CN107686850A - It is a kind of to utilize the method for co-expressing recombinant bacterial strain conversion production alpha Ketoglutarate - Google Patents
It is a kind of to utilize the method for co-expressing recombinant bacterial strain conversion production alpha Ketoglutarate Download PDFInfo
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Abstract
The invention discloses a kind of using the method for co-expressing recombinant bacterial strain conversion production alpha Ketoglutarate, belong to biological technical field.Present invention screening from streptomycete obtains a kind of new L dglutamic oxidases, and by it, induced expression purifies and carries out zymologic property research in Escherichia coli, it is found that the enzyme has greater activity at pH 5.5 ~ 7.0, optimal reactive temperature is 30 ~ 45 DEG C, VmaxFor 100 ~ 150 U/mg, KmFor 8 ~ 10 mM.The original gene sequence of L dglutamic oxidases is subjected to codon optimization, and with carrying out plasmid coexpression, structure coexpression recombinant bacterial strain from the catalase gene of Escherichia coli.L glutamic acid is converted by whole-cell catalyst of the recombinant bacterial strain（Salt）, alpha Ketoglutarate yield reaches 76.08 g/L after reacting 9 h, and molar yield is 96.8 %.The present invention solves cumbersome alpha Ketoglutarate production stage, low yield, causes the problems such as environmental pollution, realizes alpha Ketoglutarate high yield, One-step production, has higher industrial application value.
Description
Technical field
The present invention relates to a kind of using the method for co-expressing recombinant bacterial strain conversion production α-ketoglutaric acid, belong to biotechnology
Field.
Background technology
L-GLOD (L-glutamate oxidase, LGOX) is one kind with flavin adenine dinucleotide (FAD)
(FAD) it is the flavoprotein enzyme of prothetic group, paddy ammonia can be aoxidized in specific manner under conditions of exogenous confactor is not added
Acid generation hydrogen peroxide, ammonia and α-ketoglutaric acid[1].α-ketoglutaric acid (α-KG) is used as in tricarboxylic acid cycle and amino acid metabolism
Important binary acid, amino acid formed and N transformation in play key player, be widely used in medicine, fine chemistry industry,
The field such as food and animal feed[2]。
At present, α-KG production method includes chemical synthesis, fermentation method, biological catalysis.Traditional α-KG productions are adopted
With chemical synthesis, but the harmful reagent such as strong acid and strong base used in chemical synthesis process, cyanide, not only easily cause ring
Border is polluted, and more limits its application in industries such as food, cosmetics and medicine[3].Microbe fermentation method α-KG yield is reachable
186 g/L, but fermentation period is up to 117 h[4], and the generation of the accessory substance such as pyruvic acid in tunning, fumaric acid can increase
Add the difficulty and expense of follow-up α-KG extractions, be not suitable for large-scale production.Production by Enzymes α-KG have reaction time it is short, conversion
The high advantage of rate, ox are expected clear etc.[5]By mutagenic obtained high yield LGOX streptomycete mutant strain, 24 are converted in optimal conditions
H, α-KG yield are up to 38.1 g/L；Using the method for genetic engineering, by LGOX genes in Escherichia coli heterogenous expression, lead to
Cross enzyme law catalysis, the g/L of 24 h generation α-KG yield 104.7[6].Catalysed in vitro is carried out using the L-GLOD of purifying
Not only need by cumbersome protein purification steps, and course of reaction needs a large amount of expensive catalase auxiliary of external source addition
Catalysis, considerably increases industrial cost.
Catalysed in vitro is carried out compared to separation enzyme, resting cell has following advantage：Whole-cell biocatalyst is easier
Prepare, it is cost-effective；It is more stable compared to separation enzyme, it is not easy to be influenceed by factors such as ambient temperature, pH, it is easy-to-use；Conversion
During be not required to external source addition confactor, it is environment-friendly and without the generation of poisonous and harmful product, before there are industrial applications
Scape.
