CN108070581A - L-Aspartic acid β-decarboxylation the enzyme mutant and its application that a kind of enzyme activity improves - Google Patents
L-Aspartic acid β-decarboxylation the enzyme mutant and its application that a kind of enzyme activity improves Download PDFInfo
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- CN108070581A CN108070581A CN201711354029.XA CN201711354029A CN108070581A CN 108070581 A CN108070581 A CN 108070581A CN 201711354029 A CN201711354029 A CN 201711354029A CN 108070581 A CN108070581 A CN 108070581A
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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
- C12N9/88—Lyases (4.)
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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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
Abstract
The L aspartic acids β decarboxylations enzyme mutant improved the invention discloses a kind of enzyme activity and its application, belong to genetic engineering field.The mutant of the present invention is on the basis of the amino acid shown in SEQIDN0.2, and the 484th leucine is mutated into methionine.Because recombinant vector L484M pET28aN ends latter section of redundant sequence of initiation codon of structure is deleted to obtain recombinant plasmid L484M pET28a M by expression inclusion body problem.For the mutant that the present invention obtains in expression in escherichia coli, purified obtained enzyme does zymologic property research, and opposite enzyme activity has been respectively increased 40%, 36%, 42% under pH4.5, pH5.0, pH6.5.L484M and unmutated enzyme carry out resting cell 1h under identical enzyme activity condition of different pH and yield, under the conditions of optimal pH 5.5, output increased 48.5% are measured by sampling.Present invention demonstrates that 484 amino acids residues have larger impact to the catalytic action of enzyme, certain basis is provided to the research of the catalytic mechanism of the enzyme, and improves the commercial application potentiality of the enzyme.
Description
Technical field
L-Aspartic acid β-decarboxylation the enzyme mutant improved the present invention relates to a kind of enzyme activity and its application, belong to genetic engineering
Technical field.
Background technology
L-Alanine is a kind of nonessential amino acid.With constantly being researched and developed to l-Alanine, food, medicine,
The application of the industries such as chemical synthesis constantly expands.L-Alanine is a kind of amino acid being present in numerous food product, is already functioned as
The additive of diet, such as preservative, flavoring, amino acid low wine and beverage etc..Application in medicine passes through hydrolysis
Amino acid transfusion is made in albumen, and l-Alanine is in treatment protein synthesis disorder, diabetes, acute and chronic kidney function as caused by hepatopathy
Energy failure and the life aspect to maintaining the nutrition of urgent patient, rescue patient play positive effect；L-Alanine can be with
Effectively mitigate damage of the alcohol to liver；L-Alanine or the main component of blood preseration agent etc..In chemical synthesis
Using such as synthesis anticarcinogen 4- hydroxyl salicylides alanine closes zinc, synthesis VB5 etc..
L-Alanine preparation experienced by proteolysis extraction method, fermentation method to enzyme process (immobilized cell reaction or dissociate it is whole
Body cell method) process.The method of present industrialized production is exactly enzymatic conversion method, i.e., by rich in L-Aspartic acid-β-decarboxylation
Obtained from microorganism (such as Pseudomonas dacunhae etc.) cell catalysis L-Aspartic acid of enzyme activity, and L- asparagus fern ammonia
It is sour then can be efficiently catalyzed by immobilization Escherichia Coli ammonium fumarate and be made.Wherein Japan uses immobilization more
Cell method, and China uses free whole cell method.
Immobilization method is that P.dacunhae is filled column with immobilization Escherichia coli and connected respectively with carrageeenen immobilized, so as to
Reach the continuous production from ammonium fumarate to l-Alanine.M.Furui et al. pressurized reactors of sealing, make CO2It is dissolved in
In mixed liquor, ensure that flat-pushing properties of flow and prevents cell to be lost in, during solving aspartic acid decarboxylic reaction well
CO2Release causes cell stream fluid of becoming estranged air-teturning mixed phenomenon problem occur, in order to prevent L- asparagus ferns ammonia so as to destroying immobilized cell
Immobilization inactivation, pH adjustment inactivation and the inhibition alanine racemase enzyme activity of acid-β-decarboxylase activity, will be whole before immobilization
Cell is handled with acetic acid, PLP solution (pH 4.75) and glutaraldehyde.
