CN107513525A - A kind of D mandelate dehydrogenases, gene, genetic engineering bacterium and its application - Google Patents
A kind of D mandelate dehydrogenases, gene, genetic engineering bacterium and its application Download PDFInfo
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Abstract
The invention discloses one kind to have such as SEQ ID NO：The D mandelate dehydrogenases of amino acid sequence shown in 1（LhDMDH）, encode the D mandelate dehydrogenases（LhDMDH）Gene（With such as SEQ ID NO：Nucleotide sequence shown in 2）, the application of genetic engineering bacterium comprising the gene and the genetic engineering bacterium in whole-cell catalytic synthesizes benzoylformic acid.The engineering bacteria fermentation production D mandelate dehydrogenases of the present invention（LhDMDH）Level be up to 233.1 U/mL zymotic fluids, expression is 10 times or so of probe, the genetic engineering bacterium can efficiently aoxidize D mandelic acids production benzoylformic acid, based on whole-cell catalytic without additionally adding coenzyme, pass through the genetic engineering bacterium of the present invention, the conversion ratio of R mandelic acids can reach more than 98%, and the purity of benzoylformic acid can reach more than 99%, have good industrialization prospect.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of new D- mandelate dehydrogenases, to encode the D- flat
The gene of peach acidohydrogenase, the genetic engineering bacterium comprising the gene and the genetic engineering bacterium synthesize benzene in whole-cell catalytic
Application in acetonic acid.
Background technology
Benzoylformic acid (Phenylglyoxylic acid, PGA) also known as benzoyl formic acid, belong to 2-ketoacid class compound,
It is a kind of important building block, a variety of important medicine intermediates can be synthesized by raw material of benzoylformic acid.Due to acetophenone
Sour easily oxidation, decarboxylation and decarbonylation, thus its synthesis is more difficult, the synthetic method reported mainly has the hydrolysis of benzoyl nitrile
Method, styrene oxidation method, almond acid oxidation, Fu Ke are acylated method and living things catalysis synthetic method, and wherein mandelate dehydrogenase is in benzene
Played an important role in the biosynthesis of acetonic acid.By the unremitting effort of numerous researchers, D- mandelate dehydrogenases
Catalytic activity obtained huge raising, but from meeting that industrial actual demand still has a certain distance.In addition, at present
The D- mandelate dehydrogenases reported also also lack the research of temperature stability, organic solvent tolerance etc..
Be richly stored with the novel enzyme resource not being exploited in nature, how to go to excavate these unknown enzymes and adds
With appropriate transformation to meet industrial demand, the concern of vast researcher is attracted gradually.It is existing so far
Individual microbial gene group information more than 2000 is measured and openly, and these data are still constantly increasing, what these became increasingly abundant
Genomic information provides abundant resource for the excavation of new enzyme.Genome digs ore deposit（Genome mining）This term by
For multiple fields, it is primarily referred to as developing genomic information to find some new process, target spot and products.Genome digs ore deposit skill
Art can realize the leap from genome database to true enzyme database, further enrich the enzyme money that can be utilized or transform
Source.In recent years, existing many reports that the new enzyme of ore deposit scientific discovery is successfully dug using genome.
The present invention digs ore deposit technology by genome, fromLactobacillus harbinensisGenome in excavate and arrive
One new D- mandelate dehydrogenase gene（LhDMDH genes）, by the use of Escherichia coli as host, successfully construct energy
The genetic engineering bacterium of enough high efficient expression restructuring D- mandelate dehydrogenases, and carried out whole-cell catalytic D- mandelic acids synthesis acetophenone
The research of acid.
The content of the invention
It is an object of the invention to excavate a kind of new D- mandelate dehydrogenase genes, the D- of the coded by said gene is obtained
Mandelate dehydrogenase, genetic engineering bacterium is built using the D- mandelate dehydrogenase genes, so as to obtain a kind of production process it is simple,
The method for the whole-cell catalytic D- mandelic acids synthesis benzoylformic acid that working condition is gentle, production efficiency is high.
