CN108486092A - Produce the immobilization and its application of Pfansteihl recombination engineering bacteria - Google Patents
Produce the immobilization and its application of Pfansteihl recombination engineering bacteria Download PDFInfo
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Abstract
The present invention relates to the immobilizations and its application of production L lactic acid recombination engineering bacterias to be integrated into Torulopsis glabrata gene by lactate dehydrogenase gene, forms recombinant dna gene；By PCR amplification, and coli expression carrier is inserted into after modification, the engineering bacteria of structure synthesis L lactic acid；By sodium metasilicate, cetyltrimethylammonium bromide, ammonium chloride and alchlor under the conditions of water phase and Supramolecular self assembly, the Supramolecular self assembly template for the extra specific surface area that aperture is 25~45nm is prepared；By above-mentioned Supramolecular self assembly template, above-mentioned recombination engineering bacteria is fixed using physisorphtion, and fermentation prepares L lactic acid in containing glucose limited culture medium, product expression amount is up to 23.5g/L, inversion rate of glucose 68.51%, recycling 30 times, product expression amount and inversion rate of glucose are 22.6g/L and 65.87%.
Description
Technical field：The invention belongs to microbial fermentation technology, it is related to producing the immobilization of Pfansteihl recombination engineering bacteria
And its application.
Background technology
Pfansteihl is important organic synthesis intermediate, can be used for underlying biological based raw material, medical material, pesticide, makeup
The industries such as polyester and degradation plastic that the industries such as product, multifunction additive, especially developed recently are got up expand Pfansteihl
Purposes.
The preparation method of Pfansteihl mainly has（1）Fermentation method：It is with the raw material sucrose containing starch, beet sugar or its molasses
Raw material.Saccharification access lactic bacteria strain.PH controls make the lactic acid of generation with calcium carbonate in 5-5.5, temperature 50 C or so fermentation 3-4d
It is converted into calcium lactate.And neutralized, filtering, concentration, decoloration and recrystallization etc. obtain Pfansteihl, consumption of raw materials quota:Rice
2080kg/t, sulfuric acid (98%) 530kg/t.（2）Acetaldehyde hydrogen cyanide method：It is reacted as raw material with hydrogen cyanide using acetaldehyde and generates lactonitrile, then
Crude lactic acid is obtained through hydrolysis.Crude lactic acid generates lactate with ethyl alcohol esterification, then through resolving into lactic acid.Again through hydrolysis, esterification, rectifying
Etc. processes obtain product, consumption of raw materials quota:Acetaldehyde 480kg/t, hydrogen cyanide 290kg/t, sulfuric acid 1040kg/t.（3）Acrylonitrile
Method：Crude lactic acid is generated by raw material and sulfuric acid reaction of acrylonitrile, then is reacted with methanol and generates methyl lactate, through distilling to obtain thick ester,
Smart ester heat resolve is obtained into lactic acid.Acidified again, esterification, rectifying and recrystallization etc. obtain product.Consumption of raw materials quota:Acrylonitrile
780kg/t, sulfuric acid (98%) 1030kg/t.The present invention is that lactobacillus lactate dehydrogenase gene Idhl is integrated into smooth ball to intend
II a of saccharomycete gene M T form II a of recombinant dna gene Idh.The recombination is cloned by PCR, and is inserted into greatly after modification
II a of enterobacteria expression vector pTE28aT7 structure Pfansteihl engineering bacterias pTE28aT7-Idh；In isopropylthiogalactoside
（IPTG）Under induction, in the limitation culture medium containing glucose, fermented synthesis Pfansteihl；By filtering fermentating liquid, organic
Solvent extraction crystallization and combination solvent recrystallization obtain product Pfansteihl.Synthesis route is simple and direct, energy-saving and environment-friendly, is obtained
Product quality height and at low cost, gross mass yield reaches 68.49% or more.