Free whole cell method is by P.dacunhae fermented and cultureds, using culture solution as enzyme source, directly adds solid L-
Aspartic acid realizes enzymatic conversion method.P.dacunhae cells are handled to improve by Goto.Markoto with surfactants such as EDTA
The vigor of enzyme；And inhibiting racemase can carry out by adjusting the pH value of enzymic catalytic reaction, general pH control 4.5~5.5 it
Between.The key technology of free whole cell method is the stability for how improving enzyme activity and enzyme, the generation for preventing racemase.
The method of Goto.Markoto preferably resolves these problems.
In conclusion current L-Aspartic acid β-decarboxylation Enzyme catalyzed synthesis l-Alanine defect, enzyme reaction pH scopes are relatively narrow,
Can cause pH adjustment inactivation, it is more stringent to process control requirements the problems such as.It is contemplated that L-Aspartic acid β-decarboxylase
It is mutated, widens its pH sphere of action, reduced enzymatic operation difficulty, improve its industrial application value and production efficiency.
The content of the invention
First purpose of the present invention is to provide a kind of enzyme activity and improves L-Aspartic acid β-decarboxylation enzyme mutant, the mutation
The amino acid sequence of body is as shown in SEQ ID NO.1.
In one embodiment of the invention, the mutant be in amino acid as shown in sequence SEQ ID NO.2
On the basis of amino acid, 484 amino acids are mutated into methionine by leucine.
Second object of the present invention is to provide the gene for encoding the mutant, and the nucleotide sequence of the gene is such as
Shown in SEQ ID NO.3.
Third object of the present invention is to provide the recombinant expression carrier containing said gene, and the recombinant expression carrier is cut
It is except latter section of redundant sequence of carrier N-terminal initiation codon and histidine-tagged in C-terminal addition 6.
Fourth object of the present invention is to provide a kind of gene work for expressing the L-Aspartic acid β-decarboxylation enzyme mutant
Journey bacterium.
In one embodiment of the invention, the genetic engineering bacterium is using Escherichia coli as host.
The 5th purpose of the present invention is to provide the preparation method of the genetic engineering bacterium, is shown in SEQ ID NO.2
On the basis of amino acid sequence, the 484th leucine is mutated into methionine, recombination is obtained, recombination is connected to
Expression vector obtains recombinant plasmid, and recombinant plasmid transformed obtains Recombinant organism into e. coli host bacteria.
In one embodiment of the invention, the expression vector is pET28a.
In one embodiment of the invention, the method is specifically：(1) with nucleotide sequence shown in SEQ ID NO.4
For template, with primer of the sequence as shown in SEQ ID NO.5, SEQ ID NO.6, PCR, 484 bit aminos encoded are carried out
Acid is mutated into the L484M mutant gene sequences of methionine by leucine；(2) recombination sequence for obtaining step (1),
It is connected in pET28a-M expression vectors, obtains recombinant plasmid L484M-pET28a-M, recombinant plasmid is transformed into Escherichia coli
In host, recombination bacillus coli genetic engineering bacterium is obtained.
The 6th purpose of the present invention is to provide the genetic engineering bacterium resting cell L-Aspartic acid production L- third
The method of propylhomoserin, which is characterized in that the method is under conditions of pH is 4-7, using the full cell of genetic engineering bacterium as catalysis
Agent converts production l-Alanine by substrate of L-Aspartic acid.
The 7th purpose of the present invention is to provide the L-Aspartic acid β-decarboxylation enzyme mutant in field of medicine and chemical technology
Using.
In one embodiment of the invention, the application is to be used to prepare synthetic pantothenic acid calcium, carnosine, Pamidronic Acid
Sodium, Beta-alanine metal complex or Balsalazide.
The present invention transforms L- asparagus ferns on the basis of natural L-Aspartic acid β-decarboxylase, by rite-directed mutagenesis biotechnology
Propylhomoserin β-decarboxylation enzyme molecular structure, mutant enzyme opposite enzyme activity in pH4.5, the buffer solution of pH5.0, pH6.5 are divided compared with protoenzyme
It is indescribably high by 40%, 36%, 42%.Present invention demonstrates that 484 amino acids residues have larger impact to the catalytic action of enzyme, to the enzyme
The research of catalytic mechanism provide certain basis, and improve the commercial application potentiality of the enzyme.