Bibliography：
1. Bi Chun is first, Li Ling, progress [J] life sciences of Li Jing dragon L-GLODs, 2012,24 (2):
169-173
2. Chernyavskaya OG, Shishkanova NV, Il'chenko AP, et al. Synthesis of
alpha-ketoglutaric acid by Yarrowia lipolytica yeast grown on ethanol[J].Appl Microbiol Biotechnol, 2000, 53 (2): 152-158.
3. Otto C, Yovkova V, Barth G. Overproduction and Secretion of Alpha-
Ketoglutaric Acid by Microorganisms [J]. Appl. Microbiol. Biotechnol, 2011,
92 (4): 689-695.
4. Yovkova V, Otto C, Aurich A, et al. Engineering the Alpha-
Ketoglutarate Overproduction from Raw Glycerol by Overexpression of the Genes
Encoding NADP(+)-dependent Isocitrate Dehydrogenase and Pyruvate Carboxylase
in Yarrowialipolytica[J]. Appl.Microbiol. Biotechnol, 2014, 98 (5): 2003-
2013.
5. N expect clear, bright enzymatic conversion methods Pidolidone production α-ketoglutaric acid [J] biotechnologys of Zhang Zhenyu, Liu Li
Report, 2014,30 (8):1318-1322.
6. Niu P Q, Dong X X, Wang Y C, et al.Enzymatic Production of Alpha-
Ketoglutaric Acid from L-Glutamic Acid via L-Glutamate Oxidase [J].J.Biotechnol, 2014, 179: 56-62。
The content of the invention
To solve above-mentioned technical problem, produced it is an object of the invention to provide one kind using recombinant bacterial strain conversion is co-expressed
The method of α-ketoglutaric acid, use the present invention can be with Efficient Conversion Pidolidone（Salt）α-ketoglutaric acid is generated, so as to solve
The problem of industrially producing alpha -one glutaric acid production cost is high, cycle length, and efficiency is low, be advantageous to the scale metaplasia of α-ketoglutaric acid
Production.
To realize above-mentioned purpose, the present invention uses following technical scheme：
（1）The structure of recombination bacillus coli：With Escherichia coliE.coliBL21 is host, and PET21b plasmids are carrier, expression
Come fromStreptomyces mobaraensisCICC11018 L-GLOD.
（2）The induced expression purifying of L-GLOD：By recombination bacillus coli, access contains amicillin resistance
（The μ g/mL of final concentration 100）LB culture mediums, 37 DEG C are activated overnight, and the switching of 2% inoculum concentration, which is put, new contains amicillin resistance
（The μ g/mL of final concentration 100）LB culture mediums, cultivate OD600For 0.6 ~ 0.8, add 0.4 mM IPTG, 25 ~ 30 DEG C of inductions 6 ~ 8
h.Thalline is collected, carries out ultrasonication.4 DEG C, 12000 g centrifuging and taking supernatants, obtain crude enzyme liquid.Crude enzyme liquid is subjected to Ni posts parent
And chromatographic purifying, obtain the pure enzyme of L-GLOD.
（3）The measure of L-GLOD zymologic property：Choose different temperatures gradient：20 ℃、25 ℃、30 ℃、35
DEG C, 40 DEG C, 45 DEG C, 50 DEG C, pH 6.0 50 mM disodium hydrogen phosphates-citrate buffer solution, optimum temperature is surveyed
It is fixed.React, determine in pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0 respectively under the conditions of 35 DEG C
Optimal reaction pH.With the L-sodium (0.5 ~ 140 mM) of various concentrations for substrate, enter under the conditions of optimal reactive temperature and pH
Row reaction, VmaxAnd KmTried to achieve using Lineweaer-Burk graphing methods.