To achieve the above object, inventor digs ore deposit technology by genome, with current research is relatively thorough, catalytic activity compared with
High Lactobacillus brevis（Lactobacillus brevis）The D- mandelate dehydrogenases in sourceLbDMDH protein sequence is probe
BLAST analyses are carried out, found out from the result of inquiry series of genes group information source and without expression identification, the 2- assumed
dehydropantoate 2-reductase.These sequences are carried out with structure and the analysis of chadogram, then from each branch
Choose 1 ~ 2 representational gene order, for the easy acquisition of bacterial strain target gene by being obtained by template PCR amplifications of genome
Target gene is obtained, full genome synthesis is carried out after the gene codon optimization for being not easy to obtain for bacterial strain, and is screened, fromLactobacillus harbinensisGenome in excavate to a new D- mandelate dehydrogenase gene
（LhDMDH genes）, and successfully constructed using the gene being capable of high efficient expression D- mandelate dehydrogenases（LhDMDH）Gene work
Journey bacterium, and the research of whole-cell catalytic D- mandelic acids synthesis benzoylformic acid has been carried out, so as to complete the present invention.
Therefore, in one aspect, the invention provides a kind of D- mandelate dehydrogenases（LhDMDH）, it is characterised in that institute
State D- mandelate dehydrogenases（LhDMDH）With such as SEQ ID NO：Amino acid sequence shown in 1.
SEQ ID NO：1
MKIAIAGAGAMGGRFGSMLSTTENPVTFIDLWAQHVQAINAHGLLVHTDHGDERVHVPAYLPQDVHGSFDLIV
LFTKAMGIAPMLTALKPVITPQTHILVLGNGIGNIETISRFVPKDQIIAGVTVWSAALNGPGEITMKGDGGVTLEAL
GSNEAPFFKQVVQVLQDAGLKPTASHDVLAAIWRKAALNSVLNTYCTILDCNIGEFGQLPDHDAMIDGVLSEFSQIA
AKQHVHFDAADTKQLIVAQFPDGKNGLHYPSMHQDMAKGRKTEIDFLNGYIAKVGAREHIATPINAMLTALVHSKEK
LNELQSSREKVATPV
Secondly, present invention also offers encode above-mentioned D- mandelate dehydrogenases（LhDMDH）Gene, it is characterised in that the base
Because having such as SEQ ID NO：Nucleotide sequence shown in 2.
SEQ ID NO：2
GGATCCATGAAAATTGCCATTGCAGGCGCCGGTGCCATGGGCGGCCGATTCGGCAGTATGTTGTCGACGACAG
AGAATCCCGTCACGTTTATTGATTTGTGGGCGCAGCACGTTCAGGCGATTAATGCCCACGGGTTGCTGGTTCATACG
GATCATGGCGATGAACGGGTCCATGTCCCCGCGTATCTGCCGCAGGATGTCCACGGCAGTTTCGACTTGATCGTGTT
ATTCACCAAAGCCATGGGCATTGCGCCGATGCTGACGGCGCTGAAGCCGGTCATCACGCCCCAGACGCATATTTTGG
TGCTGGGCAACGGGATCGGCAACATTGAGACGATCAGTCGCTTTGTACCTAAAGATCAAATTATCGCCGGAGTGACG
GTCTGGTCGGCCGCTTTGAACGGTCCTGGCGAAATCACCATGAAGGGTGACGGCGGCGTGACGCTGGAAGCCCTCGG
CAGCAATGAGGCCCCATTCTTCAAGCAAGTCGTTCAAGTTTTGCAGGATGCCGGGTTGAAGCCGACTGCCTCCCATG
ATGTGCTGGCCGCTATCTGGCGTAAAGCCGCATTGAACTCGGTATTGAATACGTATTGCACGATCCTGGACTGCAAT
ATCGGCGAATTCGGGCAATTGCCGGACCACGATGCCATGATCGACGGCGTTCTGAGCGAATTCAGCCAAATTGCTGC
CAAGCAGCACGTGCATTTCGACGCGGCCGACACGAAACAATTGATCGTGGCGCAATTCCCAGATGGCAAGAATGGGC
TGCATTATCCGTCCATGCACCAAGACATGGCCAAAGGCCGTAAGACAGAAATTGATTTCTTGAATGGCTATATTGCC
AAGGTTGGCGCGCGGGAGCATATTGCCACGCCAATCAACGCAATGCTGACCGCACTGGTGCACAGCAAAGAGAAGTT
GAACGAATTACAAAGCAGTCGGGAGAAAGTCGCGACACCGGTTCTCGAG
Again, present invention also offers a kind of above-mentioned D- mandelate dehydrogenases of heterogenous expression（LhDMDH）Genetic engineering bacterium, its
In, the genetic engineering bacterium includes such as SEQ ID NO：Nucleotide sequence shown in 2.
