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 PDF

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CN107513525A
CN107513525A CN201710969932.0A CN201710969932A CN107513525A CN 107513525 A CN107513525 A CN 107513525A CN 201710969932 A CN201710969932 A CN 201710969932A CN 107513525 A CN107513525 A CN 107513525A
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mandelate
genetic engineering
gene
dehydrogenases
engineering bacterium
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CN107513525B (en
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唐存多
史红玲
阚云超
姚伦广
和子涵
焦铸锦
史鸿飞
刘飞
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Nanyang Normal University
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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

A kind of D- mandelate dehydrogenases, gene, genetic engineering bacterium and its application
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
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<120>A kind of D- mandelate dehydrogenases, gene, genetic engineering bacterium and its application
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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
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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
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Val Val Gln Val Leu Gln Asp Ala Gly Leu Lys Pro Thr Ala Ser His
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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
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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
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Thr Pro Ile Asn Ala Met Leu Thr Ala Leu Val His Ser Lys Glu Lys
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ggaccagaaa agccagcagg cgccgggcca gggcggccga cggcagaggc gacgacagag 60
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gagaacgggc cagccccgcg acgccgcagg agccacggca gcgacgacgg acaccaaagc 180
cagggcagcg ccgagcgacg gcgcgaagcc ggcacacgcc ccagacgcaa gggcgggcaa 240
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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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Cited By (2)

* Cited by examiner, † Cited by third party
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

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GENBANK: "2-dehydropantoate 2-reductase [Lactobacillus harbinensis],NCBI Reference Sequence: WP_027828400.1", 《GENBANK》 *
唐存多等: "新型 R-扁桃酸脱氢酶的基因挖掘及表达鉴定", 《中国生物工程杂志》 *
曾贞等: "S-扁桃酸脱氢酶基因的克隆及表达", 《中国生物工程杂志》 *
范长伟: "D-扁桃酸脱氢酶的基因挖掘及在多酶偶联合成L-苯甘氨酸的应用", 《中国优秀硕士学位论文全文数据库 基础科学辑》 *

Cited By (2)

* Cited by examiner, † Cited by third party
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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