEG C, 120r/min cultivates 15h.Front culture 0.5ml is seeded in the 500ml triangular flask containing 100ml enrich media, 30 DEG C, 130r/min shaking culture 24h.4 DEG C, 6000*g sterile centrifugation 10min, abandons supernatant, and with sterile phosphate buffer vibration mixing in centrifuge tube, then at 4 DEG C, 6000*g sterile centrifugation 12min, abandons supernatant.Front culture 0.5ml is seeded in the 500ml triangular flask containing 100ml enrich media, 35 DEG C, 130r/min shaking culture 24h.4 DEG C, 6000*g sterile centrifugation 10min, abandons supernatant, and with sterile phosphate buffer vibration mixing in centrifuge tube, again at 4 DEG C, 6000*g sterile centrifugation 12min, abandons supernatant.Respectively the aseptic access of thalline 10 bottles not containing enrich media is being contained in the 500ml triangular flask of 100m fermention medium, 30 DEG C ~ 37 DEG C, 130r/min shaking culture 72h.
Immobilization recombination bacillus coli fermentative production 1,3-Dihydroxyacetone:
The immobilization recombination bacillus coli of-70 DEG C of preservations activates on LB culture medium flat plate, be connected in 20mlLB substratum with sterilizing toothpick picking list bacterium colony, 30 DEG C ~ 37 DEG C overnight incubation, inoculum size with 2% is inoculated in fermention medium, the IPTG adding 1 ~ 2mmol/L induces the expression of gldA, fermentation culture 72h.
The separation of tunning: after fermentation harvest, centrifugation and reclaim katalaze enzyme, fermentation mother liquor uses n-butyl acetate extraction, by extraction gained organic concentrated after, use butylacetate and sherwood oil (1:3 ~ 4 volume) recrystallization to obtain product.
Immobilization bacterial strain of the present invention, Product Expression amount is high, reaches as high as 120g/L, and reusing is good, and after reusing 15 times, Product Expression amount still can reach 100g/L, has positive effect, have a extensive future to the biological fermentation preparation of 1,3-Dihydroxyacetone.
Embodiment
Below in conjunction with specific embodiment, the invention will be further elaborated, but be not limited to these specific embodiments, and all embodiments all operate by above-mentioned operation steps.
Embodiment 1,
Plasmid and bacterial strain:
Cloning vector PET28a, intestinal bacteria E.Coli JM109 available from commercial company.
Restriction enzyme, Taq enzyme, T4 DNA ligase is all purchased from the precious biotech firm in Dalian.MCM-41 is from Southern Yangtze University.Other chemical reagent are domestic analytical pure chemical reagent.
Klebsiella spp isolation medium: distilled water 1000mL, glycerine 5g, NaCl 5g, yeast powder 5 g, glucose 20g, agar powder 15g, adjust pH7.0,0.1MPa sterilizing 20min. to be cooled to 35 DEG C, add Neil red (Nile Red) 2mL/L (0.30mg Nile Red is dissolved in 100mL dimethyl sulfoxide (DMSO)), culture dish is poured under aseptic condition, for subsequent use after cooling.
Enrich media: distilled water 1000mL, yeast powder 10 g, agar powder 10g, glycerine 3g, (NH
4)
2sO
45g, adjusts pH7.0,0.1MPa sterilizing 10min.
product fermention medium:
The preparation of phosphate buffered saline buffer: distilled water 1000mL, NaH
2pO
4.12H
2o 8.95g, KH
2pO
41.5g, pH7.0,0.1MPa sterilizing, 15min ~ 20min, for subsequent use.
Genetic engineering bacterium enrichment medium is LB substratum: fermention medium is the LB substratum of interpolation 15.0% glycerine, to blast air as genetic engineering bacterium oxygenant; Culture temperature is 35 DEG C.