In addition, present invention also offers the construction method of said gene engineering bacteria, methods described comprises the following steps：
Step 1: structure mandelate dehydrogenase containing D-（LhDMDH）Gene recombinant plasmid：By D- mandelate dehydrogenase genes
（LhDMDH genes）It is connected on pET28a plasmids, obtains recombinant plasmid；
Step 2: recombinant plasmid transformed enters host cell：The recombinant plasmid is utilized heat-shock transformed to e. coli bl21 sense
In by state cell, 0.4 mL LB fluid nutrient mediums of addition are coated on containing 50 μ g/mL after 37 DEG C, 220 rpm are incubated 1 h
On the LB solid plates of kalamycin, 12 ~ 16 h of culture obtain monoclonal bacterium colony at 37 DEG C；
Step 3: the screening and identification of recombinant bacterium：Picking monoclonal bacterium colony contains the LB liquid of 50 μ g/mL kalamycins to 4 mL
In body culture medium, the overnight incubation under 37 DEG C, 220 rpm, extraction plasmid carries out double digestion identification using BamH I and Xho I,
Confirm to obtain target gene engineering bacteria.
Moreover, it relates to above-mentioned D- mandelate dehydrogenase genes engineering bacteria is in the biosynthesis of benzoylformic acid
Using and benzoylformic acid biological synthesis method, methods described comprises the following steps：
Above-mentioned D- mandelate dehydrogenase genes engineering bacteria single bacterium colony is seeded to the LB liquid that 4 mL contain 50 μ g/mL kalamycins
In body culture medium, the overnight incubation under 37 DEG C, 220 rpm；The product pipettor being incubated overnight is inoculated with by 2% inoculum concentration
Into 100 mL LB fluid nutrient mediums；Then, it is placed at 37 DEG C, 200 rpm, 2.5 h of culture, is eventually adding final concentration of 0.1
The mmol/L IPTG aqueous solution, it is placed at 16 DEG C, 220 rpm, 20 h of culture, obtains zymotic fluid；
D- mandelic acids are added in above-mentioned zymotic fluid, make final concentration of 1 ~ 100 mM of D- mandelic acids, maintenance reaction temperature is 4
~ 50 DEG C, 2 ~ 48 h are reacted, detect the growing amount of benzoylformic acid in course of reaction with HPLC methods, rate to be transformed reaches more than 90%
When, activated carbon is added, 10 ~ 15min is reacted at 20 ~ 50 DEG C, adsorpting pigment, filters and removes activated carbon and absorption on the activated carbon
Thalline, extracted 1 ~ 3 time with isometric ethyl acetate, rotation is isolated and purified after being evaporated with silicagel column, obtains chromatographically pure
Benzoylformic acid.
Beneficial effect is：The genetic engineering bacterium production D- mandelate dehydrogenases that the present invention is built（LhDMDH）Level it is high, D-
Mandelate dehydrogenase（LhDMDH）Expression be up to 233.1 U/mL zymotic fluids, expression is 10 times or so of probe.
The genetic engineering bacterium of the present invention can efficiently aoxidize D- mandelic acids production benzoylformic acid, based on whole-cell catalytic without additionally adding
Add coenzyme, by the genetic engineering bacterium of the present invention, the conversion ratio of D- mandelic acids can reach more than 98%, the purity of benzoylformic acid
More than 99% can be reached, there is good industrialization prospect.
Brief description of the drawings
Fig. 1 is the SDS-PAGE collection of illustrative plates of enzyme involved in the present invention.
Marked in figure：1st, BL21/pET28a, 2, BL21/ pET28a-LhThe thick enzymes of DMDH, 3, BL21/ pET28a-LhThe pure enzymes of DMDH.
Fig. 2 is that D- is flat during synthesizing benzoylformic acid using the genetic engineering bacterium whole-cell catalytic D- mandelic acids of the present invention
Peach acid（D-MA）In the conversion ratio variation diagram of different time points.