（4）The codon optimization of L-GLOD gene：By the original gene sequence of L-GLOD, according to
E. coli codon Preference carries out codon optimization.
（5）Co-express the structure of recombinant bacterial strain:Primer is designed according to catalase of Escherichia coli gene order, with big
Enterobacteria genome is template, and PCR obtains catalase gene fragment.With catalase gene fragment and codon optimization
L-GLOD genetic fragment afterwards is template, and design primer carries out fusion DNA vaccine, using pET21b as carrier, builds table altogether
Up to recombinant plasmid, Escherichia coli are importedE.coliBL21, obtain coexpression restructuring coliform.
（6）Resting cell production α-ketoglutaric acid is carried out using recombinant bacterial strain is co-expressed：Pidolidone（Salt） 100g/
L, whole-cell catalyst：30 g/L, react and carried out in the mM disodium hydrogen phosphates of pH 7.0 200-citrate buffer solution, 35 DEG C
200 rpm convert 9 h.
Effective benefit of the present invention is to be successfully realized L-GLOD gene with catalase gene in large intestine
Co-expressed in bacillus, and utilize and co-express recombinant bacterial strain resting cell Pidolidone（Salt）Produce α-ketoglutaric acid, conversion effect
Rate is 96.8%.Resting cell system of the present invention, solve chemical synthesis α-ketoglutaric acid complex steps, pollute asking for environment
Topic, and enzymatic conversion method are produced the problem of needing external source to add a large amount of catalases, realize high yield, the nothing of α-ketoglutaric acid
Pollution, One-step production, to meet that the large-scale production of α-ketoglutaric acid is provided fundamental basis.
Brief description of the drawings
Figure of description 1 is SmLGOX optimum temperatures；
Figure of description 2 is SmLGOX optimal pHs；
Figure of description 3 is to calculate Enzyme kinetic parameter using Lineweaer-Burk graphing methods；
Figure of description 4 is that SmLGOX codon optimizations are front and rear schemes with SDS-PAGE after coexpression；
Figure of description 5 is that whole-cell catalyst converts Pidolidone（Salt）Produce the Yield mapping of α-ketoglutaric acid.
Specific implementation method
Illustrate specific implementation method of the present invention below by way of specific embodiment, but these embodiments are not formed to the present invention
The restriction of mode, scope and effect.
A detailed description is done to the specific implementation method of the present invention below.The present invention is retouched by following steps
State：
The structure of step 1, recombination bacillus coli
The primer ,-CATG of sense primer 5 ' are designed according to L-GLOD original gene sequenceCATATGGTGCCCGCCAAG
The TCCACCGC-3 ,-CTGA of anti-sense primer 5 'CTCGAGGGCGAGG
TGCGCCTCCAGC-3 ', wherein sense primer contain the restriction enzyme sites of Nde I, and anti-sense primer contains the restriction enzyme sites of Xho I.It is logical
Cross PCR and obtain LGOX genetic fragments, using pET21b as carrier, construction recombination plasmid pET21b-LGOX, by recombinant plasmid transformedE.coliDH5 α, through PCR, digestion verification correctly and after sequencing, conversionE.coliBL21, build recombinant bacterial strainE.coliBL21
（pET21b-LGOX）.
Step 2, the purifying of LGOX induced expression
By -80 DEG C of preservationsE.coliBL21（pET21b- LGOX）Access contains amicillin resistance（The μ g/ of final concentration 100
mL）LB culture mediums, 37 DEG C are activated overnight, and the switching of 2% inoculum concentration, which is put, new contains amicillin resistance（The μ of final concentration 100
g/mL）LB culture mediums, cultivate OD600For 0.6 ~ 0.8, add 0.2 ~ 0.6 mmol/L IPTG, 30 DEG C of 6 ~ 8 h of induction.Collect
Thalline, carry out ultrasonication.Ni post affinitive layer purifications are finally carried out, obtain pure enzyme LGOX.
