CN108660165A - The method that recombination engineering bacterium fermentation synthesizes Pfansteihl - Google Patents
The method that recombination engineering bacterium fermentation synthesizes Pfansteihl Download PDFInfo
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- CN108660165A CN108660165A CN201810376571.3A CN201810376571A CN108660165A CN 108660165 A CN108660165 A CN 108660165A CN 201810376571 A CN201810376571 A CN 201810376571A CN 108660165 A CN108660165 A CN 108660165A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01027—L-Lactate dehydrogenase (1.1.1.27)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01028—D-Lactate dehydrogenase (1.1.1.28)
Abstract
It is that lactobacillus lactate dehydrogenase gene Idhl is integrated into II a of Torulopsis glabrata gene M T the invention discloses the method that recombination engineering bacterium fermentation synthesizes L lactic acid, forms II a of recombinant dna gene Idh.It is cloned by PCR, and is inserted into II a of coli expression carrier pTE28aT7 structure L lactic acid engineering bacteria pTE28aT7 Idh after modification；In isopropylthiogalactoside（IPTG）Under induction, in the limitation culture medium containing glucose, fermented synthesis L lactic acid；It is recrystallized by filtering fermentating liquid, organic solvent extractive crystallization and combination solvent and obtains product L lactic acid.The method of the recombination engineering bacterium fermentation synthesis L lactic acid of the present invention is concise, yield is high, of low cost, plays an important role, has a extensive future in prepared by the microbial method of L lactic acid.
Description
Technical field：The invention belongs to technical field of microbial fermentation, are related to recombination engineering bacterium fermentation synthesis Pfansteihl
Method.
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.
Invention content
The purpose of the present invention is to provide the methods that recombination engineering bacterium fermentation synthesizes Pfansteihl.
The present invention is realized by following methods：
Lactobacillus lactate dehydrogenase gene Idhl is integrated into II a of Torulopsis glabrata gene M T, forms recombinant dna gene
IdhⅡa.The recombination is cloned by PCR, and is inserted into coli expression carrier pTE28aT7 after modification and builds Pfansteihl
II a of engineering bacteria pTE28aT7-Idh；In isopropylthiogalactoside（IPTG）Under induction, in the limitation culture containing glucose
In base, fermented synthesis Pfansteihl；It is recrystallized by filtering fermentating liquid, organic solvent extractive crystallization and combination solvent and obtains product
Pfansteihl.
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~37 DEG C, 110r/min shaken cultivations 48h.
Recombination engineering bacterium fermentation produces 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, 30 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.
The method of the recombination engineering bacterium fermentation synthesis Pfansteihl of the present invention is concise, yield is high, of low cost.Based on upper
Advantage is stated, engineering bacteria of the invention will play an important role in the preparation of the bioanalysis of Pfansteihl, have 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.
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.32 DEG C, 120r/min cultures 15h.Preceding culture 0.5ml is seeded to containing 100ml eutrophy culture mediums
In 500ml triangular flasks, 32 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, 32 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.
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.
Recombination engineering bacterium fermentation produces 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, 30 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 L- breasts
Sour 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.
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, 110r/min cultures 15h.Preceding culture 0.5ml is seeded to containing 100ml eutrophy culture mediums
In 500ml triangular flasks, 33 DEG C, 120r/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, 120r/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, 120r/min shaken cultivations 48h.
Recombination engineering bacterium fermentation produces 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, 30 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 52h.
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（1:3~3.5 volumes）It is recrystallized to give L-
Lactic acid 9.64g (51.7~52.4 DEG C of fusing point, content 99.35%, yield 69.35%).
Claims (6)
1. the present invention relates to the methods that recombination engineering bacterium fermentation synthesizes Pfansteihl, including：
（1）It is that lactobacillus lactate dehydrogenase gene Idhl is integrated into II a of Torulopsis glabrata gene M T, forms recombination
II a of DNA genes Idh；
（2）By PCR amplification, and it is inserted into coli expression carrier pTE28aT7 after modification, builds Pfansteihl engineering bacteria
pTE28aT7-IdhⅡa；
（3）In isopropylthiogalactoside（IPTG）Under induction, high efficient strain is screened using high flux screening method；
（4）In the limitation culture medium containing glucose, fermented synthesis Pfansteihl is extracted by filtering fermentating liquid, organic solvent
It takes crystallization and combination solvent to recrystallize and obtains product Pfansteihl.
2. according to the method described in claim 1, it is characterized in that lactobacillus lactate dehydrogenase gene Idhl be integrated into it is smooth
II a of torulopsis bacterium gene M T form II a of recombinant dna gene Idh.
3. according to the method described in claim 1, it is characterized in that PCR amplification condition is：95 DEG C of pre-degeneration 10min, 91 DEG C of changes
Shape 60s, 56 DEG C of annealing 30s, 70 DEG C extend 80-120s, extend 15min for 70 DEG C after 30 cycles.
4. the method for recombination engineering fermentation synthesis Pfansteihl according to claim 1, including：
（1）It ferments in containing glucose limited culture medium to the recombination engineering, using high flux screening；
（2）Filtering fermentating liquid, organic solvent extractive crystallization and combination solvent recrystallization obtain product Pfansteihl.
5. according to the method described in claim 4, it is characterized in that the fermentation rank of recombination engineering bacterium fermentation synthesis Pfansteihl
Section fermentation condition is 30 DEG C~35 DEG C of temperature, 110r/min shaken cultivation 68h, when a concentration of 27.8 % of substrate glucose, fermentation
Liquid pH5.5~6.0, temperature are 25-35 DEG C, and enzyme concentration is every gram of glucose 33U, adds the K of 1 mmol/L+, the transformation period is
68 h realize Pfansteihl overexpression 23.50g/L.
6. according to according to the method for claim 4, it is characterised in that zymotic fluid first passes through filter separation catalyzing enzyme and reuses,
Zymotic fluid after being filtered using n-butyl acetate extraction, then by after extract liquor condensing crystallizing, by volume using butyl acetate and petroleum ether
1：3~5 combination solvent recrystallization, obtains product Pfansteihl.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111821892A (en) * | 2020-07-21 | 2020-10-27 | 李伟 | Preparation method of immobilized lactobacillus fermentation |
| CN115011537A (en) * | 2022-06-14 | 2022-09-06 | 湖北工业大学 | Engineering bacterium for inducing double anaerobic promoters to produce high-optical-purity L-lactic acid and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101007756A (en) * | 2006-01-25 | 2007-08-01 | 上海同杰良生物材料有限公司 | Technical process of preparing high-purity L-lactic acid |
| 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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2018
- 2018-04-25 CN CN201810376571.3A patent/CN108660165A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101007756A (en) * | 2006-01-25 | 2007-08-01 | 上海同杰良生物材料有限公司 | Technical process of preparing high-purity L-lactic acid |
| 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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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111821892A (en) * | 2020-07-21 | 2020-10-27 | 李伟 | Preparation method of immobilized lactobacillus fermentation |
| CN115011537A (en) * | 2022-06-14 | 2022-09-06 | 湖北工业大学 | Engineering bacterium for inducing double anaerobic promoters to produce high-optical-purity L-lactic acid and preparation method and application thereof |
| CN115011537B (en) * | 2022-06-14 | 2023-06-23 | 湖北工业大学 | Engineering bacterium for producing high optical purity L-lactic acid by double anaerobic promoters and preparation method and application thereof |
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