Fig. 3 is the HPLC detection collection of illustrative plates of benzoylformic acid.
Embodiment
According to following embodiments, the present invention may be better understood.However, it will be readily appreciated by those skilled in the art that
Content described by following examples is merely to illustrate the present invention, and is understood not to the limit to the scope of the present invention
System.
Embodiment 1：The gene excavating of D- mandelate dehydrogenase encoding genes.
To study the D- mandelate dehydrogenases in the Lactobacillus brevis source relatively more thorough, catalytic activity is higherLbDMDH albumen
Sequence is that probe carries out BLAST analyses, found out from the result of inquiry series of genes group information source and is reflected without expression
2-dehydropantoate 2-reductase that are fixed, assuming.These sequences are carried out with structure and the analysis of chadogram, then
From each branch choose 1 ~ 2 representational gene order, for the easy acquisition of bacterial strain target gene by using genome as
Template PCR amplifications obtain target gene, and full genome synthesis is carried out after the gene codon optimization for being not easy to obtain for bacterial strain,
Selected by comparative analysisLactobacillus harbinensisThe 2-dehydropantoate 2- of genomic source
Reductase carries out next step research as potential D- mandelate dehydrogenases.
WithE.coliK12 bacterial strains codon usage frequency is reference, using OPTIMIZER servers to potentialLhDMDH gene orders carry out codon optimization, after gene upstream and downstream adds BamH I and Xho I restriction enzyme sites respectively, commission
Suzhou Hong Xun bio tech ltd carries out the synthesis of full genome, and obtaining has such as SEQ ID NO：Nucleotides sequence shown in 2
The D- mandelate dehydrogenase encoding genes of row.
Embodiment 2：Produce D- mandelate dehydrogenases（LhDMDH）Genetic engineering bacterium structure.
Step 1: construction recombination plasmid：D- mandelate dehydrogenase encoding genes in embodiment 1 are connected to pET28a matter
On grain, recombinant plasmid pET28a- is obtainedLbDMDH；
Step 2: recombinant plasmid transformed enters host cell：By recombinant plasmid pET28a-LbDMDH utilizes heat-shock transformed to large intestine bar
Bacterium BL21（Purchased from Invitrogen companies）In competent cell, 0.4 mL LB fluid nutrient mediums are added, in 37 DEG C, 220 rpm
After 1 h of lower incubation, it is coated on the LB solid plates containing 50 μ g/mL kalamycins, 12 ~ 16 h is cultivated at 37 DEG C, are obtained
Monoclonal bacterium colony；
Step 3: the screening and identification of recombinant bacterium：Picking monoclonal bacterium colony contains the LB liquid of 50 μ g/mL kalamycins to 4 mL
In body culture medium, the overnight incubation under 37 DEG C, 220 rpm, extraction plasmid utilizesBamH I andXhoI carries out double digestion identification,
Judge that it is positive colony to contain with target gene genetic fragment of the same size, the Preliminary Identification clone according to electrophoresis result；So
Positive colony is delivered into Shanghai Sangon Biological Engineering Technology And Service Co., Ltd afterwards and carries out sequencing, the further table of sequencing result
The bright colonies are target gene engineering bacteria.
Embodiment 3：D- mandelate dehydrogenases（LhDMDH）Induced expression and analysis.
The identified genetic engineering bacterium single bacterium colony for selecting the acquisition of embodiment 2 is seeded to 4 mL to contain 50 μ g/mL OK a karaoke clubs mould
In the LB fluid nutrient mediums of element, the overnight incubation under 37 DEG C, 220 rpm；The product pipettor that is incubated overnight is connect by 2%
Kind amount is inoculated into 100 mL LB fluid nutrient mediums；Then, it is placed at 37 DEG C, 200 rpm, 2.5 h of culture, is eventually adding end
Concentration is the 0.1 mmol/L IPTG aqueous solution, is placed at 16 DEG C, 220 rpm, 20 h of culture, obtains the bacterium solution through induced expression
（Zymotic fluid）.