Step 3, LGOX zymologic properties measure
LGOX enzyme activity determination methods：In 3 mL reaction systems, 11 mg/mL Pidolidone solution are contained in reaction solution,
121.5 μ g/mL 4- amino antipyrine solution, 0.26 μ L/mL DMAs, 20 U/mL horseradish peroxides
Compound enzyme, and appropriate enzyme liquid, face used time mixing, 35 DEG C of 10 min of reaction, reaction assay is carried out using spectrophotometer.Enzyme activity
It is defined as：1 μm of ol α-ketoglutaric acid of catalysis generation per minute is defined as an enzyme-activity unit U, specific enzyme activity U/mg.
Different temperatures gradient is set：20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, adopt
With pH 6.0 50 mM disodium hydrogen phosphates-citrate buffer solution, optimum temperature is measured.Respectively in pH under the conditions of 35 DEG C
4.0th, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5 50 mM disodium hydrogen phosphates-citric acid
Buffer solution is reacted, measure optimal reaction pH.The measure of kinetic constant be with the L-sodium of various concentrations (0.5 ~
140 mM) it is substrate, reacted under conditions of optimal reactive temperature and pH, VmaxAnd KmMade using Lineweaer-Burk
Figure method is tried to achieve.
Step 4, codon optimization and the structure for co-expressing recombinant bacterial strain
According to e. coli codon Preference, codon optimization is carried out to L-GLOD gene, and re-start base
Because of sequent synthesis.Primer, using genome of E.coli as template, PCR are designed according to catalase of Escherichia coli gene order
Obtain catalase gene fragment.With glucose oxidation enzyme gene piece after the genetic fragment of catalase and codon optimization
Section is template, and design primer carries out fusion DNA vaccine, using pET21b as carrier, builds co-expression plasmid pET21b-LGOX-C-katE,
ConversionE.coliDH5 α checkings are correctly and after sequencing, conversionE.coliBL21, structure coexpression bacterial strainE.coliBL21
（pET21b- LGOX-C-katE）.
Step 5, using co-express recombinant bacterial strain carry out resting cell production α-ketoglutaric acid
Disodium hydrogen phosphate-citrate buffer solution that buffer solution is the mM of pH 7.0 200 is converted, conversion temperature is 35 DEG C, and substrate is dense
Spend for 100 g/L, dense bacterium is 30 g/L.Sampled when 1 h, 3 h, 6 h, 9 h, α -one is carried out using high performance liquid chromatography
