CN109136205B - L-amino acid deaminase mutant with improved heat resistance and preparation method thereof - Google Patents
L-amino acid deaminase mutant with improved heat resistance and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology, and relates to a method for improving the thermal stability of L-amino acid deaminase, which is to proteus mirabilis (A)Proteus mirabilis) An enzyme mutant obtained by site-directed mutagenesis of L-amino acid deaminase, wherein the site of mutation is Trp at position 120. The invention analyzes the space environment around the special amino acid sites in the structure and the interaction between the amino acids, such as hydrogen bonds, ionic bonds, Van der Waals and the like, combines the molecular biology technology to modify the special amino acid sites, obtains the L-amino acid deaminase mutant by rationally designing mutation sites and applying a site-directed mutation method based on the protein space structure information, can improve the probability of obtaining the forward mutated enzyme mutant, and simultaneously greatly reduces the workload of mutant screening. The invention improves the structural stability of the enzyme, and is further beneficial to prolonging the service life of the enzyme.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for improving the thermal stability of L-amino acid deaminase.
Background
L-amino acid deaminase exists in fungi, actinomycetes and bacteria, is a FAD dependent flavoenzyme, and directly oxidizes L-amino acid to generate corresponding keto acid and ammonia under aerobic conditions. Is widely used in the fields of daily chemicals, medicine synthesis, food processing and the like. The corresponding keto acid is catalytically synthesized by the L-amino acid deaminase, so that the oxidation process can be efficiently synthesized under relatively mild conditions, the high-temperature, high-pressure and strong-acid catalysis process is avoided, and the efficient expression can be realized under the condition of not using or using few inducers. The whole process is efficient, and the product quality is stable. More importantly, the ketonic acid product produced by the enzyme method does not contain toxic and harmful strong acid, heavy metal and other components, has good biological safety and can be used in the fields with high added values, such as cosmetics, medicines and the like. However, the prior L-amino acid deaminase has low enzyme specific activity, poor thermal stability and short enzyme service cycle, and thus the industrial application of the L-amino acid deaminase is influenced.
Disclosure of Invention
The invention aims to overcome the defects of low specific activity, poor thermal stability and short enzyme using period of the existing L-amino acid deaminase and provide an L-amino acid deaminase mutant with improved heat resistance.
The invention adopts the following technical scheme: an L-amino acid deaminase mutant with improved heat resistance is prepared from proteus mirabilisProteus mirabilis) An enzyme mutant obtained by site-directed mutagenesis of L-amino acid deaminase, wherein the site of mutation is Trp at position 120.
The invention reasonably selects mutation sites, namely proteus mirabilis (A.mirabilis)Proteus mirabilis) An L-amino acid deaminase (LAAD) gene (genbank accession No. EU 669819.1) whose protein structure (PDB: 5 FJM), and a lipase mutant obtained by site-directed mutagenesis. The amino acid sequence of the original L-amino acid deaminase LAAD is SEQ ID NO: 1, the mutant amino acid in the L-amino acid deaminase mutant is expressed by using 'original amino acid-position-substituted amino acid'.
Preferably, Trp at position 120 is mutated to Ala or Leu.
Preferably, the DNA sequence of the mutant is SEQ NO.2 or SEQ NO. 3.
The invention also provides a preparation method of the mutant, which comprises the following steps:
(1) the whole gene synthesis is from proteus mirabilis (Proteus mirabilis) An L-amino acid deaminase (LAAD) gene (genbank accession number EU 669819.1),Ndei andHindIII ligation into pET24a, construction of plasmid LAAD, transformation intoE.coli BL21(DE3) to construct recombinant bacteria PMLAAD;
(2) introducing a target mutant amino acid site by using a PCR (polymerase chain reaction) method through overlap extension by using a plasmid LAAD (pET 24a as an expression vector for expressing an L-amino acid deaminase gene) as a template, carrying out segmented amplification on upstream and downstream fragments containing the mutant amino acid site, and splicing the gene fragments with the mutant site into a full-length gene fragment containing the mutant site;
(3) purifying the amplified product by DNA, digesting the purified DNA fragment by restriction enzyme Dpn I, purifying, and transforming to escherichia coli E.coli DH5 alpha competent cell to obtain mutant plasmid;
(4) electrically transferring the mutant plasmid to a host cell, coating the transformation liquid in an LB plate containing the kanamycin, and culturing for 16h at 37 ℃;
(5) and (2) selecting a single colony on the plate, placing the single colony in an LB culture medium, culturing the single colony in a shaking table at 37 ℃, culturing the single colony at 220rpm until OD600=0.5-1, adding IPTG (isopropyl-beta-thiogalactoside) until the final concentration is 0.1mM, cooling to 25 ℃, inducing for 20 hours, centrifugally collecting thalli, performing ultrasonic cell breaking, and performing enzyme activity determination to determine to obtain the L-amino acid deaminase mutant.
