CN108624576B - Mutant of L-amino acid deaminase and preparation method and application thereof - Google Patents
Mutant of L-amino acid deaminase and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an L-amino acid deaminase mutant and a preparation method and application thereof, belonging to the field of genetic engineering. The mutant T436A of the L-amino acid deaminase comprises a substitution of a corresponding amino acid residue site on L-amino acid deaminase Loops from Proteus mirabilis, the catalytic efficiency of the mutant T436A on L-leucine is improved by 72.7 percent compared with that of the original L-amino acid deaminase, and each liter of conversion solution contains 106.2g/L of alpha-ketoisocaproic acid. The molar conversion rate of the L-leucine reaches 97.7 percent.
Description
Technical Field
The invention relates to an L-amino acid deaminase mutant and a preparation method and application thereof, belonging to the field of genetic engineering.
Background
The L-amino acid deaminase is an oxidoreductase that catalyzes the production of an alpha-keto acid from an L-amino acid. There have been many studies and reports on the enzyme, and most of them are about the three-dimensional structure and substrate specificity of the enzyme. In recent years, the production of alpha-keto acids by biotransformation of the enzyme has attracted the attention of researchers, and has a wide application prospect. Because the enzyme is positioned in a cell membrane and is related to an electron transfer chain on the cell membrane, in the process of converting L-amino acid into alpha-keto acid, electrons are transferred to cytochrome oxidase through the electron transfer chain, so that molecular oxygen is reduced into water, hydrogen peroxide is not generated in the process, the conversion is kept to be continuously carried out under a mild environment, and the possibility of heterologously expressing amino acid deaminase to convert L-leucine into alpha-ketoisocaproic acid is provided.
However, the yield and the conversion rate of the alpha-ketoisocaproic acid produced by the enzyme conversion are low at present, and the requirement of industrial production cannot be met, so that the yield and the conversion rate of the alpha-ketoisocaproic acid need to be further improved. A method for optimizing and improving the yield of the alpha-ketoisocaproic acid by RBS (RBS) developed by related research teams can improve the yield of the alpha-ketoisocaproic acid, but the conversion rate is too low (Liulong, Chengkai, Guoguang, Lijiang Hua and Song Yang). Therefore, in order to improve the yield and conversion rate of the alpha-ketoisocaproic acid and enhance the industrial application value of the alpha-ketoisocaproic acid, the catalytic efficiency of the L-amino acid deaminase on the L-leucine is urgently needed to be improved.
Disclosure of Invention
The first technical problem to be solved by the invention is to provide an L-amino acid deaminase mutant, which contains an amino acid sequence shown as SEQ ID NO. 1.
It is a second object of the present invention to provide a gene encoding the mutant of the L-amino acid deaminase.
In one embodiment of the invention, the gene comprises the nucleotide sequence shown in SEQ ID NO. 2.
It is a third object of the present invention to provide a method for improving the catalytic efficiency of an L-amino acid deaminase for L-leucine, which comprises mutating the amino acid threonine Thr at position 436 of an L-amino acid deaminase derived from Proteus mirabilis (Proteus mirabilis) to alanine Ala.
In one embodiment of the present invention, the L-amino acid deaminase derived from Proteus mirabilis has Genbank accession AAA8675 comprising 474 amino acids.
The fourth purpose of the invention is to provide a method for constructing the mutant, which is to determine the mutation site through structural analysis on the basis of the proteus mirabilis (Proteusmirabilis) L-amino acid deaminase homologous modeling; designing a mutation primer of site-directed mutagenesis, constructing a mutant plasmid T436A-pET20b by using a pET20b vector carrying an L-amino acid deaminase gene as a template for site-directed mutagenesis, transforming Escherichia coli BL21(DE3) cells by using the mutated plasmid, and selecting and verifying to obtain a positive monoclonal T436A.
The fifth purpose of the invention is to provide a method for improving the yield of the alpha-ketoisocaproic acid, which uses the L-amino acid deaminase mutant strain as a whole-cell catalyst to convert a substrate containing L-leucine to obtain the alpha-ketoisocaproic acid.
In one embodiment of the invention, a system containing 20-25 g/L of whole cells and 100-120 g/L of substrate is reacted at 30-35 ℃ for 18-22 h to generate alpha-ketoisocaproic acid.