The measure of glutaric acid content.α-ketoglutaric acid yield reaches 76.08 g/L after reacting 9 h, and molar yield is 96.8 %.
SEQ NO.1
GCCGTGCCCGCCAAGTCCACCGCCGACTGGGACACGTGCTTGGAAGTGGCAAGAGCCCTGCTCGTGGTCGACG
AGCACGACCGGCCGCTGGTCCCCGAGTACAAGAAGATCCTTGACGACGGGCTGCCGCGCACCGGGAAGAAGGCGGGG
CGGAAGGTGCTCGTCGTGGGCGCCGGGCCGGCCGGGCTGGTGGCGGCTTGGCTGCTGAAGCGGGCCGGGCACCACGT
GACGCTGCTGGAGGCCAACGGGAACCGGGTCGGCGGGCGGATCAAGACGTTCCGTAAGGGTGGGCACGAGCACGCGG
TGCAGCCGTTCGCGGACCCCCGGCAGTACGCCGAGGCCGGGGCCATGCGCATCCCGGGCAGTCACCCGCTCGTGATG
AGCCTGATCGACGGGCTCGGGGTGAAGCGCCGGCCCTTCTACCTCGTGGACGTGGACGGGCAGGGCAAGCCCGTCAA
TCATGCCTGGCTGCACGTCAACGGGGTGCGGGTACGGCGTGCCGACTACGTCAAGGATCCTCGGAAGGTCAACCGGT
CGTTCGGCGTTCCCCGGGAGCTGTGGGACACGCCGTCGTCCGTCATCCTGCGGCGCGTGCTCGACCCCGTCCGCGAC
GAGTTCAGCACTGCCGGGGCCGACGGTAAGCGGGTCGACAAGCCGATGCCCGAGCGGGTGAAGGGGTGGGCCCGGGT
CATCCAGAAGTACGGTGACTGGTCGATGTACCGGTTCCTCACGGAGGAGGCCGGGTTCGACGAGCGCACCCTCGATC
TCGTCGGGACGCTGGAGAATCTCACGTCCCGGCTGCCGCTGTCGTTCGTGCACAGCTTCATCAGCCAGTCGCTGATC
AGCCCGGACACGGCGTTCTGGGAGCTGGTCGGCGGCACCGCGTCGCTGCCCGACGCGCTGTTGAAGAAGGTCGACGA
CGTGCTGCGGCTCGACCGGCGGGCGACGCGCATCGAGTACTGGTCGCCGGACCGGACGGGAGCCGACCGGGCGACAC
ACGTCCGCGAGGGCGGCCCGCACGTGTGGATCGACACCGTGTCGGAGGGCCGGGACGGCAAGGTCGTCCGCGAGCAG
TTCACCGGCGACCTCGCGATCGTCACCGTGCCGTTCACGGGGCTGCGCCACGTGCAGGTCAGCCCGCTGATGTCGTA
CGGCAAACGGCGCGCCGTCACCGAGCTGCACTACGACAGCGCCACCAAGGTGCTGCTCGAATTCAGCCGGCGCTGGT
GGGAGTTCACCGAGGAGGACTGGAAGCGGGAGCTGGAGGACGTGCGGCCGGGGCTGTACGCCGCGTACCGGGACGGC
AAGGCGCCCGCCGACGGCAGTCTCCTCGGCACCCACCCCTCCGTCCCGCACGGCCACATCAGCCAGGCCCAGCGCGC
CCACTACGCCGCCAACTACTGGGAGGGGCGCGACCAGCCCGAGGCGGCGCACATCGTGGGCGGCGGGTCGGTCTCCG
ACAACCCCAACCGGTTCATGTTCAACCCCTCCCATCCCGTACCCGGCAGCGAGGGCGGCGTCGTCCTCGCCGTCTAC
TGCTGGGCCGACGACGCCTCGCGCTGGGACTCCCTCGACGACGAGGCCCGCTACCCGCACGCCCTCTGCGGGCTGCA
ACAGGTCTACGGACAGCGCGTCGAGGTCTTCTACACCGGCGCCGGCCGCACCCAGAGCTGGCTGCGCGACCCGTACG
CGTACGGCGAGGCGTCCGTCCTCCTGCCCGGCCAGCACACGGAACTGCTCGGCGCCATTCGGGAACCCGAGGGCCCG
CTGCACTTCGCGGGGGACCACACATCCGTCAAACCGTCCTGGATCGAGGGCGCCGTCGAATCCGGCGTCCGGGCGGC
GCTGGAGGCGCACCTCGCC
SEQ NO.2
GCGGTTCCTGCGAAAAGCACCGCGGATTGGGATACCTGTCTGGAAGTGGCGCGCGCGTTATTAGTGGTGGATG
AACATGATCGTCCGCTGGTGCCGGAATATAAGAAGATTCTGGATGATGGCCTGCCTCGCACCGGTAAAAAAGCGGGC
CGCAAAGTGTTAGTTGTTGGTGCGGGTCCTGCAGGTTTAGTTGCGGCGTGGCTGTTAAAACGTGCGGGTCATCATGT
GACTCTGCTGGAAGCGAACGGCAATCGTGTTGGCGGCCGCATTAAAACCTTTCGCAAAGGCGGCCATGAACATGCGG
TTCAGCCGTTTGCAGATCCGCGTCAGTATGCAGAAGCAGGCGCGATGCGTATTCCTGGCAGCCATCCGTTAGTGATG
AGCCTGATTGATGGCCTGGGTGTTAAACGCCGCCCGTTTTATCTGGTGGATGTGGATGGCCAAGGCAAACCGGTTAA