The primer sequences of the PCR are as follows:
the host cell is escherichia coli (e.
Through the implementation of the technical scheme, the invention has the following beneficial effects:
(1) the invention analyzes the space environment around the special amino acid sites in the structure and the interaction between the amino acids, such as hydrogen bonds, ionic bonds, Van der Waals and the like, combines the molecular biology technology to modify the special amino acid sites, obtains the L-amino acid deaminase mutant by rationally designing mutation sites and applying a site-directed mutation method based on the protein space structure information, can improve the probability of obtaining the forward mutated enzyme mutant, and simultaneously greatly reduces the workload of mutant screening. The invention improves the structural stability of the enzyme, and is further beneficial to prolonging the service life of the enzyme.
(2) The mutants obtained by the invention are Trp120Ala mutants and Trp120Leu mutants with improved thermal stability, and are more suitable for application of industrial biotransformation.
Detailed Description
Example 1:
the invention will be further illustrated with reference to the following specific examples.
The media formulations referred to in the examples are as follows:
LB liquid medium: yeast extract 0.5%, peptone 1%, NaCl 1%, pH7.0. Sterilizing at 121 deg.C for 20 min. When preparing the plate, 2% of agar is added, and the plate is sterilized at 121 ℃ for 20 min.
The final concentration was 50mg/L for kanamycin antibiotic selection.
The unit in the above medium is% (W/V).
Example 1:
construction of L-amino acid deaminase 120 amino acid site mutant
The whole gene synthesis is from proteus mirabilis (Proteus mirabilis) An L-amino acid deaminase (LAAD) gene (genbank accession number EU 669819.1),Ndei andHindIII ligation into pET24a, construction of plasmid LAAD, transformation intoE.coli BL21(DE3) to construct recombinant PMLAAD.
Extracting plasmid LAAD of recombinant bacteria PMLAAD, using the plasmid LAAD as a template, adopting a site-directed mutagenesis technology to construct mutants Trp120Ala and Trp120Leu, introducing a target mutation site by using an overlap extension PCR method, and carrying out site-directed mutagenesis reaction conditions:
primers used, see table below:
PCR amplification conditions: 94 ℃ for 2 min; at 94 ℃ for 20s, at 55 ℃ for 30s, at 68 ℃ for 6min, for 25 cycles; 10min at 68 ℃, 5min at 4 ℃ and end. The products of the overlap extension PCR amplification are digested by restriction enzyme DpnI, and are electrically transformed into E.coli DH5 alpha competent cells. Spread on LB plates (containing 50mg/L Kan). After 16h of growth, 10 single colonies on the plate were picked for colony PCR, and positive clones were picked for gene sequencing, indicating that the correct mutant plasmid was obtained.
2. L-amino acid deaminase and mutant recombinant strain thereof
Electrotransfer of the mutant plasmid toE.coliBL21(DE3) competent cells. Coating the transformation liquid on an LB (50 mg/L Kan) plate, culturing for 16h at 37 ℃, selecting a single colony on the plate to be placed in 100ml of LB culture medium, culturing at 37 ℃ and 220rpm on a shaking table until OD600=0.5-1, (OD 600=0.5 in the embodiment) is added with IPTG until the final concentration is 0.1mM, cooling to 25 ℃, inducing for 20 hours, centrifugally collecting thalli, performing ultrasonic cell breaking, and performing enzyme activity measurement to determine to obtain the L-amino acid deaminase recombinant strain.
The original strain collects thalli according to the same process and breaks the cells by ultrasound.
The amino acid sequence of the original L-amino acid deaminase LAAD is SEQ ID NO: 1 at the amino acid substitution position.
The DNA sequence of the mutant is SEQ NO.2 or SEQ NO. 3.
3. Determination of thermal stability of L-amino acid deaminase mutant
L-amino acid deaminase activity assay: the total reaction system is 200 mu l, the enzyme activity is expressed by relative enzyme activity, and the concentration of products detected by HPLC is used for expressing the magnitude of the enzyme activity.