In one embodiment of the invention, the conversion is carried out in 20mM Tris buffer.
In one embodiment of the present invention, whole cells of the mutant strain are collected as follows: inoculating the seed culture solution into fermentation medium according to the inoculation amount of 2%, and culturing at 37 deg.C to OD6000.6-0.8, adding IPTG with a final concentration of 0.4mM, inducing at 25 deg.C for 12h, and collecting cells.
In one embodiment of the invention, the seed medium used to culture the strain contains per liter: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and distilled water for constant volume.
In one embodiment of the invention, the fermentation medium used for the fermentation contains per liter: peptone 12g, yeast extract 24g, glycerin 4mL, potassium dihydrogen phosphate 2.31g, dipotassium hydrogen phosphate 16.42 g.
The invention also claims the use of the mutants in fermentation production.
The invention has the beneficial effects that: the invention provides an L-amino acid deaminase mutant for efficiently catalyzing L-leucine to produce alpha-ketoisocaproic acid, and a preparation method and application thereof. The catalytic efficiency of the mutant T436A to L-leucine is improved by 72.7 percent compared with that of the original L-amino acid deaminase, the yield of alpha-ketoisocaproic acid reaches 106.2g/L, and the molar conversion rate of a substrate is 97.7 percent. Compared with a chemical method, the conversion method has the advantages of mild reaction conditions, single target product, easiness in separation, environmental friendliness and the like, and is simple in process, convenient to control and easy to popularize and apply.
Drawings
FIG. 1 is a SDS-PAGE electrophoresis of L-amino acid deaminase mutant purification from Proteus mirabilis (Proteus mirabilis); lane 1: a standard molecular weight protein; lane 2: blank control; lane 3, T436A purified preparation.
FIG. 2 yield of alpha-ketoisocaproic acid catalyzed by Proteus mirabilis L-amino acid deaminase mutant. □: starting enzyme wt; ■: mutant T436A.
Detailed Description
LB culture medium: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and distilled water to a constant volume of 1L.
Fermentation medium: (TB Medium): peptone 12g, yeast extract 24g, glycerin 4mL, potassium dihydrogen phosphate 2.31g, dipotassium hydrogen phosphate 16.42g, distilled water to constant volume of 1L.
Sample preparation: 1mL of the transformed transformation solution was centrifuged at 12,000rpm for 5min, the supernatant was diluted and filtered through a 0.45 μm filter, and the filtrate was subjected to liquid chromatography.
Content determination of alpha-ketoisocaproic acid: a high performance liquid chromatograph (equipped with ultraviolet visible detector) is prepared by adopting a Berloe AminexHPX-87H (300 × 7.8mm, 9 μm) chromatographic column, filtering with a 0.22 μm filter membrane to obtain a mobile phase with concentration of 2.5mmol/L, ultrasonic degassing at flow rate of 0.6mL/min and column temperature of 40 deg.C, and detecting at ultraviolet detection wavelength of 205 nm.
Example 1 preparation of Proteus mirabilis (Proteus mirabilis) L-amino acid deaminase mutants
pET20b plasmid (abbreviated as pma-pET20b) connected with proteus mirabilis L-amino acid deaminase coding gene pma (shown in a gene sequence SEQ ID NO. 3) is used as a template, a mutation primer of T436A is used for obtaining a product with the size of about 1422bp through one-time PCR, the PCR product is converted into escherichia coli JM109 competent cells after being treated by Dpn I, and a transformant is selected for sequencing verification.
The mutation primers are shown below (boxes are mutation sites): T436A mutant primer pair:
the PCR systems are all as follows:
the PCR conditions were all as follows:
pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 3min and then entering the following cycle: denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 5s, and extension at 72 deg.C for 1min for 40 s; 34 cycles of treatment; extending for 10min at 72 ℃, and keeping the temperature at 4 ℃.
Sequencing results show that random mutation does not occur except the desired mutation site, so that the mutant plasmid T436A-pET20b is successfully constructed.
Example 2: a method for expressing and purifying Proteus mirabilis (Proteus mirabilis) L-amino acid deaminase mutant.