CCATGCGTGGCTGCATGTGAATGGTGTTCGCGTTCGCCGTGCGGATTATGTGAAAGATCCGCGCAAAGTGAACCGCA
GCTTTGGTGTGCCGCGTGAATTATGGGATACCCCGAGCAGCGTTATTCTGCGCCGCGTTTTAGATCCTGTGCGCGAT
GAATTTTCAACCGCGGGCGCGGATGGTAAACGCGTGGATAAACCGATGCCGGAACGCGTTAAAGGTTGGGCGCGCGT
GATTCAGAAATATGGCGACTGGAGCATGTATCGCTTTCTGACCGAAGAAGCGGGCTTTGATGAACGCACCCTGGATT
TAGTTGGCACCCTGGAAAACTTAACCAGCCGCCTGCCGTTAAGCTTTGTGCATAGCTTTATTAGCCAGAGCCTGATT
TCACCGGATACCGCGTTTTGGGAACTGGTTGGTGGCACCGCGAGCTTACCTGATGCGCTGCTGAAAAAAGTGGATGA
TGTGCTGCGCTTAGATCGTCGTGCGACCCGCATTGAATATTGGAGCCCGGATCGTACTGGTGCGGATCGTGCGACCC
ATGTTCGTGAAGGTGGTCCGCATGTGTGGATTGATACCGTGAGCGAAGGCCGCGATGGCAAAGTTGTGCGCGAACAG
TTTACTGGCGATCTGGCGATTGTGACCGTGCCGTTTACCGGTCTGCGCCATGTTCAAGTGAGCCCGCTGATGAGCTA
TGGTAAACGTCGCGCGGTGACCGAACTGCATTATGATAGCGCGACCAAAGTGCTGCTGGAATTTAGCCGCCGCTGGT
GGGAATTTACCGAAGAAGATTGGAAACGCGAACTGGAAGATGTTCGCCCGGGCTTATATGCAGCGTATCGCGATGGT
AAAGCGCCTGCGGATGGTAGCTTATTAGGCACCCATCCGTCAGTTCCGCATGGCCATATTAGCCAAGCGCAGCGTGC
ACATTATGCGGCGAACTATTGGGAAGGCCGCGATCAACCTGAAGCGGCGCATATTGTTGGTGGTGGCAGCGTTAGCG
ATAACCCGAACCGCTTTATGTTTAACCCGAGCCATCCGGTTCCTGGTAGCGAAGGTGGTGTTGTGCTGGCGGTTTAT
TGCTGGGCGGATGATGCAAGCCGTTGGGATAGCCTGGATGATGAAGCGCGCTATCCGCATGCACTGTGTGGTCTGCA
ACAGGTTTATGGCCAGCGCGTGGAAGTGTTTTATACCGGCGCGGGTCGTACTCAATCATGGCTGCGTGATCCGTATG
CGTATGGCGAAGCGAGCGTTTTATTACCTGGCCAGCATACCGAATTACTGGGCGCGATTCGCGAACCTGAAGGCCCT
TTACATTTTGCGGGCGATCATACCTCAGTGAAACCGAGCTGGATTGAAGGTGCGGTGGAAAGCGGTGTTCGTGCGGC
GTTAGAAGCGCATTTAGCG
Claims (7)
1. a kind of utilize the method for co-expressing recombinant bacterial strain conversion production α-ketoglutaric acid, it is characterised in that comprises the following steps：
（1）The present invention obtains a kind of new L-GLOD gene by bioinformatics method, screening；
（2）By the L-GLOD gene obtained in above-mentioned steps in expression in escherichia coli, and carry out Pidolidone oxygen
The induced expression purifying of hdac protein；
（3）The L-GLOD of the purifying obtained in above-mentioned steps is subjected to zymologic property measure；
（4）The L-GLOD gene obtained in above-mentioned steps is subjected to codon optimization, and induced in Escherichia coli
Expression；
（5）By the L-GLOD gene and catalase gene after the codon optimization obtained in above-mentioned steps
KateE carries out plasmid coexpression, and builds coexpression recombinant bacterial strain；
（6）The coexpression recombinant bacterial strain built by the use of in above-mentioned steps converts Pidolidone as whole-cell catalyst（Salt）Production
α-ketoglutaric acid.