The acquisition of klebsiella spp dehydrogenase gene and the structure of recombinant plasmid:
By carrying out sequential analysis to 1,3-Dihydroxyacetone synthetic enzyme gldA, devise conservative primer: primer-I:5`-GGTGGGATCCTACATGCGCACTTATTTGAG-3`primer-II:5 `-AATGCTCGAGCGAATTAACGCGCCAGCCAC-3`.Be that masterplate amplification obtains a large fragment with klebsiella spp (Klebsiella pneumoniae) genomic dna, therefrom analyze the open reading frame obtaining DHA synthase gene 1 133bp size, according to the amplimer of the sequences Design DHA synthase gene gldA obtained, introduce XbaI and EcoRI position respectively in upstream and downstream.The two ferment treatment of XbaI and EcoRI is carried out to PET28a and pcr amplification product, after recovery under the effect of T4DNA ligase enzyme 16 DEG C spend the night continuously, in order to transforming.
Pcr amplification: 97 DEG C of denaturation 10min, 94 DEG C of distortion 60s, 58 DEG C of annealing 30s, 72 DEG C extend 60-120s, 30 rear 72 DEG C of extension 10min of circulation.
GldA gene and vector plasmid PET28a are carried out XbaI and EcoRI double digestion simultaneously, and enzyme utilizes T4 ligase enzyme to be connected in plasmid PET28a by gldA gene after cutting, obtain recombinant plasmid PET-gldA.
The preparation of competent escherichia coli cell and conversion:
According to " Molecular Cloning: A Laboratory guide ", calcium legal system is for competent escherichia coli cell and transform, be connected to 1mlLB after conversion and cultivate concentrated 36 DEG C, 150r/min shaking culture 2h, be spread evenly across on the LB flat board containing penbritin and screen recombination bacillus coli.Screen the bacterial strain obtained and utilize bacterium colony PCR and digestion with restriction enzyme recombinant plasmid checking recon.
Supermolecular module is fixed:
The phosphoric acid buffer of 3.5gMCM41 and 5mLPH7.0 is added in the Erlenmeyer flask of 25mL, and then adds the activator of 2mL and the colibacillus engineering solution of 2mL, drip the glutaraldehyde of 7.5% in the most backward solution until liquid quality fraction is 0.75%.Immobilized reactant, at 180r/min, carries out 2h under the condition of 15 degree, more after filtration, 50mL PH be 7.0 phosphoric acid liquid washing after being fixed colibacillus engineering.
The extraction of klebsiella spp fermentation and DHA:
Front cultivation is in L-test tube, adds 5ml enrich media with aseptic technique, with single bacterium colony of sterile toothpick access klebsiella spp (Klebsiella pneumoniae).35 DEG C, 120r/min cultivates 15h.Front culture 0.5ml is seeded in the 500ml triangular flask containing 100ml enrich media, 30 DEG C, 130r/min shaking culture 24h.4 DEG C, 6000*g sterile centrifugation 10min, abandons supernatant, and with sterile phosphate buffer vibration mixing in centrifuge tube, then at 4 DEG C, 6000*g sterile centrifugation 12min, abandons supernatant.Front culture 0.5ml is seeded in the 500ml triangular flask containing 100ml enrich media, 35 DEG C, 130r/min shaking culture 24h.4 DEG C, 6000*g sterile centrifugation 10min, abandons supernatant, and with sterile phosphate buffer vibration mixing in centrifuge tube, again at 4 DEG C, 6000*g sterile centrifugation 12min, abandons supernatant.Respectively the aseptic access of thalline 10 bottles not containing enrich media is being contained in the 500ml triangular flask of 100m fermention medium, 30 DEG C, 130r/min shaking culture 72h.
Immobilization recombination bacillus coli fermentative production 1,3-Dihydroxyacetone:
The immobilization recombination bacillus coli of-70 DEG C of preservations activates on LB culture medium flat plate, be connected in 20mlLB substratum with sterilizing toothpick picking list bacterium colony, 30 DEG C of overnight incubation, the inoculum size with 2% is inoculated in fermention medium, the IPTG adding 1mmol/L induces the expression of gldA, fermentation culture 72h.
The separation of tunning: after fermentation harvest, centrifugation and reclaim katalaze enzyme, fermentation mother liquor uses n-butyl acetate extraction, by extraction gained organic concentrated after, use butylacetate and sherwood oil (1:4 volume) recrystallization to obtain product.Product Expression amount reaches 120g/L.Katalaze enzyme recycles and reuses 20 times, and Product Expression amount reaches 107g/L.