The above-mentioned bacterium solution through induced expression is distributed into 50 mL centrifuge tube, 5 min are centrifuged in 8000 rpm, 4 DEG C,
Thalline is collected, respectively with the washing of 50 mL deionized waters twice, thalline is collected under similarity condition.Above-mentioned every kind of thalline is split with 10 mL
Solve buffer solution（20 mmol/L Tris-HCl pH 7.9,500 mmol/L NaCl, 1.2114 g Tris, 14.61 g
NaCl, add the mL of water 400, adjust pH to 7.9 with hydrochloric acid after dissolving, be settled to 500 mL）Suspend, 50% dutycycle, super 5s, stop
5s, the min of ice-water bath ultrasonication 20.12000 rpm, 4 DEG C of 15 min of centrifugation, collect supernatant, SDS-PAGE analysis purpose eggs
White expression form（As shown in Figure 1）.
It is as follows to lysate measure D- mandelate dehydrogenase activity, the method for enzyme activity determination：Reaction system is 1 mL, comprising
1 mM NAD+, 6 mM substrate D- mandelic acids, the 100 mM glycine-NaOH buffers of pH 9.5, after 30 DEG C are incubated 2 min
The enzyme liquid of 100 μ L dilution suitable multiples is added, determines the change of light absorption value under 340 nm in 1 min.Under this condition, every point
The enzyme amount that clock produces needed for 1 μm of ol NADH is defined as 1 U.The enzyme activity for measuring lysate is 2331 U/mL, is converted into zymotic fluid
Enzyme activity be 233.1 U/mL, expression is about 10 times of probe.
Enzyme activity：U = EW*V*1000/6220*L=EW/6.22
EW：A340 changing value, V under 1 min：The volume of reaction solution（mL）, 6220：Molar extinction coefficient（L*mol-1*cm-1）,
L：Optical path length（cm）.
D- mandelate dehydrogenases will be recombinated using nickel post and carry out affinity chromatography, and the ratio for then determining pure enzyme is lived, the ratio of pure enzyme
Work is 126 U/mg, is 4 times of probe.
Embodiment 4：The biosynthesis of benzoylformic acid.
D- mandelic acids are added in the engineering bacteria fermentation liquid that embodiment 3 obtains, make final concentration of the 1 of D- mandelic acids
~ 100 mM, maintenance reaction temperature are 4 ~ 50 DEG C, react 2 ~ 48 h, detect the generation of benzoylformic acid in course of reaction with HPLC methods
Amount, when rate to be transformed reaches more than 90% (as shown in Figure 2), adds activated carbon, and 10 ~ 15min absorption colors are reacted at 20 ~ 50 DEG C
Element, filter and remove the thalline of activated carbon and absorption on the activated carbon, extracted 1 ~ 3 time with isometric ethyl acetate, rotation is evaporated
Isolated and purified afterwards with silicagel column, obtain the benzoylformic acid (as shown in Figure 3) of chromatographically pure.
By the genetic engineering bacterium of the present invention, the conversion ratio of D- mandelic acids can reach more than 98%, the purity of benzoylformic acid
More than 99% can be reached, there is good industrialization prospect.
Sequence table
<110>Nanyang Normal College
<120>A kind of D- mandelate dehydrogenases, gene, genetic engineering bacterium and its application
<130> 2017
<160> 2
<170> SIPOSequenceListing 1.0
<210> 3
<211> 319
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 3
Met Lys Ile Ala Ile Ala Gly Ala Gly Ala Met Gly Gly Arg Phe Gly
1 5 10 15
Ser Met Leu Ser Thr Thr Glu Asn Pro Val Thr Phe Ile Asp Leu Trp
20 25 30
Ala Gln His Val Gln Ala Ile Asn Ala His Gly Leu Leu Val His Thr
35 40 45
Asp His Gly Asp Glu Arg Val His Val Pro Ala Tyr Leu Pro Gln Asp
50 55 60
Val His Gly Ser Phe Asp Leu Ile Val Leu Phe Thr Lys Ala Met Gly