2. according to claim 1 a kind of using the method for co-expressing recombinant bacterial strain conversion production α-ketoglutaric acid, it is special
Sign is：The step（1）Middle L-GLOD gene source inStreptomyces mobaraensis。
3. according to claim 1 a kind of using the method for co-expressing recombinant bacterial strain conversion production α-ketoglutaric acid, it is special
Sign is：The step（2）Middle expression condition is：By the recombinant plasmid transformed large intestine bar containing L-GLOD gene
Bacterium, 37 DEG C are activated overnight, and LB culture mediums are put in the switching of 2% inoculum concentration, cultivate OD600For 0.6 ~ 0.8, add 0.4 mM IPTG, 25 ~
30 DEG C of 6 ~ 8 h of induction；Thalline is collected, with 20 mM pH8.0 disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution (500 mM
NaCl thalline) is resuspended, carries out ultrasonication；4 DEG C, 12000g centrifuging and taking supernatants, obtain crude enzyme liquid；Crude enzyme liquid is subjected to Ni posts
Affinitive layer purification, obtain the L-GLOD of purifying.
4. according to claim 1 a kind of using the method for co-expressing recombinant bacterial strain conversion production α-ketoglutaric acid, it is special
Sign is：The step（3）The L-GLOD of the purifying of middle acquisition respectively in different temperatures gradient, different pH gradients and
Zymologic property measure is carried out under different concentration of substrate gradient conditions；The L-GLOD molecular weight of albumen is about 66 after measured
KDa, optimal reactive temperature are 35 DEG C, and optimal reaction pH is 6.0, VmaxFor 100 ~ 150 U/mg, KmFor 8 ~ 10 mM.
5. according to claim 1 a kind of using the method for co-expressing recombinant bacterial strain conversion production α-ketoglutaric acid, it is special
Sign is：The step（4）The middle original gene sequence by L-GLOD enters according to e. coli codon Preference
Row codon optimization；L-GLOD original gene sequence and L-GLOD gene order point after codon optimization
Not as shown in SEQ ID NO.1 and SEQ ID NO.2.
6. according to claim 1 a kind of using the method for co-expressing recombinant bacterial strain conversion production α-ketoglutaric acid, it is special
Sign is：The step（5）Middle as expression original paper is L-GLOD gene is total to catalase gene KateE
The recombinant bacterial strain of expression.
7. a kind of method that production α-ketoglutaric acid is converted using recombination bacillus coli according to claim 1, its feature
It is：The step（6）Middle resting cell system is：Pidolidone（Salt）100 g/L, the g/L of whole-cell catalyst 30,
Reaction is carried out in pH 7.0 200 mM disodium hydrogen phosphates-citrate buffer solution, 35 DEG C, and 200 rpm convert 9 h.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486173A (en) * | 2018-03-27 | 2018-09-04 | 河南巨龙生物工程股份有限公司 | A kind of preparation method of α-ketoglutaric acid |
| CN108949654A (en) * | 2018-04-19 | 2018-12-07 | 江南大学 | A kind of engineering bacteria and its application in production α-ketoglutaric acid |
| CN109679979A (en) * | 2019-01-11 | 2019-04-26 | 四川吉晟生物医药有限公司 | The production method of the recombinant vector of expression L-GLOD and catalase, engineering bacteria and application and α-ketoglutaric acid |
| CN111261220A (en) * | 2018-11-30 | 2020-06-09 | 中国科学院天津工业生物技术研究所 | Implementation method for efficiently screening, designing and modifying electronic carrier |