With the development of enzyme immobilization technology, more and more researchers focus on immobilization Escherichia coli enzyme.Immobilization large intestine
Bacillus enzyme has following advantage：Compared to resolvase, immobilised enzymes is in catalytic reaction process, and plasmid is more stablized, purpose
Gene outcome vigor is higher, and product is easily isolated purifying.Escherichia coli enzyme is adsorbed on resin for the first time from nineteen fifty-nine Hattori
Since realizing Escherichia coli enzyme immobilization, fixed Escherichia coli work enzyme or the research work for fixed hyperplasia Escherichia coli enzyme
Make expansion in succession.
Supermolecule typically refers to be combined together by intermolecular interaction by two or more molecule, and composition is multiple
Miscellaneous, organized aggregation, and certain integrality is kept to make it have specific microstructure and macroscopic properties.People can
With according to Supramolecular self assembly principle, using intermolecular interaction force as tool, having specific structure and function
Component or building block, new super molecular compound is assembled into according to certain mode.These new compounds are not only only capable of
Show the special property not available for individual molecule, moreover it is possible to greatly increase the type and number of compound.If people can
Control Supramolecular self assembly process well, so that it may with it is anticipated that target is simpler, more reliable obtains with specific structure
With the compound of function.The present invention, by sodium metasilicate, cetyltrimethylammonium bromide, ammonium chloride and alchlor in water phase and
Under the conditions of Supramolecular self assembly, the aperture of preparation is the extra specific surface area of 25~45nm（Not less than 1000m2/g）Supermolecule
Self assembly template.By above-mentioned Supramolecular self assembly template, it is used for fixing above-mentioned recombination engineering bacteria using physisorphtion, and
For in containing glucose limited culture medium fermentation prepare Pfansteihl.The immobilization bacterial strain of the present invention, product expression amount highest
Up to 23.5g/L, inversion rate of glucose is higher than 68.51%；Reusing is good, and after reusing 30 times, product expression amount still may be used
Up to 22.6g/L, inversion rate of glucose is higher than 65.87%, and will be prepared to the microbial fermentation of Pfansteihl have the function of positive, answer
With having a extensive future.
Invention content
Present invention aims at the immobilizations and its application that provide production Pfansteihl recombination engineering bacteria.
The present invention is realized by following methods：
The recombination engineering bacteria that Bacillus coli expression produces Pfansteihl is fixed with Supramolecular self assembly template.The structure of engineering bacteria is
Lactobacillus lactate dehydrogenase gene Idhl is integrated into II a of Torulopsis glabrata gene M T, forms recombinant dna gene Idh
Ⅱa.It is cloned by PCR, and is inserted into coli expression carrier pTE28aT7 after modification and builds Pfansteihl engineering bacteria
pTE28aT7-IdhⅡa。
Plasmid and bacterial strain：
Bacterial strain and plasmid:Lactobacillus lactic dehydrogenase（Purchased from Shanghai Sheng Gong bioengineering Co., Ltd）, torulopsis glabrata
Bacterium（Purchased from upper sea base rice Bioisystech Co., Ltd）, Escherichia coli (Escherichia coli) JM109, Escherichia coli
Plasmid pTE28aT7（Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences professor Wang Yucheng provides）.
Culture medium:LB, YEPD, YNB culture medium, bacterium often use culture medium：MD, YPD, BMGY, BMMY culture medium, recombination
Yeast often uses culture medium.
Reagent and enzyme:Small amount plasmid extraction kit is purchased from Shanghai Sheng Gong bioengineering Co., Ltd, plastic recovery kit
Purchased from TaKaRa companies, restriction endonuclease BamHI, Eco RI and T4 ligase ligases are purchased from Promega companies.
Yeast is purchased from Shanghai Sheng Gong bioengineering Co., Ltd without the basic nitrogen source medium of amino (YNB), Geneticin G418.Other examinations
Agent is that domestic analysis is pure.
Key instrument:PCR instrument, gel imager and protein electrophoresis instrument are purchased from BIO-RAD companies of the U.S., DYY-6C type electrophoresis
Instrument is purchased from Liuyi Instruments Plant, Beijing, and high performance liquid chromatograph and LC-MS instrument are purchased from Waters of the U.S..