Specific embodiment
In order to be more clearly understood that the technology contents of the present invention, spy lifts following embodiment and is described in detail, and purpose is only
It is to be best understood from the protection domain that present disclosure is not intended to limit the present invention.
Enzyme activity defines：Under 45 DEG C of reaction conditions, 1 μm of required enzyme amount of ol l-Alanine of generation per minute is one
Enzyme-activity unit.
L-Aspartic acid β-decarboxylase enzyme activity assay method：Using L-Aspartic acid as substrate, measured and be catalyzed by HPLC methods
The amount of the l-Alanine generated in reaction.Enzyme activity determination system (1mL)：830 μ L 0.2M pH5.5 acetate buffer solutions, 1mM α -one
Glutaric acid, 0.1mM PLP, 40mM L-Aspartic acids solution (adjust pH to neutrality) with sodium hydroxide, the enzyme solution of debita spissitudo.
Reaction mixture is at 45 DEG C, and under the conditions of pH5.5, after reacting 30min, boiling water bath terminates reaction.Reaction mixture is in 14000g items
10min is centrifuged under part, supernatant is taken to dilute 10 times, by 0.2mm filter membranes, the sample handled well measures product the third ammonia of L- by HPLC
The concentration of acid.
Embodiment 1：The structure of the recombinant vector of β containing L-Aspartic acid-decarboxylation enzyme mutant
(1) acquisition of L484M mutant：It is arranged with nucleotides sequence shown in SEQ ID NO.4 as template, (sequence is such as by Fprimer
Shown in SEQ ID NO.5), Rprimer (sequence is as shown in SEQ ID NO.6) be primer, carry out PCR i.e. obtain SEQ ID
Recombination shown in NO.3.
(2) structure of plasmid pET28a-M：By latter section of redundancy of recombinant expression carrier L484M-pET28a initiation codons
Sequence is cut off, and new expression vector is denoted as L484M-pET28a-M.
(3) recombinant plasmid L484M-pET28a-Mization is gone into JM109 competent cells, conversion fluid coating is containing kanamycins
(50 μ g/L) LB tablets extract plasmid, send to Shanghai life work sequencing.
Embodiment 2：Produce L-Aspartic acid β-decarboxylase Escherichia coli structure
The recombinant plasmid L484M-pET28a-Mization that embodiment 1 obtains is gone into E.coli BL21 competent cells, specifically
Method is as follows：
(1) reagent needed for transformation experiment is as follows：
LB culture mediums：Every liter of culture medium is prepared, tryptone 10g, yeast extract should be added in 950ml deionized waters
5g, NaCl 10g, for shake container until dissolving, it is 1L to add in deionized water to total volume, is dispensed, high pressure sterilization 20min.
LB agar mediums：Fluid nutrient medium first is prepared by above-mentioned formula, agar 1%-2% is added in before high pressure sterilization.
100mmol/L CaCl2(sterilizing), 50% glycerine (sterilizing), 50EP pipes, 1.5mL EP pipes
(2) picking Escherichia coli single bacterium colony is inoculated in 10mL LB culture mediums, and 37 DEG C of shaken cultivations are stayed overnight.
(3) be seeded to by 1% inoculum concentration in 50mL LB culture mediums, 37 DEG C of 180r/min shaken cultivations 1.5-2h to some
Germ filament generates.
(4) by 100mmol/LCaCl2, 50% glycerine, 50EP pipes, 1.5mL EP, which run affairs, first to be put cooled on ice and is placed in surpassing
It sterilizes in net platform.
(5) bacterium solution is dispensed in super-clean bench into 2 50EP pipes, trim, in 5000r/min 5min..