Embodiment 2,
Composition and the culture condition of each substratum are the same.Recombination bacillus coli is prepared the same.
Supermolecular module is fixed:
The phosphoric acid buffer of 2.7g Dealuminated USY and 5mLPH7.0 is added in the Erlenmeyer flask of 25mL, and then add the activator of 2mL and the colibacillus engineering solution of 2mL, drip the glutaraldehyde of 7.5% in the most backward solution until liquid quality fraction is 0.70%.Immobilized reactant, at 170r/min, carries out 2h under the condition of 15 degree, more after filtration, 50mL PH be 7.0 phosphoric acid liquid washing after being fixed colibacillus engineering.
The extraction of klebsiella spp fermentation and DHA:
Front cultivation is in L-test tube, adds 5ml enrich media with aseptic technique, with single bacterium colony of sterile toothpick access klebsiella spp (Klebsiella pneumoniae).35 DEG C, 120r/min cultivates 15h.Front culture 0.5ml is seeded in the 500ml triangular flask containing 100ml enrich media, 30 DEG C, 130r/min shaking culture 24h.4 DEG C, 6000*g sterile centrifugation 10min, abandons supernatant, and with sterile phosphate buffer vibration mixing in centrifuge tube, then at 4 DEG C, 6000*g sterile centrifugation 12min, abandons supernatant.Front culture 0.5ml is seeded in the 500ml triangular flask containing 100ml enrich media, 35 DEG C, 130r/min shaking culture 24h.4 DEG C, 6000*g sterile centrifugation 10min, abandons supernatant, and with sterile phosphate buffer vibration mixing in centrifuge tube, again at 4 DEG C, 6000*g sterile centrifugation 12min, abandons supernatant.Respectively the aseptic access of thalline 10 bottles not containing enrich media is being contained in the 500ml triangular flask of 100m fermention medium, 35 DEG C, 130r/min shaking culture 72h.
Immobilization recombination bacillus coli fermentative production 1,3-Dihydroxyacetone:
The immobilization recombination bacillus coli of-70 DEG C of preservations activates on LB culture medium flat plate, be connected in 20mlLB substratum with sterilizing toothpick picking list bacterium colony, 36 DEG C of overnight incubation, inoculum size with 2% is inoculated in fermention medium, the IPTG adding 1.5mmol/L induces the expression of gldA, fermentation culture 72h.
The separation of tunning: after fermentation harvest, centrifugation and reclaim katalaze enzyme, fermentation mother liquor uses n-butyl acetate extraction, by extraction gained organic concentrated after, use butylacetate and sherwood oil (1:3 volume) recrystallization to obtain product.Product Expression amount reaches 108g/L.Katalaze enzyme recycles and reuses 20 times, and Product Expression amount reaches 87g/L.
Embodiment 3,
Composition and the culture condition of each substratum are the same.Recombination bacillus coli is prepared the same.
Supermolecular module is fixed
The phosphoric acid buffer of 3.3g mesoporous carbon and 5mLPH7.0 is added in the Erlenmeyer flask of 25mL, and then adds the activator of 2mL and the colibacillus engineering solution of 2mL, drip the glutaraldehyde of 7.5% in the most backward solution until liquid quality fraction is 0.85%.Immobilized reactant, at 170r/min, carries out 2h under the condition of 15 degree, more after filtration, 50mL PH be 7.0 phosphoric acid liquid washing after being fixed colibacillus engineering.