The structure of expression vector：According to the cDNA sequence of the lactobacillus lactate dehydrogenase gene Idhl delivered, design
Positive anti-primer:Forward primer Pl (5'-ACGAGGAATTCATGAATTGGCCTACTCG-3');Reverse primer P2 (5'-
ATATGCGGCCGCGTGAACAGTAGGCAGAG-3').Forward primer carries EcoRI restriction enzyme sites, and reverse primer carries Not works
Restriction enzyme site obtains Idhl genetic fragments with primer Pl, P2 to II a PCR amplifications of recombinant plasmid pTE28aT7-Idh.Equally,
II a of gene M T of Torulopsis glabrata are designed, forward primer carries BamHI restriction enzyme sites, and reverse primer carries Xoh work enzymes
Enzyme site obtains II a genetic fragments of MT with primer Pl ', P2 ' to II a PCR amplifications of recombinant plasmid pTE28aT7-Idh.It will be upper
2 kinds of genetic fragments are stated, in the effect of ligase T4 ligase, are connected to become II a of gene Idh of recombinant DNA.Through recombinant DNA matter
After the extraction of grain, screening and sequencing, using CaCl2 plasmid recombinant techniques, by II a of gene Idh and carrier of recombinant DNA,
It is transferred in the competent cell of E.coli JM109, transformant sequencing obtains II a of expression plasmid pTE28aT7-Idh.
The screening of the structure of engineering bacteria and high copy recon：
By II a of expression vector pTE28aT7-Idh, the band containing target gene, electricity are recycled after hxjy II linearization for enzyme restriction
It hits and is transferred in P.pastoris kM71 competent cells, grow monoclonal in MD tablets, then copy more through YPD/G418 plate screenings
Shellfish transformant, G418 concentration screening gradients are followed successively by 0.5,1,2 mg/mL, and height copy transformant is identified through PCR.PPIC9K is empty
Plasmid processing is same as above, and transformed bacteria is as negative control.
The production Pfansteihl Activity determination of BGL：The supernatant for collecting recombinant bacterium fermented and cultured is concentrated through PEG, ammonium sulfate precipitation,
Crude enzyme liquid is made in dialysis, examines production hexanol activity as follows:27.8% glucose solution 10 is prepared with the acetate buffer solution of pH5.5
ML, 400 uL crude enzyme liquids, 30 DEG C of 24 h of reaction, then 35 DEG C of 24 h of reaction, then boil 10 min, micro porous filtration, HPLC and
LC-MS detects product.
The extraction of recombination engineering bacteria：
Preceding culture is that 5ml eutrophy culture mediums are added with sterile working in L- test tubes, and Aerobacter aerogenes is accessed with sterile toothpick
Single bacterium colony.35 DEG C, 120r/min cultures 15h.Preceding culture 0.5ml is seeded to containing 100ml eutrophy culture mediums
In 500ml triangular flasks, 30 DEG C, 110r/min shaken cultivations for 24 hours.4 DEG C, 6000*g sterile centrifugation 10min abandon supernatant, are centrifuging
Mixing is vibrated with sterile phosphate buffer in pipe, then at 4 DEG C, 6000*g sterile centrifugation 12min abandon supernatant.By preceding culture
Object 0.5ml is seeded in the 500ml triangular flasks containing 100ml eutrophy culture mediums, 35 DEG C, 110r/min shaken cultivations for 24 hours.4
DEG C, 6000*g sterile centrifugation 10min abandon supernatant, vibrate mixing in centrifuge tube with sterile phosphate buffer, again at 4 DEG C,
6000*g sterile centrifugation 12min, abandon supernatant.Sterile 10 bottles of the access of the thalline without containing eutrophy culture medium is being contained respectively
In the 500ml triangular flasks of 100m fermentation mediums, 31 DEG C~35 DEG C, 110r/min shaken cultivations 48h.