(6) supernatant is removed in super-clean bench, with 0.1M CaCl2Suspension cell places 15min on ice, centrifuges (5000r/min
5min), then equally operate 1-2 times.Last time 0.1M CaCl2With 50% glycerine according to 2:1 ratio removes suspension cell, most
After dispense to 1.5mL EP and manage, every pipe 150-200 μ L.5 μ L recombinant plasmids L484M-pET28a-M are added in into a pipe competence,
Gently mixing by placing 45min, 42 DEG C of water-bath 90s on ice, places 5min, adds in 800 μ L LB, 37 DEG C, 180r/ on ice
Min cultivates 1.5-2h, and bacterium solution is taken to be coated with resistant panel.37 DEG C of culture 12h, the verification of picking positive transformant.Obtain recombinant bacterium
L484M-pET28a-M/BL21。
Embodiment 3：Recombinant bacterium L484M-pET28a-M/BL21L- aspartic acids β-decarboxylase high efficient expression and enzyme activity determination
The recombinant bacterium L484M-pET28a-M/BL21 that embodiment 2 is built and the control strain for expressing unmutated enzyme
Asd-pET28a-M/BL21 is inoculated in respectively in LB culture mediums of the l0mL containing kanamycins, and 37 DEG C of shaken cultivations are stayed overnight, and next day presses
1% inoculum concentration is transferred in LB culture mediums, and 37 DEG C of culture 1.5-2h add in 16 DEG C of IPTG, the 12h induction of final concentration 0.4mM
Expression.Cell after induction is in 4 DEG C, 10000r/min centrifugation 5min, buffer solution suspension cell ultrasonication, 4 DEG C, 10000r/
Min centrifuges 30min, and supernatant is intracellular crude enzyme liquid, for the measure of enzyme activity.
Embodiment 4：Mutant enzyme is compared with the zymologic property of unmutated enzyme
L-Aspartic acid β-decarboxylase L484M is obtained after crude enzyme liquid that embodiment 3 obtains is purified, analyzes its zymology
Matter, 6.5 times pH4.5, pH 5.0, pH opposite enzyme activity have been respectively increased 40%, 36%, 42%.During microbe conversion, companion
With CO2Generation, system pH can rise, so conversion process needs acid adding to adjust.Enzyme activity rise can in the range of the pH widened
The amount of acid reagent is effectively reduced, so as to cost-effective.
Embodiment 5：Resting cell compares under condition of different pH
L484M and unmutated enzyme carry out resting cell under identical enzyme activity condition of different pH, and pH is set as 4.5,5.0,
5.5,6.0,6.5,7.0, yield is measured by sampling in conversion 1h.The result shows that：Under the conditions of optimal pH 5.5, output increased 48.5%.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention
Enclosing be subject to what claims were defined.
Sequence table
<110>Southern Yangtze University
<120>L-Aspartic acid β-decarboxylation the enzyme mutant and its application that a kind of enzyme activity improves
<160> 6
<170> PatentIn version 3.3
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Met Ser Lys Asp Tyr Gln Ser Leu Ala Asn Leu Ser Pro Phe Glu Leu
1 5 10 15
Lys Asp Glu Leu Ile Lys Ile Ala Ser Gly Asp Gly Asn Arg Leu Met
20 25 30
Leu Asn Ala Gly Arg Gly Asn Pro Asn Phe Leu Ala Thr Thr Pro Arg
35 40 45
Arg Ala Phe Phe Arg Leu Gly Leu Phe Ala Ala Ala Glu Ser Glu Leu
50 55 60
Ser Tyr Ser Tyr Met Asn Thr Val Gly Val Gly Gly Leu Ala Lys Ile
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Glu Gly Ile Glu Gly Arg Phe Glu Arg Tyr Ile Ala Glu Asn Arg Asp
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Gln Glu Gly Val Arg Phe Leu Gly Lys Ser Leu Ser Tyr Val Arg Asp