The extraction of klebsiella spp fermentation and DHA:
Front cultivation is in L-test tube, adds 5ml enrich media with aseptic technique, with single bacterium colony of sterile toothpick access klebsiella spp (Klebsiella pneumoniae).35 DEG C, 120r/min cultivates 15h.Front culture 0.5ml is seeded in the 500ml triangular flask containing 100ml enrich media, 30 DEG C, 130r/min shaking culture 24h.4 DEG C, 6000*g sterile centrifugation 10min, abandons supernatant, and with sterile phosphate buffer vibration mixing in centrifuge tube, then at 4 DEG C, 6000*g sterile centrifugation 12min, abandons supernatant.Front culture 0.5ml is seeded in the 500ml triangular flask containing 100ml enrich media, 35 DEG C, 130r/min shaking culture 24h.4 DEG C, 6000*g sterile centrifugation 10min, abandons supernatant, and with sterile phosphate buffer vibration mixing in centrifuge tube, again at 4 DEG C, 6000*g sterile centrifugation 12min, abandons supernatant.Respectively the aseptic access of thalline 10 bottles not containing enrich media is being contained in the 500ml triangular flask of 100m fermention medium, 35 DEG C, 130r/min shaking culture 72h.
Immobilization recombination Escherichia coli fermentation produces 1,3-Dihydroxyacetone:
The immobilization recombination bacillus coli of-70 DEG C of preservations activates on LB culture medium flat plate, be connected in 20mlLB substratum with sterilizing toothpick picking list bacterium colony, 36 DEG C of overnight incubation, the inoculum size with 2% is inoculated in fermention medium, the IPTG adding 1mmol/L induces the expression of gldA, fermentation culture 72h.
The separation of tunning: after fermentation harvest, centrifugation and reclaim katalaze enzyme, fermentation mother liquor uses n-butyl acetate extraction, by extraction gained organic concentrated after, use butylacetate and sherwood oil (1:4 volume) recrystallization to obtain product.Product Expression amount reaches 95g/L.Katalaze enzyme recycles and reuses 15 times, and Product Expression amount reaches 65g/L.
Claims (8)
1. the present invention relates to the immobilization and application of producing 1,3-Dihydroxyacetone recombination engineering bacteria, it is characterized in that supermolecular module fixes recombination engineering strain.
2. method according to claim 1, is characterized in that: the Supramolecular self assembly template mean pore size adopted is 15 ~ 35 nanometers, and in acidic aqueous solution Stability Analysis of Structures.
3. method according to claim 1, is characterized in that: supermolecular module is MCM-41, Dealuminated USY, mesoporous carbon.
4. method according to claim 1, is characterized in that: being prepared as follows of recombination engineering bacteria:
(1) with klebsiella spp (Klebsiella pneumoniae) genomic dna for template, use pcr amplification to obtain the gene (gldA) of encoding glycerol desaturase (GDH);
(2) be cloned on coli expression carrier PET 28a, build cloning vector PET-gldA;
(3) PET-gldA expresses in E.Coli JM109.
5. method according to claim 4, is characterized in that pcr amplification condition is: 97 DEG C of denaturation 10min, 94 DEG C of distortion 60s, 58 DEG C of annealing 30s, and 72 DEG C extend 60-120s, 30 rear 72 DEG C of extension 10min of circulation.
6. utilize the immobilization recombination engineering bacteria described in claim 1 to prepare the method for 1,3-Dihydroxyacetone, comprising:
(1) described immobilization recombination engineering bacteria is fermented;
(2) separate fermentation liquid, extracts object product 1,3-Dihydroxyacetone.
7. method according to claim 6, wherein restructuring product 1, the fermentation stage fermentation condition of 3-otan is fermentation synthesis 1,3-Dihydroxyacetone in containing the substratum of glycerine, and the technology condition of fermentation is temperature 30 DEG C ~ 37 DEG C, 130r/min shaking culture 72h.
8. basis in accordance with the method for claim 6, it is characterized in that fermented liquid is first separated by filtration katalaze enzyme and reuses, n-butyl acetate extraction is utilized to filter secondary fermentation liquid, after again extraction liquid being concentrated, utilize the combination solvent recrystallization of butylacetate and sherwood oil 1:3 ~ 4 by volume, obtain product 1,3-Dihydroxyacetone.
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| CN106497957A (en) * | 2016-11-14 | 2017-03-15 | 安徽瑞赛生化科技有限公司 | The method that recombination engineering bacterium fermentation synthesizes 2,5 furandicarboxylic acids |
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| 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 |
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