The preparation of Supramolecular self assembly template：
By 25g sodium metasilicate（NaSiO3.9H2O) heating is dissolved in 50ml distilled water, controls 40~50 DEG C, is 8 with 5M sulfuric acid tune pH
~9, it stirs 10 minutes, cetyltrimethylammonium bromide (CTAB) 6.4g, alchlor is added（AlCl3）2.7g and ammonium chloride
2.1g, it is SiO to make the molar ratio of each substance in mixture system2:CTAB:AlCl3:NH4Cl：H2O=1.0:0.16~0.2:0.5
~0.6:1.0~2.2:40~60,0.5h is stirred at room temperature, forms white gels shape.Mixture is put into reaction kettle, 130
~150 DEG C of self-assembling reaction 72h, are cooled to room temperature, filtering and washing, after drying, 2h are roasted at 260 DEG C, is roasted at 540 DEG C
6h is cooled to room temperature, closed to preserve for use.
Recombination engineering bacteria immobilization：
The phosphate buffer of appropriate Supramolecular self assembly template and 5mLPH7.0 are added in the conical flask of 25mL, are then added
7.5% glutaraldehyde is finally added dropwise until solution matter in the activator of 2mL and the colibacillus engineering solution of 2mL into solution
It is 0.75% to measure score.Immobilized reactant carries out 2h under conditions of 180r/min, 15 degree, is 7.0 using filter, 50mL PH
Phosphoric acid liquid washing after obtain immobilization colibacillus engineering.
Recombination engineering bacterium fermentation synthesizes Pfansteihl：
The recombination bacillus coli of -70 DEG C of preservations activates on LB culture medium flat plates, is connected to sterilizing toothpick picking single bacterium colony
In 20mlLB culture mediums, 31 DEG C~37 DEG C overnight incubations are inoculated in 2% inoculum concentration in fermentation medium, and addition 1~
The expression of II a of IPTG induction Idh of 2mmol/L, fermented and cultured 48h.
The separation of tunning：After fermentation harvest, centrifuges and recycling catalyzing enzyme, fermentation mother liquor are extracted using butyl acetate
It takes, after organic condensing crystallizing of extraction gained, uses butyl acetate and petroleum ether（1:3~4 volumes）It is recrystallized to give product.
The immobilization bacterial strain of the present invention, product expression amount is high, reaches as high as 23.5g/L, inversion rate of glucose is higher than
68.51%；Reusing is good, and after reusing 30 times, still up to 22.6g/L, inversion rate of glucose is higher than product expression amount
65.87%, will be prepared to the microbial fermentation of Pfansteihl has the function of positive, has a extensive future.
Specific implementation mode
With reference to specific embodiment, the invention will be further elaborated, but is not limited to these specific embodiments,
And all embodiments are pressed above-mentioned operating procedure and are operated.
Embodiment 1,
Plasmid and bacterial strain：
Bacterial strain and plasmid:Lactobacillus lactic dehydrogenase（Purchased from Shanghai Sheng Gong bioengineering Co., Ltd）, torulopsis glabrata
Bacterium（Purchased from upper sea base rice Bioisystech Co., Ltd）, Escherichia coli (Escherichia coli) JM109, Escherichia coli
Plasmid pTE28aT7（Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences professor Wang Yucheng provides）.
Culture medium:LB, YEPD, YNB culture medium, bacterium often use culture medium：MD, YPD, BMGY, BMMY culture medium, recombination
Yeast often uses culture medium.
The structure of expression vector：According to the cDNA sequence of the lactobacillus lactate dehydrogenase gene Idhl delivered, design
Positive anti-primer:Forward primer Pl (5'-ACGAGGAATTCATGAATTGGCCTACTCG-3');Reverse primer P2 (5'-
ATATGCGGCCGCGTGAACAGTAGGCAGAG-3').Forward primer carries EcoRI restriction enzyme sites, and reverse primer carries Not works
Restriction enzyme site obtains Idhl genetic fragments with primer Pl, P2 to II a PCR amplifications of recombinant plasmid pTE28aT7-Idh.Equally,
II a of gene M T of Torulopsis glabrata are designed, forward primer carries BamHI restriction enzyme sites, and reverse primer carries Xoh work enzymes
Enzyme site obtains II a genetic fragments of MT with primer Pl ', P2 ' to II a PCR amplifications of recombinant plasmid pTE28aT7-Idh.It will be upper
2 kinds of genetic fragments are stated, in the effect of ligase T4 ligase, are connected to become II a of gene Idh of recombinant DNA.Through recombinant DNA matter
After the extraction of grain, screening and sequencing, using CaCl2 plasmid recombinant techniques, by II a of gene Idh and carrier of recombinant DNA,
It is transferred in the competent cell of E.coli JM109, transformant sequencing obtains II a of expression plasmid pTE28aT7-Idh.