65 70 75 80
Ile Ala Pro Met Leu Thr Ala Leu Lys Pro Val Ile Thr Pro Gln Thr
85 90 95
His Ile Leu Val Leu Gly Asn Gly Ile Gly Asn Ile Glu Thr Ile Ser
100 105 110
Arg Phe Val Pro Lys Asp Gln Ile Ile Ala Gly Val Thr Val Trp Ser
115 120 125
Ala Ala Leu Asn Gly Pro Gly Glu Ile Thr Met Lys Gly Asp Gly Gly
130 135 140
Val Thr Leu Glu Ala Leu Gly Ser Asn Glu Ala Pro Phe Phe Lys Gln
145 150 155 160
Val Val Gln Val Leu Gln Asp Ala Gly Leu Lys Pro Thr Ala Ser His
165 170 175
Asp Val Leu Ala Ala Ile Trp Arg Lys Ala Ala Leu Asn Ser Val Leu
180 185 190
Asn Thr Tyr Cys Thr Ile Leu Asp Cys Asn Ile Gly Glu Phe Gly Gln
195 200 205
Leu Pro Asp His Asp Ala Met Ile Asp Gly Val Leu Ser Glu Phe Ser
210 215 220
Gln Ile Ala Ala Lys Gln His Val His Phe Asp Ala Ala Asp Thr Lys
225 230 235 240
Gln Leu Ile Val Ala Gln Phe Pro Asp Gly Lys Asn Gly Leu His Tyr
245 250 255
Pro Ser Met His Gln Asp Met Ala Lys Gly Arg Lys Thr Glu Ile Asp
260 265 270
Phe Leu Asn Gly Tyr Ile Ala Lys Val Gly Ala Arg Glu His Ile Ala
275 280 285
Thr Pro Ile Asn Ala Met Leu Thr Ala Leu Val His Ser Lys Glu Lys
290 295 300
Leu Asn Glu Leu Gln Ser Ser Arg Glu Lys Val Ala Thr Pro Val
305 310 315
<210> 3
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<213>Artificial sequence (Artificial Sequence)
<400> 3
ggaccagaaa agccagcagg cgccgggcca gggcggccga cggcagaggc gacgacagag 60
aacccgcacg agaggggcgc agcacgcagg cgaaagccca cggggcggca acggacaggc 120
gagaacgggc cagccccgcg acgccgcagg agccacggca gcgacgacgg acaccaaagc 180
cagggcagcg ccgagcgacg gcgcgaagcc ggcacacgcc ccagacgcaa gggcgggcaa 240
cgggacggca acagagacga cagcgcgacc aaagacaaaa cgccggagga cggcggcggc 300
cgcgaacggc cggcgaaaca ccagaagggg acggcggcgg acgcggaagc cccggcagca 360
agaggcccca ccaagcaagc gcaaggcagg agccggggaa gccgacgccc ccagaggcgg 420
ccgcacggcg aaagccgcag aaccggagaa acgagcacga ccggacgcaa acggcgaacg 480
ggcaagccgg accacgagcc agacgacggc gcgagcgaac agccaaagcg ccaagcagca 540
cggcacgacg cggccgacac gaaacaagac gggcgcaacc cagaggcaag aagggcgcaa 600
ccgccagcac caagacaggc caaaggccga agacagaaag acgaaggcaa gccaaggggc 660
gcgcgggagc aagccacgcc aacaacgcaa gcgaccgcac gggcacagca aagagaagga 720
acgaaacaaa gcagcgggag aaagcgcgac accggccgag 760
Claims (6)
1. a kind of D- mandelate dehydrogenases, the D- mandelate dehydrogenases have such as SEQ ID NO：Amino acid sequence shown in 1
Row.
2. encoding the gene of the D- mandelate dehydrogenases described in claim 1, the gene has such as SEQ ID NO：Shown in 2
Nucleotide sequence.
3. include the genetic engineering bacterium of gene as claimed in claim 2.
4. the construction method of the genetic engineering bacterium described in claim 3, methods described comprise the following steps：
Step 1: construction recombination plasmid, the gene described in claim 2 is connected on pET28a plasmids, recombinant plasmid is obtained；
Step 2: recombinant plasmid transformed enters host cell：The recombinant plasmid is utilized heat-shock transformed to e. coli bl21 sense
In by state cell, after 0.4 mL LB fluid nutrient mediums of addition are incubated 1 h under 37 DEG C, 220 rpm, it is coated on containing 50 μ g/
On the LB solid plates of mL kalamycins, 12 ~ 16 h are cultivated at 37 DEG C, obtain monoclonal bacterium colony；
Step 3: the screening and identification of recombinant bacterium：Monoclonal bacterium colony described in picking contains 50 μ g/mL kalamycins to 4 mL
In LB fluid nutrient mediums, the overnight incubation under 37 DEG C, 220 rpm, extraction plasmid carries out double digestion mirror using BamH I and Xho I
It is fixed, confirm to obtain the genetic engineering bacterium.