Experimental groups: mu.l of 500mM pH6.5 phosphate buffer and then 50. mu.l of the cell-breaking enzyme solution (mutant strain) were added to each test tube, and the tubes were preheated for 30min in a 30 ℃ shaker. Then, 130. mu.l of L-Phe substrate solution (10 g/L, pH6.5,30 ℃ C. pre-heated for 30 min) was added thereto, the mixture was put on a 30 ℃ shaker and reacted at 250rpm for 30min, and after the reaction was completed, 1.8ml of an aqueous phosphoric acid solution (pH 3) was added thereto, and water bath treatment was carried out at 100 ℃ for 3 min. Centrifuging to take the supernatant, and detecting the concentration of the product ketophenylalanine by HPLC.
Control group: mu.l of 500mM phosphate buffer pH6.5 and then 50. mu.l of the cell-breaking enzyme solution (original strain) were added to the tube and preheated in a shaker at 30 ℃ for 30 min. Then, 130. mu.l of L-Phe substrate solution (10 g/L, pH6.5,30 ℃ C. pre-heated for 30 min) was added thereto, the mixture was put on a 30 ℃ shaker and reacted at 250rpm for 30min, and after the reaction was completed, 1.8ml of an aqueous phosphoric acid solution (pH 3) was added thereto, and water bath treatment was carried out at 100 ℃ for 3 min. Centrifuging to take the supernatant, and detecting the concentration of the product ketophenylalanine by HPLC.
HPLC detection conditions:
mobile phase A: phosphate buffer (10 mmol/L potassium dihydrogen phosphate solution, pH adjusted to 3.5 with phosphoric acid);
mobile phase B: methanol;
and (3) detecting the column: c18, 250 × 4.6mm × 4.5 μm;
detection wavelength: 210 nm;
flow rate: 1 ml/min;
column temperature: 35 ℃;
sample introduction amount: 10 mu L of the solution;
gradient elution procedure:
determination of the remaining enzyme activity rate of L-amino acid deaminase:
and (3) incubating the ultrasonic cell-breaking enzyme solution at 60 ℃ for 1h, standing at 4 ℃ for 20min, detecting the residual activity of the enzyme, and comparing the relative activity and the residual activity percentage (%) of the enzyme with the initial activity (0 h) according to the difference of data of an experimental group and a control group. The test data are shown in the table below.
The results showed that the heat resistance of both Trp120 mutants was improved to different extents. Wherein, the Trp120Ala mutant is treated for 1h at 60 ℃, the enzyme activity residue is 75 percent, which is improved by 3.3 times compared with the wild L-amino acid deaminase, and the Trp120Ala mutant has better thermal stability. Meanwhile, the enzyme activity of the mutant Trp120Ala is slightly reduced, and the enzyme activity of the mutant Trp120Leu is improved.
Sequence listing
<110> Zhejiang Shungshu Biotech Co., Ltd
<120> L-amino acid deaminase mutant with improved heat resistance and preparation method thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 471
<212> PRT
<213> Proteus mirabilis (Proteus mirabilis)
<220>
<222> (1)..(471)
<223> amino acid sequence of original LAAD
<400> 1
Met Ala Ile Ser Arg Arg Lys Phe Ile Leu Gly Gly Thr Val Val Ala
1 5 10 15
Val Ala Ala Gly Ala Gly Val Leu Thr Pro Met Leu Thr Arg Glu Gly
20 25 30
Arg Phe Val Pro Gly Thr Pro Arg His Gly Phe Val Glu Gly Thr Gly
35 40 45
Gly Pro Leu Pro Lys Gln Asp Asp Val Val Val Ile Gly Ala Gly Ile