The screening of the structure of engineering bacteria and high copy recon：
By II a of expression vector pTE28aT7-Idh, the band containing target gene, electricity are recycled after hxjy II linearization for enzyme restriction
It hits and is transferred in P.pastoris kM71 competent cells, grow monoclonal in MD tablets, then copy more through YPD/G418 plate screenings
Shellfish transformant, G418 concentration screening gradients are followed successively by 0.5,1,2 mg/mL, and height copy transformant is identified through PCR.PPIC9K is empty
Plasmid processing is same as above, and transformed bacteria is as negative control.
The production Pfansteihl Activity determination of BGL：The supernatant for collecting recombinant bacterium fermented and cultured is concentrated through PEG, ammonium sulfate precipitation,
Crude enzyme liquid is made in dialysis, examines production hexanol activity as follows:27.8% glucose solution 10 is prepared with the acetate buffer solution of pH5.5
ML, 400 uL crude enzyme liquids, for 24 hours, then then 35 DEG C of 24 h of reaction boil 10 min, micro porous filtration for 30 DEG C of reactions, HPLC and
LC-MS detects product.
The extraction of recombination engineering bacteria：
Preceding culture is that 5ml eutrophy culture mediums are added with sterile working in L- test tubes, and Aerobacter aerogenes is accessed with sterile toothpick
Single bacterium colony.33 DEG C, 120r/min cultures 15h.Preceding culture 0.5ml is seeded to containing 100ml eutrophy culture mediums
In 500ml triangular flasks, 30 DEG C, 110r/min shaken cultivations for 24 hours.4 DEG C, 6000*g sterile centrifugation 10min abandon supernatant, are centrifuging
Mixing is vibrated with sterile phosphate buffer in pipe, then at 4 DEG C, 6000*g sterile centrifugation 12min abandon supernatant.By preceding culture
Object 0.5ml is seeded in the 500ml triangular flasks containing 100ml eutrophy culture mediums, 33 DEG C, 110r/min shaken cultivations for 24 hours.4
DEG C, 6000*g sterile centrifugation 10min abandon supernatant, vibrate mixing in centrifuge tube with sterile phosphate buffer, again at 4 DEG C,
6000*g sterile centrifugation 12min, abandon supernatant.Sterile 10 bottles of the access of the thalline without containing eutrophy culture medium is being contained respectively
In the 500ml triangular flasks of 100m fermentation mediums, 31 DEG C~35 DEG C, 110r/min shaken cultivations 48h.
PCR amplification：95 DEG C of pre-degeneration 10min, 91 DEG C of deformation 60s, 58 DEG C of annealing 30s, 70 DEG C of extension 60-120s, 30
70 DEG C of extension 10min after cycle.
The preparation and conversion of competent escherichia coli cell：
Prepare competent escherichia coli cell according to calcium method and converted, conversion be followed by 1mlLB culture concentrate 36 DEG C,
130r/min shaken cultivation 2h are spread evenly across on the LB tablets containing ampicillin and screen recombination bacillus coli.It screens
The bacterial strain arrived verifies recon using bacterium colony PCR and digestion with restriction enzyme recombinant plasmid.