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Gln Leu Gly Leu Asp Pro Ala Ala Phe Leu His Glu Met Val Asp Gly
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Leu Lys Asp Pro Ala Ile Lys Ile Phe Phe Cys Val Asn Pro Ser Asn
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Pro Pro Ser Val Lys Met Asp Glu Arg Ser Leu Glu Arg Val Arg Lys
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Met Ser Lys Asp Tyr Gln Ser Leu Ala Asn Leu Ser Pro Phe Glu Leu
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Ala Gly Asp Lys Val Ala Ile Gly Met Pro Val Phe Thr Pro Tyr Ile
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Glu Ile Pro Glu Leu Ala Gln Tyr Ala Leu Glu Glu Val Ala Ile Asn
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Ala Asp Pro Ala Leu Asn Trp Gln Tyr Pro Asp Ser Glu Leu Asp Lys
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Leu Lys Asp Pro Ala Ile Lys Ile Phe Phe Cys Val Asn Pro Ser Asn
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Pro Pro Ser Val Lys Met Asp Glu Arg Ser Leu Glu Arg Val Arg Lys
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Ile Val Ala Glu His Arg Pro Asp Leu Met Ile Leu Thr Asp Asp Val
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Tyr Gly Thr Phe Ala Asp Gly Phe Gln Ser Leu Phe Ala Ile Cys Pro
290 295 300
Ala Asn Thr Leu Leu Val Tyr Ser Phe Ser Lys Tyr Phe Gly Ala Thr
305 310 315 320
Gly Trp Arg Leu Gly Val Val Ala Ala His Lys Glu Asn Ile Phe Asp
325 330 335
Leu Ala Leu Gly Arg Leu Pro Glu Ser Glu Lys Thr Ala Leu Asp Asp
340 345 350
Arg Tyr Arg Ser Leu Leu Pro Asp Val Arg Ser Leu Lys Phe Leu Asp
355 360 365
Arg Leu Val Ala Asp Ser Arg Ala Val Ala Leu Asn His Thr Ala Gly
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Leu Ser Thr Pro Gln Gln Val Gln Met Thr Leu Phe Ser Leu Phe Ala
385 390 395 400
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Arg Arg Arg Glu Ala Thr Leu Tyr Arg Glu Leu Gly Thr Pro Pro Gln
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Arg Asp Glu Asn Ala Val Asp Tyr Tyr Thr Leu Ile Asp Leu Gln Asp
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Arg Ala Ser Leu Ala Asn Leu Asn Glu Tyr Glu Tyr Ala Ala Ile Gly
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<210> 3
<211> 1602
<212> DNA
<213>Artificial sequence
<400> 3
atgtctaaag actaccagtc tctggctaac ctgtctccgt tcgaactgaa agacgaactg 60
atcaaaatcg cttctggtga cggtaaccgt ctgatgctga acgctggtcg tggtaacccg 120
aacttcctgg ctaccacccc gcgtcgtgct ttcttccgtc tgggtctgtt cgctgctgct 180
gaatctgaac tgtcttactc ttacatgaac accgttggtg ttggtggtct ggctaaaatc 240
gaaggtatcg aaggtcgttt cgaacgttac atcgctgaaa accgtgacca ggaaggtgtt 300
cgtttcctgg gtaaatctct gtcttacgtt cgtgaccagc tgggtctgga cccggctgct 360
ttcctgcacg aaatggttga cggtatcctg ggttgcaact acccggttcc gccgcgtatg 420
ctgaacatct ctgaaaaaat cgttcgtcag tacatcatcc gtgaaatggg tgctgacgct 480
atcccgtctg aatctgttaa cctgttcgct gttgaaggtg gtaccgctgc tatggcttac 540
atcttcgaat ctatgaaagt taacggtctg ctgaaagctg gtgacaaagt tgctatcggt 600
atgccggttt tcaccccgta catcgaaatc ccggaactgg ctcagtacgc tctggaagaa 660
gttgctatca acgctgaccc ggctctgaac tggcagtacc cggactctga actggacaaa 720
ctgaaagacc cggctatcaa aatcttcttc tgcgttaacc cgtctaaccc gccgtctgtt 780
aaaatggacg aacgttctct ggaacgtgtt cgtaaaatcg ttgctgaaca ccgtccggac 840
ctgatgatcc tgaccgacga cgtttacggt accttcgctg acggtttcca gtctctgttc 900
gctatctgcc cggctaacac cctgctggtt tactctttct ctaaatactt cggtgctacc 960
ggttggcgtc tgggtgttgt tgctgctcac aaagaaaaca tcttcgacct ggctctgggt 1020