| CN112625993A (en) * | 2021-01-05 | 2021-04-09 | 雅本化学股份有限公司 | Preparation of alpha-ketoglutaric acid by microbial conversion method |
| CN113817761A (en) * | 2020-06-19 | 2021-12-21 | 中国科学院微生物研究所 | Escherichia coli chassis bacteria without tricarboxylic acid cycle and construction method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001079503A1 (en) * | 2000-04-19 | 2001-10-25 | Yamasa Corporation | L-glutamate oxidase |
| CN104152498A (en) * | 2014-07-31 | 2014-11-19 | 洛阳华荣生物技术有限公司 | Method for producing alpha-ketoglutaric acid by virtue of enzymic method |
| CN105331642A (en) * | 2015-11-30 | 2016-02-17 | 浙江汇宁生物科技有限公司 | Method for producing alpha-oxoglutarate under catalysis of L-glutamate oxidase |
| CN105821066A (en) * | 2016-05-26 | 2016-08-03 | 江南大学 | Dual-enzyme co-expression strain for producing alpha-oxoglutarate |
-
2016
- 2016-08-04 CN CN201610637736.9A patent/CN107686850B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001079503A1 (en) * | 2000-04-19 | 2001-10-25 | Yamasa Corporation | L-glutamate oxidase |
| CN104152498A (en) * | 2014-07-31 | 2014-11-19 | 洛阳华荣生物技术有限公司 | Method for producing alpha-ketoglutaric acid by virtue of enzymic method |
| CN105331642A (en) * | 2015-11-30 | 2016-02-17 | 浙江汇宁生物科技有限公司 | Method for producing alpha-oxoglutarate under catalysis of L-glutamate oxidase |
| CN105821066A (en) * | 2016-05-26 | 2016-08-03 | 江南大学 | Dual-enzyme co-expression strain for producing alpha-oxoglutarate |
Non-Patent Citations (1)
| Title |
|---|
| YANG H.L.ET AL.: "Full=L-glutamate oxidase", 《UNIPROT》 * |
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| CN108486173A (en) * | 2018-03-27 | 2018-09-04 | 河南巨龙生物工程股份有限公司 | A kind of preparation method of α-ketoglutaric acid |
| CN108486173B (en) * | 2018-03-27 | 2022-04-01 | 河南巨龙生物工程股份有限公司 | Preparation method of alpha-ketoglutaric acid |
| CN108949654A (en) * | 2018-04-19 | 2018-12-07 | 江南大学 | A kind of engineering bacteria and its application in production α-ketoglutaric acid |
| CN108949654B (en) * | 2018-04-19 | 2020-12-01 | 江南大学 | Engineering bacterium and application thereof in production of alpha-ketoglutaric acid |
| CN111261220A (en) * | 2018-11-30 | 2020-06-09 | 中国科学院天津工业生物技术研究所 | Implementation method for efficiently screening, designing and modifying electronic carrier |
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| CN109679979A (en) * | 2019-01-11 | 2019-04-26 | 四川吉晟生物医药有限公司 | The production method of the recombinant vector of expression L-GLOD and catalase, engineering bacteria and application and α-ketoglutaric acid |
| CN109679979B (en) * | 2019-01-11 | 2023-03-17 | 四川吉晟生物医药有限公司 | Recombinant vector for expressing L-glutamate oxidase and catalase, engineering bacteria and application thereof, and production method of alpha-ketoglutaric acid |
| CN113817761A (en) * | 2020-06-19 | 2021-12-21 | 中国科学院微生物研究所 | Escherichia coli chassis bacteria without tricarboxylic acid cycle and construction method and application thereof |
| CN113817761B (en) * | 2020-06-19 | 2023-08-11 | 中国科学院微生物研究所 | Escherichia coli chassis bacteria without tricarboxylic acid cycle, construction method and application thereof |
| CN112625993A (en) * | 2021-01-05 | 2021-04-09 | 雅本化学股份有限公司 | Preparation of alpha-ketoglutaric acid by microbial conversion method |
| CN112625993B (en) * | 2021-01-05 | 2023-01-20 | 雅本化学股份有限公司 | Preparation of alpha-ketoglutaric acid by microbial conversion method |
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