The extraction of recombination engineering bacteria：
Preceding culture is that 5ml eutrophy culture mediums are added with sterile working in L- test tubes, and Aerobacter aerogenes is accessed with sterile toothpick
Single bacterium colony.35 DEG C, 120r/min cultures 15h.Preceding culture 0.5ml is seeded to containing 100ml eutrophy culture mediums
In 500ml triangular flasks, 30 DEG C, 110r/min shaken cultivations for 24 hours.4 DEG C, 6000*g sterile centrifugation 10min abandon supernatant, are centrifuging
Mixing is vibrated with sterile phosphate buffer in pipe, then at 4 DEG C, 6000*g sterile centrifugation 12min abandon supernatant.By preceding culture
Object 0.5ml is seeded in the 500ml triangular flasks containing 100ml eutrophy culture mediums, 35 DEG C, 110r/min shaken cultivations for 24 hours.4
DEG C, 6000*g sterile centrifugation 10min abandon supernatant, vibrate mixing in centrifuge tube with sterile phosphate buffer, again at 4 DEG C,
6000*g sterile centrifugation 12min, abandon supernatant.Sterile 10 bottles of the access of the thalline without containing eutrophy culture medium is being contained respectively
In the 500ml triangular flasks of 100m fermentation mediums, 30 DEG C~35 DEG C, 110r/min shaken cultivations 48h.
The preparation of Supramolecular self assembly template：
By 25g sodium metasilicate（NaSiO3.9H2O) heating is dissolved in 50ml distilled water, controls 40~50 DEG C, is 8 with 5M sulfuric acid tune pH
~9, it stirs 10 minutes, cetyltrimethylammonium bromide (CTAB) 6.4g, alchlor is added（AlCl3）2.7g and ammonium chloride
2.1g, it is SiO to make the molar ratio of each substance in mixture system2:CTAB:AlCl3:NH4Cl：H2O=1.0:0.16~0.2:0.5
~0.6:1.0~2.0:40~60,0.5h is stirred at room temperature, forms white gels shape.Mixture is put into reaction kettle, 130
~150 DEG C of self-assembling reaction 72h, are cooled to room temperature, filtering and washing, after drying, 2h are roasted at 260 DEG C, is roasted at 540 DEG C
6h is cooled to room temperature, closed Supramolecular self assembly template（Aperture：27.5~31.3nm；Specific surface area 1230m2/g）Preservation waits for
With.
Recombination engineering bacteria immobilization：
The phosphate buffer of appropriate Supramolecular self assembly template and 5mLPH7.0 are added in the conical flask of 25mL, are then added
7.5% glutaraldehyde is finally added dropwise until solution matter in the activator of 2mL and the colibacillus engineering solution of 2mL into solution
It is 0.75% to measure score.Immobilized reactant carries out 2h under conditions of 180r/min, 15 degree, is 7.0 using filter, 50mL PH
Phosphoric acid liquid washing after obtain immobilization colibacillus engineering.
Recombination engineering bacterium fermentation synthesizes Pfansteihl：
The recombination bacillus coli of -70 DEG C of preservations activates on LB culture medium flat plates, is connected to sterilizing toothpick picking single bacterium colony
In 20mlLB culture mediums, 32 DEG C~35 DEG C overnight incubations are inoculated in 2% inoculum concentration in fermentation medium, and addition 1~
The expression of II a of IPTG induction Idh of 2mmol/L, fermented and cultured 48h.
The separation of tunning：After fermentation harvest, centrifuges and recycling catalyzing enzyme, fermentation mother liquor are extracted using butyl acetate
It takes, after organic condensing crystallizing of extraction gained, uses butyl acetate and petroleum ether（1:3~4 volumes）It is recrystallized to give product
Pfansteihl 9.52g (51.2~52.8 DEG C of fusing point, content 99.27%, yield 68.49%).
Embodiment 2,
The composition and condition of culture of each culture medium are same as above, and recombination bacillus coli preparation is same as above, the preparation of Supramolecular self assembly template
Method and condition is same as above.
Go out the fixed recombination engineering bacteria of the Supramolecular self assembly template after the 5th use from filtering fermentation liquor, carries out
Following fermenting experiment.
Recombination engineering bacterium fermentation synthesizes Pfansteihl：
The recombination bacillus coli of -70 DEG C of preservations activates on LB culture medium flat plates, is connected to sterilizing toothpick picking single bacterium colony
In 20mlLB culture mediums, 33 DEG C~35 DEG C overnight incubations are inoculated in 2% inoculum concentration in fermentation medium, and addition 1~
The expression of II a of IPTG induction Idh of 2mmol/L, fermented and cultured 48h.