50 55 60
Leu Gly Ile Met Thr Ala Ile Asn Leu Ala Glu Arg Gly Leu Ser Val
65 70 75 80
Thr Ile Val Glu Lys Gly Asn Ile Ala Gly Glu Gln Ser Ser Arg Phe
85 90 95
Tyr Gly Gln Ala Ile Ser Tyr Lys Met Pro Asp Glu Thr Phe Leu Leu
100 105 110
His His Leu Gly Lys His Arg Trp Arg Glu Met Asn Ala Lys Val Gly
115 120 125
Ile Asp Thr Thr Tyr Arg Thr Gln Gly Arg Val Glu Val Pro Leu Asp
130 135 140
Glu Glu Asp Leu Glu Asn Val Arg Lys Trp Ile Asp Ala Lys Ser Lys
145 150 155 160
Asp Val Gly Ser Asp Ile Pro Phe Arg Thr Lys Met Ile Glu Gly Ala
165 170 175
Glu Leu Lys Gln Arg Leu Arg Gly Ala Thr Thr Asp Trp Lys Ile Ala
180 185 190
Gly Phe Glu Glu Asp Ser Gly Ser Phe Asp Pro Glu Val Ala Thr Phe
195 200 205
Val Met Ala Glu Tyr Ala Lys Lys Met Gly Ile Lys Ile Phe Thr Asn
210 215 220
Cys Ala Ala Arg Gly Leu Glu Thr Gln Ala Gly Val Ile Ser Asp Val
225 230 235 240
Val Thr Glu Lys Gly Pro Ile Lys Thr Ser Arg Val Val Val Ala Gly
245 250 255
Gly Val Gly Ser Arg Leu Phe Met Gln Asn Leu Asn Val Asp Val Pro
260 265 270
Thr Leu Pro Ala Tyr Gln Ser Gln Gln Leu Ile Ser Ala Ala Pro Asn
275 280 285
Ala Pro Gly Gly Asn Val Ala Leu Pro Gly Gly Ile Phe Phe Arg Asp
290 295 300
Gln Ala Asp Gly Thr Tyr Ala Thr Ser Pro Arg Val Ile Val Ala Pro
305 310 315 320
Val Val Lys Glu Ser Phe Thr Tyr Gly Tyr Lys Tyr Leu Pro Leu Leu
325 330 335
Ala Leu Pro Asp Phe Pro Val His Ile Ser Leu Asn Glu Gln Leu Ile
340 345 350
Asn Ser Phe Met Gln Ser Thr His Trp Asp Leu Asn Glu Glu Ser Pro
355 360 365
Phe Glu Lys Tyr Arg Asp Met Thr Ala Leu Pro Asp Leu Pro Glu Leu
370 375 380
Asn Ala Ser Leu Glu Lys Leu Lys Lys Glu Phe Pro Ala Phe Lys Glu
385 390 395 400
Ser Thr Leu Ile Asp Gln Trp Ser Gly Ala Met Ala Ile Ala Pro Asp
405 410 415
Glu Asn Pro Ile Ile Ser Asp Val Lys Glu Tyr Pro Gly Leu Val Ile
420 425 430
Asn Thr Ala Thr Gly Trp Gly Met Thr Glu Ser Pro Val Ser Ala Glu
435 440 445
Ile Thr Ala Asp Leu Leu Leu Gly Lys Lys Pro Val Leu Asp Ala Lys
450 455 460
Pro Phe Ser Leu Tyr Arg Phe
465 470
<210> 2
<211> 1416
<212> DNA
<213> Artificial sequence (Artificial sequence)
<220>
<222> (1)..(1416)
<223> Trp120Ala DNA sequence of mutant strain
<400> 2
atggcaataa gtagaagaaa atttattctt ggtggcacag tggttgctgt tgctgcaggc 60
gctggggttt taacacctat gttaacgcga gaagggcgtt ttgttcctgg tacgccgaga 120
catggttttg ttgagggaac tggcggtcca ttaccgaaac aagatgatgt tgttgtaatt 180
ggtgcgggta ttttaggtat catgaccgcg attaaccttg ctgagcgtgg cttatctgtc 240
acaatcgttg aaaaaggaaa tattgccggc gaacaatcat ctcgattcta tggtcaagct 300
attagctata aaatgccaga tgaaaccttc ttattacatc acctcgggaa gcaccgcgcg 360
cgtgagatga acgctaaagt tggtattgat accacttatc gtacacaagg tcgtgtagaa 420
gttcctttag atgaagaaga tttagaaaac gtaagaaaat ggattgatgc taaaagcaaa 480
gatgttggct cagacattcc atttagaaca aaaatgattg aaggcgctga gttaaaacag 540
cgtttacgtg gcgctaccac tgattggaaa attgctggtt tcgaagaaga ctcaggaagc 600
ttcgatcctg aagttgcgac ttttgtgatg gcagaatatg ccaaaaaaat gggtatcaaa 660
attttcacaa actgtgcagc ccgtggttta gaaacgcaag ctggtgttat ttctgatgtt 720
gtaacagaaa aaggaccaat taaaacctct cgtgttgttg tcgccggtgg tgttgggtca 780
cgtttattta tgcagaacct aaatgttgat gtaccaacat tacctgctta tcaatcacag 840
caattaatta gcgcagcacc aaatgcgcca ggtggaaacg ttgctttacc cggcggaatt 900
ttctttcgtg atcaagcgga tggaacgtat gcaacttctc ctcgtgtcat tgttgctccg 960