5. application of the genetic engineering bacterium in the biosynthesis of benzoylformic acid described in claim 3.
6. a kind of biological synthesis method of benzoylformic acid, methods described comprise the following steps：
Genetic engineering bacterium single bacterium colony described in claim 3 is seeded into the LB liquid that 4 mL contain 50 μ g/mL kalamycins to train
Support in base, the overnight incubation under 37 DEG C, 220 rpm；The product being incubated overnight is inoculated into 100 with pipettor by 2% inoculum concentration
In mL LB fluid nutrient mediums；Then, it is placed at 37 DEG C, 200 rpm, 2.5 h of culture, is eventually adding final concentration of 0.1 mmol/
The L IPTG aqueous solution, it is placed at 16 DEG C, 220 rpm, 20 h of culture, obtains zymotic fluid；
D- mandelic acids are added in the zymotic fluid, make final concentration of 1 ~ 100 mM of D- mandelic acids, maintenance reaction temperature is 4
~ 50 DEG C, 2 ~ 48 h are reacted, detect the growing amount of benzoylformic acid in course of reaction with HPLC methods, rate to be transformed reaches more than 90%
When, activated carbon is added, 10 ~ 15min adsorpting pigments are reacted at 20 ~ 50 DEG C, filters and removes activated carbon and absorption on the activated carbon
Thalline, extracted 1 ~ 3 time with isometric ethyl acetate, rotation is isolated and purified after being evaporated with silicagel column, obtains chromatographically pure
Benzoylformic acid.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710969932.0A CN107513525B (en) | 2017-10-18 | 2017-10-18 | A kind of D- mandelate dehydrogenase, gene, genetic engineering bacterium and its application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710969932.0A CN107513525B (en) | 2017-10-18 | 2017-10-18 | A kind of D- mandelate dehydrogenase, gene, genetic engineering bacterium and its application |
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| Publication Number | Publication Date |
|---|---|
| CN107513525A true CN107513525A (en) | 2017-12-26 |
| CN107513525B CN107513525B (en) | 2019-09-24 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108504642A (en) * | 2018-04-11 | 2018-09-07 | 南阳师范学院 | A kind of genetic engineering compound bacteria and its application in benzoylformic acid biosynthesis |
| CN110452920A (en) * | 2019-07-11 | 2019-11-15 | 南阳师范学院 | A kind of genetic engineering bacterium and with D, L- mandelic acid is the method that substrate prepares L- phenylglycine |
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| CN102660625A (en) * | 2012-04-23 | 2012-09-12 | 郑州市拓信生物研究所 | Preparation method of R-o-chloromandelic acid and alcohol ester thereof |
| CN103088076A (en) * | 2013-01-29 | 2013-05-08 | 南京理工大学 | Method for preparing benzoyl formic acid and R-mandelic acid by coupling reaction of S- mandelic acid dehydrogenase and laccase |
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| CN102660625A (en) * | 2012-04-23 | 2012-09-12 | 郑州市拓信生物研究所 | Preparation method of R-o-chloromandelic acid and alcohol ester thereof |
| CN103088076A (en) * | 2013-01-29 | 2013-05-08 | 南京理工大学 | Method for preparing benzoyl formic acid and R-mandelic acid by coupling reaction of S- mandelic acid dehydrogenase and laccase |
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| GENBANK: "2-dehydropantoate 2-reductase [Lactobacillus harbinensis]，NCBI Reference Sequence: WP_027828400.1", 《GENBANK》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108504642A (en) * | 2018-04-11 | 2018-09-07 | 南阳师范学院 | A kind of genetic engineering compound bacteria and its application in benzoylformic acid biosynthesis |
| CN110452920A (en) * | 2019-07-11 | 2019-11-15 | 南阳师范学院 | A kind of genetic engineering bacterium and with D, L- mandelic acid is the method that substrate prepares L- phenylglycine |
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| CN107513525B (en) | 2019-09-24 |
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