The separation of tunning：After fermentation harvest, centrifuges and recycling catalyzing enzyme, fermentation mother liquor are extracted using butyl acetate
It takes, after organic condensing crystallizing of extraction gained, uses butyl acetate and petroleum ether（1:3~4 volumes）It is recrystallized to give product
Pfansteihl 9.58g (51.6~52.6 DEG C of fusing point, content 99.35%, yield 68.92%).
Embodiment 3,
The composition and condition of culture of each culture medium are same as above, and recombination bacillus coli preparation is same as above, the preparation of Supramolecular self assembly template
Method and condition is same as above.
Go out the fixed recombination engineering bacteria of Supramolecular self assembly template after the 30th use from filtering fermentation liquor, carries out
Following fermenting experiment.
Recombination engineering bacterium fermentation synthesizes Pfansteihl：
The recombination bacillus coli of -70 DEG C of preservations activates on LB culture medium flat plates, is connected to sterilizing toothpick picking single bacterium colony
In 20mlLB culture mediums, 31 DEG C~35 DEG C overnight incubations are inoculated in 2% inoculum concentration in fermentation medium, and addition 1~
The expression of II a of IPTG induction Idh of 2mmol/L, fermented and cultured 48h.
The separation of tunning：After fermentation harvest, centrifuges and recycling catalyzing enzyme, fermentation mother liquor are extracted using butyl acetate
It takes, after organic condensing crystallizing of extraction gained, uses butyl acetate and petroleum ether（1:3~4 volumes）It is recrystallized to give product
Pfansteihl 9.64g (51.7~52.4 DEG C of fusing point, content 99.35%, yield 69.35%).
Claims (4)
1. the present invention relates to the immobilizations and its application of production Pfansteihl recombination engineering bacteria, it is characterised in that Supramolecular self assembly
Template fixes production Pfansteihl recombination engineering bacteria.
2. according to the method described in claim 1, it is characterized in that by sodium metasilicate, cetyltrimethylammonium bromide (CTAB),
Under the conditions of water phase and Supramolecular self assembly, the aperture of preparation is the extra specific surface area of 25~45nm for ammonium chloride and alchlor
（Not less than 1000m2/g）Supramolecular self assembly template, and the stable structure in acidic aqueous solution, each object in synthetic system
The molar ratio of matter is SiO2:CTAB:AlCl3:NH4Cl：H2O=1.0:0.16~0.2:0.5~0.6:1.0~2.2:40~60.
3. according to the method described in claim 1, it is characterized in that the construction method of recombination engineering bacteria is as follows：
（1）Lactobacillus lactate dehydrogenase gene Idhl is integrated into II a of Torulopsis glabrata gene M T, forms recombinant DNA
II a of gene Idh；
（2）It is cloned by PCR, and is inserted into coli expression carrier pTE28aT7 after modification and builds Pfansteihl engineering bacteria
pTE28aT7-IdhⅡa。
4. according to the method described in claim 1, it is characterized in that by above-mentioned Supramolecular self assembly template, using physisorphtion
Fix above-mentioned recombination engineering bacteria, and in containing glucose limited culture medium fermentation prepare Pfansteihl, work of fermenting
Skill condition is 30 DEG C~35 DEG C of temperature, 110r/min shaken cultivation 48h, and it can be used repeatedly 30 times or more for immobilised enzymes.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102250970A (en) * | 2011-05-26 | 2011-11-23 | 徐州恒源生物工程有限公司 | Method for synthesizing 1,3-dioxyacetone by glycerol fermentation |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250970A (en) * | 2011-05-26 | 2011-11-23 | 徐州恒源生物工程有限公司 | Method for synthesizing 1,3-dioxyacetone by glycerol fermentation |
| 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 |
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| 田康明等: "代谢工程大肠杆菌利用甘油高效合成L-乳酸", 《生物工程学报》 * |
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Application publication date: 20180904 |