gtagtaaaag aatcatttac ttacggctat aaatatttac ctctgctggc tttacctgat 1020
ttcccagtac atatttcgtt aaatgagcag ttgattaatt cctttatgca atcaacacat 1080
tgggatctta atgaagagtc gccatttgaa aaatatcgtg atatgaccgc tctgcctgat 1140
ctgccagaat taaatgcctc actggaaaaa ctgaaaaaag agttcccagc atttaaagaa 1200
tcaacgttaa ttgatcagtg gagtggtgcg atggcgattg caccagatga aaacccaatt 1260
atctctgatg ttaaagagta tccaggtcta gttattaata ctgcaacagg ttggggaatg 1320
actgaaagcc ctgtatcagc agaaattaca gcagatttat tattaggcaa aaaaccagta 1380
ttagatgcca aaccatttag tctgtatcgt ttctaa 1416
<210> 3
<211> 1416
<212> DNA
<213> Artificial sequence (Artificial sequence)
<220>
<222> (1)..(1416)
<223> mutant Trp120Leu DNA sequence
<400> 3
atggcaataa gtagaagaaa atttattctt ggtggcacag tggttgctgt tgctgcaggc 60
gctggggttt taacacctat gttaacgcga gaagggcgtt ttgttcctgg tacgccgaga 120
catggttttg ttgagggaac tggcggtcca ttaccgaaac aagatgatgt tgttgtaatt 180
ggtgcgggta ttttaggtat catgaccgcg attaaccttg ctgagcgtgg cttatctgtc 240
acaatcgttg aaaaaggaaa tattgccggc gaacaatcat ctcgattcta tggtcaagct 300
attagctata aaatgccaga tgaaaccttc ttattacatc acctcgggaa gcaccgcctg 360
cgtgagatga acgctaaagt tggtattgat accacttatc gtacacaagg tcgtgtagaa 420
gttcctttag atgaagaaga tttagaaaac gtaagaaaat ggattgatgc taaaagcaaa 480
gatgttggct cagacattcc atttagaaca aaaatgattg aaggcgctga gttaaaacag 540
cgtttacgtg gcgctaccac tgattggaaa attgctggtt tcgaagaaga ctcaggaagc 600
ttcgatcctg aagttgcgac ttttgtgatg gcagaatatg ccaaaaaaat gggtatcaaa 660
attttcacaa actgtgcagc ccgtggttta gaaacgcaag ctggtgttat ttctgatgtt 720
gtaacagaaa aaggaccaat taaaacctct cgtgttgttg tcgccggtgg tgttgggtca 780
cgtttattta tgcagaacct aaatgttgat gtaccaacat tacctgctta tcaatcacag 840
caattaatta gcgcagcacc aaatgcgcca ggtggaaacg ttgctttacc cggcggaatt 900
ttctttcgtg atcaagcgga tggaacgtat gcaacttctc ctcgtgtcat tgttgctccg 960
gtagtaaaag aatcatttac ttacggctat aaatatttac ctctgctggc tttacctgat 1020
ttcccagtac atatttcgtt aaatgagcag ttgattaatt cctttatgca atcaacacat 1080
tgggatctta atgaagagtc gccatttgaa aaatatcgtg atatgaccgc tctgcctgat 1140
ctgccagaat taaatgcctc actggaaaaa ctgaaaaaag agttcccagc atttaaagaa 1200
tcaacgttaa ttgatcagtg gagtggtgcg atggcgattg caccagatga aaacccaatt 1260
atctctgatg ttaaagagta tccaggtcta gttattaata ctgcaacagg ttggggaatg 1320
actgaaagcc ctgtatcagc agaaattaca gcagatttat tattaggcaa aaaaccagta 1380
ttagatgcca aaccatttag tctgtatcgt ttctaa 1416
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<220>
<222> (1)..(23)
<223> Trp120Ala For
<400> 5
gaagcaccgc gcgcgtgaga tga 23
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<220>
<222> (1)..(23)
<223> Trp120Ala Rev
<400> 5
cttcgtggcg cgcgcactct act 23
<210> 6
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<220>
<222> (1)..(23)
<223> Trp120Leu For
<400> 6
gaagcaccgc ctgcgtgaga tga 23
<210> 7
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<220>
<222> (1)..(23)
<223> Trp120Leu Rev
<400> 7
cttcgtggcg gacgcactct act 23
Claims (4)
1. An L-amino acid deaminase mutant with improved heat resistance, which is an enzyme mutant obtained by site-directed mutagenesis of proteus mirabilis L-amino acid deaminase, wherein the proteus mirabilis L-amino acid deaminase LAAD amino acid sequence is SEQ ID NO: 1, wherein Trp with the mutation site of 120 is mutated into Ala or Leu; the DNA sequence of the mutant is shown as SEQ NO.2 or SEQ NO. 3.