cgtctgccgg aatctgaaaa aaccgctctg gacgaccgtt accgttctct gctgccggac 1080
gttcgttctc tgaaattcct ggaccgtctg gttgctgact ctcgtgctgt tgctctgaac 1140
cacaccgctg gtctgtctac cccgcagcag gttcagatga ccctgttctc tctgttcgct 1200
ctgatggacg aatctgacca gtacaaacac accctgaaac agctgatccg tcgtcgtgaa 1260
gctaccctgt accgtgaact gggtaccccg ccgcagcgtg acgaaaacgc tgttgactac 1320
tacaccctga tcgacctgca ggacgttacc tctaaactgt acggtgaagc tttctctaaa 1380
tgggctgtta aacagtcttc taccggtgac atgctgttcc gtatcgctga cgaaaccggt 1440
atcgttctga tgccgggtcg tggtttcggt tctgaccgtc cgtctggtcg tgcttctctg 1500
gctaacctga acgaatacga atacgctgct atcggtcgtg ctctgcgtca gatggctgac 1560
gaactgtacg ctcagtacac ccagcagggt aacaaacgtt aa 1602
<210> 4
<211> 1602
<212> DNA
<213>Artificial sequence
<400> 4
atgtctaaag actaccagtc tctggctaac ctgtctccgt tcgaactgaa agacgaactg 60
atcaaaatcg cttctggtga cggtaaccgt ctgatgctga acgctggtcg tggtaacccg 120
aacttcctgg ctaccacccc gcgtcgtgct ttcttccgtc tgggtctgtt cgctgctgct 180
gaatctgaac tgtcttactc ttacatgaac accgttggtg ttggtggtct ggctaaaatc 240
gaaggtatcg aaggtcgttt cgaacgttac atcgctgaaa accgtgacca ggaaggtgtt 300
cgtttcctgg gtaaatctct gtcttacgtt cgtgaccagc tgggtctgga cccggctgct 360
ttcctgcacg aaatggttga cggtatcctg ggttgcaact acccggttcc gccgcgtatg 420
ctgaacatct ctgaaaaaat cgttcgtcag tacatcatcc gtgaaatggg tgctgacgct 480
atcccgtctg aatctgttaa cctgttcgct gttgaaggtg gtaccgctgc tatggcttac 540
atcttcgaat ctatgaaagt taacggtctg ctgaaagctg gtgacaaagt tgctatcggt 600
atgccggttt tcaccccgta catcgaaatc ccggaactgg ctcagtacgc tctggaagaa 660
gttgctatca acgctgaccc ggctctgaac tggcagtacc cggactctga actggacaaa 720
ctgaaagacc cggctatcaa aatcttcttc tgcgttaacc cgtctaaccc gccgtctgtt 780
aaaatggacg aacgttctct ggaacgtgtt cgtaaaatcg ttgctgaaca ccgtccggac 840
ctgatgatcc tgaccgacga cgtttacggt accttcgctg acggtttcca gtctctgttc 900
gctatctgcc cggctaacac cctgctggtt tactctttct ctaaatactt cggtgctacc 960
ggttggcgtc tgggtgttgt tgctgctcac aaagaaaaca tcttcgacct ggctctgggt 1020
cgtctgccgg aatctgaaaa aaccgctctg gacgaccgtt accgttctct gctgccggac 1080
gttcgttctc tgaaattcct ggaccgtctg gttgctgact ctcgtgctgt tgctctgaac 1140
cacaccgctg gtctgtctac cccgcagcag gttcagatga ccctgttctc tctgttcgct 1200
ctgatggacg aatctgacca gtacaaacac accctgaaac agctgatccg tcgtcgtgaa 1260
gctaccctgt accgtgaact gggtaccccg ccgcagcgtg acgaaaacgc tgttgactac 1320
tacaccctga tcgacctgca ggacgttacc tctaaactgt acggtgaagc tttctctaaa 1380
tgggctgtta aacagtcttc taccggtgac atgctgttcc gtatcgctga cgaaaccggt 1440
atcgttctgc tgccgggtcg tggtttcggt tctgaccgtc cgtctggtcg tgcttctctg 1500
gctaacctga acgaatacga atacgctgct atcggtcgtg ctctgcgtca gatggctgac 1560
gaactgtacg ctcagtacac ccagcagggt aacaaacgtt aa 1602
<210> 5
<211> 33
<212> DNA
<213>Artificial sequence
<400> 5
accggtatcg ttctgatgcc gggtcgtggt ttc 33
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence
<400> 6
gccttactgg ttagcagaat g 21
Claims (10)
- A kind of 1. L-Aspartic acid β-decarboxylation enzyme mutant, which is characterized in that the amino acid sequence of the mutant such as SEQ ID Shown in NO.1.
- 2. encode the gene of mutant described in claim 1.
- A kind of 3. recombinant expression carrier containing gene described in claim 2, which is characterized in that the recombinant expression carrier excision Carrier N-terminal initiation codon latter section of redundant sequence and C-terminal add 6 it is histidine-tagged.
- 4. a kind of genetic engineering bacterium for expressing L-Aspartic acid β-decarboxylation enzyme mutant described in claim 1.