2. The method for preparing an L-amino acid deaminase mutant with improved thermostability according to claim 1, comprising the steps of:
(1) the whole gene synthesis is derived from the proteus mirabilis L-amino acid deaminase LAAD gene,Ndei andHindIII, connecting pET24a, constructing a plasmid LAAD, transforming the plasmid LAAD to E.Coli BL21(DE3), and constructing a recombinant bacterium PMLAAD;
(2) introducing a target mutant amino acid site by using a PCR (polymerase chain reaction) method through overlap extension by taking a plasmid LAAD (amplified polymorphic DNA) as a template, amplifying upstream and downstream fragments containing the mutant amino acid site in a segmented manner, and splicing gene fragments with the mutant site into a full-length gene fragment containing the mutant site;
(3) after the full-length gene fragment containing the mutation site is purified by DNA, restriction endonuclease is addedDpnI, digesting the purified DNA fragment, purifying, and transforming into Escherichia coliE.coliDH5 alpha competent cell, get the mutant plasmid;
(4) electrically transferring the mutant plasmid to a host cell, coating the transformation liquid in an LB plate containing 50mg/L kanamycin, and culturing at 37 ℃ for 16 h;
(5) and (2) selecting a single colony on the plate, placing the single colony in an LB culture medium, culturing the single colony in a shaking table at 37 ℃ and 220rpm until OD600=0.5-1, adding IPTG (isopropyl-beta-thiogalactoside) until the final concentration is 0.1mM, cooling to 25 ℃, inducing for 20 hours, centrifugally collecting thalli, ultrasonically breaking the cells, and performing enzyme activity determination to determine to obtain the L-amino acid deaminase mutant.
3. The method according to claim 2, wherein the primer used in the overlap extension PCR is:
Trp120Ala For 5’- gaagcaccgcgcgcgtgagatga -3’
Trp120Ala Rev 5’- cttcgtggcgcgcgcactctact -3’
Trp120Leu For 5’- gaagcaccgcctgcgtgagatga -3’
Trp120Leu Rev 5’- cttcgtggcggacgcactctact -3’。
4. the method according to claim 2, wherein the PCR amplification conditions are: 94 ℃ for 2 min; at 94 ℃ for 20s, at 55 ℃ for 30s, at 68 ℃ for 6min, for 25 cycles; 10min at 68 ℃ and 5min at 4 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810905502.7A CN109136205B (en) | 2018-08-10 | 2018-08-10 | L-amino acid deaminase mutant with improved heat resistance and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810905502.7A CN109136205B (en) | 2018-08-10 | 2018-08-10 | L-amino acid deaminase mutant with improved heat resistance and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
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| CN103911400A (en) * | 2014-04-02 | 2014-07-09 | 江南大学 | Method for efficiently producing alpha-oxoglutarate by adopting whole-cell transformation |
| CN107904222A (en) * | 2017-11-30 | 2018-04-13 | 江南大学 | The L amino acid deaminase mutant and its construction method that a kind of heat endurance improves |
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| EP1326993A2 (en) * | 2000-10-17 | 2003-07-16 | PCBU Services, Inc. | PRODUCTION OF g(a)-HYDROXY-CARBOXYLIC ACIDS USING A COUPLED ENZYME SYSTEM |
| CN103911400A (en) * | 2014-04-02 | 2014-07-09 | 江南大学 | Method for efficiently producing alpha-oxoglutarate by adopting whole-cell transformation |
| CN107904222A (en) * | 2017-11-30 | 2018-04-13 | 江南大学 | The L amino acid deaminase mutant and its construction method that a kind of heat endurance improves |
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