- 5. genetic engineering bacterium according to claim 4, which is characterized in that the genetic engineering bacterium is using Escherichia coli as place It is main.
- A kind of 6. method for preparing genetic engineering bacterium described in claim 4, which is characterized in that be in ammonia shown in SEQ ID NO.2 On the basis of base acid sequence, the 484th leucine is mutated into methionine, recombination is obtained, recombination is connected to table Recombinant plasmid is obtained up to carrier, recombinant plasmid transformed obtains Recombinant organism into e. coli host bacteria.
- 7. preparation method according to claim 6, which is characterized in that the method is specifically：(1) with SEQ ID NO.4 Shown nucleotide sequence is template, with primer of the sequence as shown in SEQ ID NO.5, SEQ ID NO.6, carries out PCR, is encoded 484 amino acids the L484M mutant gene sequences of methionine are mutated by leucine；(2) weight for obtaining step (1) Group gene order, is connected in pET28a-M expression vectors, obtains recombinant plasmid L484M-pET28a-M, recombinant plasmidization conversion Into escherichia coli host, recombination bacillus coli genetic engineering bacterium is obtained.
- 8. a kind of side of genetic engineering bacterium resting cell L-Aspartic acid production l-Alanine using described in claim 4 Method, which is characterized in that the method is under conditions of pH is 4-7, using the full cell of genetic engineering bacterium as catalyst, with L- days Winter propylhomoserin is substrate conversion production l-Alanine.
- 9. L-Aspartic acid β-decarboxylation enzyme mutant is in the application of field of medicine and chemical technology described in claim 1.
- 10. application according to claim 9, which is characterized in that the application be used to prepare synthetic pantothenic acid calcium, carnosine, Pamidronate disodium, Beta-alanine metal complex or Balsalazide.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804602A (en) * | 2019-11-18 | 2020-02-18 | 江南大学 | L-aspartic acid β -decarboxylase mutant and application thereof |
| CN112175916A (en) * | 2020-09-10 | 2021-01-05 | 济宁医学院 | L-amino acid ligase mutant, recombinant vector, recombinant bacterium and application thereof |
| CN112941003A (en) * | 2021-04-19 | 2021-06-11 | 江南大学 | Method for synthesizing L-alanine by catalyzing maleic acid through double-enzyme coupling whole cells |
| CN113106082A (en) * | 2021-05-27 | 2021-07-13 | 云南师范大学 | Alanine racemase from animal manure metagenome as well as preparation and application thereof |
| CN114107270A (en) * | 2021-12-07 | 2022-03-01 | 江南大学 | L-aspartic acid beta-decarboxylase mutant |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105062997A (en) * | 2015-08-25 | 2015-11-18 | 江南大学 | L-asparaginase mutant with improved enzyme activity and construction method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105062997A (en) * | 2015-08-25 | 2015-11-18 | 江南大学 | L-asparaginase mutant with improved enzyme activity and construction method thereof |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804602A (en) * | 2019-11-18 | 2020-02-18 | 江南大学 | L-aspartic acid β -decarboxylase mutant and application thereof |
| CN110804602B (en) * | 2019-11-18 | 2021-01-29 | 江南大学 | L-aspartic acid beta-decarboxylase mutant and application thereof |
| CN112175916A (en) * | 2020-09-10 | 2021-01-05 | 济宁医学院 | L-amino acid ligase mutant, recombinant vector, recombinant bacterium and application thereof |
| CN112941003A (en) * | 2021-04-19 | 2021-06-11 | 江南大学 | Method for synthesizing L-alanine by catalyzing maleic acid through double-enzyme coupling whole cells |
| CN113106082A (en) * | 2021-05-27 | 2021-07-13 | 云南师范大学 | Alanine racemase from animal manure metagenome as well as preparation and application thereof |
| CN113106082B (en) * | 2021-05-27 | 2022-11-04 | 云南师范大学 | Animal waste metagenome-derived alanine racemase and preparation and application thereof |
| CN114107270A (en) * | 2021-12-07 | 2022-03-01 | 江南大学 | L-aspartic acid beta-decarboxylase mutant |
| CN114107270B (en) * | 2021-12-07 | 2023-07-18 | 江南大学 | L-aspartic acid beta-decarboxylase mutant |
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