The mutant T436A-pET20b was transformed into E.coli BL21(DE3) cells, and transformants were selected for sequencing verification. And culturing the verified positive transformant in an LB culture medium at 37 ℃ overnight, inoculating the positive transformant into a TB fermentation culture medium at 37 ℃ for culturing for about 3h, inducing by using IPTG (isopropyl thiogalactoside) with the final concentration of 0.4mM, and cooling to 25 ℃ for culturing for 12 h.
Collecting fermentation liquor, centrifuging at 8000rpm for 20min at 4 deg.C, and collecting thallus. Sonicating the collected cells in a wash solution, wash solution components: 500mM NaCI, 20mM Tris pH8.0, 0.05% w/v Triton X-100, 20mM imidazole. Cell lysate 16,000 g is centrifuged for 30min, the supernatant is applied to a nickel chelating column pre-equilibrated with washing solution after passing through a 0.45 μm membrane, then the column is washed with 20 column volumes of washing solution to remove impurities, and then the target protein is eluted with 30mL of eluent, the eluent composition: 500mM NaCI, 20mM Tris pH8.0, 500mM imidazole. And then further purifying the eluted protein by using a desalting column, and then carrying out centrifugal concentration by using a 30KDa ultrafiltration tube to obtain the purified L-amino acid deaminase mutant. The purification result is shown in FIG. 1, the purified L-amino acid mutant reaches electrophoretic purity, and the apparent molecular weight is 52 KDa.
Example 3: catalytic efficiency of Proteus mirabilis (Proteus mirabilis) L-amino acid deaminase mutant on L-leucine
The catalytic reaction system is 20mL, comprises recombinant Escherichia coli whole cells with the final concentration of 20g/L, 115g/L substrate L-leucine, 20mM Tris buffer solution (pH 8.0), samples are taken for HPLC analysis after being incubated for 20h at 200rpm and 30 ℃. The catalytic efficiency is calculated by the amount of product formed. The results are shown in FIG. 2. The mutant T436A can reach 87.5g/L after catalyzing for 12h, the yield can reach 106.2g/L after catalyzing for 20h, and the conversion rate reaches 97.7%.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> mutant of L-amino acid deaminase, preparation method and application thereof
<160>5
<170>PatentIn version 3.3
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Gly Ala Leu Pro Lys Glu Ala Asp Val Val Ile Ile Gly Ala Gly Ile
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Gln Gly Ile Met Thr Ala Ile Asn Leu Ala Glu Arg Gly Met Ser Val
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Thr Ile Leu Glu Lys Gly Gln Ile Ala Gly Glu Gln Ser Gly Arg Ala
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Tyr Ser Gln Ile Ile Ser Tyr Gln Thr Ser Pro Glu Ile Phe Pro Leu
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His His Tyr Gly Lys Ile Leu Trp Arg Gly Met Asn Glu Lys Ile Gly
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Ala Asp Thr Ser Tyr Arg Thr Gln Gly Arg Val Glu Ala Leu Ala Asp
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Glu Lys Ala Leu Asp Lys Ala Gln Ala Trp Ile Lys Thr Ala Lys Glu
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Ala Ala Gly Phe Asp Thr Pro Leu Asn Thr Arg Ile Ile Lys Gly Glu
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Glu Leu Ser Asn Arg Leu Val Gly Ala Gln Thr Pro Trp Thr Val Ala
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Ala Phe Glu Glu Asp Ser Gly Ser Val Asp Pro Glu Thr Gly Thr Pro
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Ala Leu Ala Arg Tyr Ala Lys Gln Ile Gly Val Lys Ile Tyr Thr Asn
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Cys Ala Val Arg Gly Ile Glu Thr Ala Gly Gly Lys Ile Ser Asp Val
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Gly Ile Trp Ser Arg Leu Phe Met Gly Asn Met Gly Ile Asp Ile Pro
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ggtgccggta ttcaagggat catgaccgct attaaccttg ctgaacgtgg tatgagtgtc 240
actatcttag aaaagggtca gattgccggt gagcaatcag gccgtgcata cagccaaatt 300
attagttacc aaacatcgcc agaaatcttc ccattacacc attatgggaa aatattatgg 360
cgtggcatga atgagaaaat tggtgcggat accagttatc gtactcaagg tcgtgtagaa 420
gcgctggcag atgaaaaagc attagataaa gctcaagcgt ggatcaaaac agctaaagaa 480
gcggcaggtt ttgatacacc attaaatact cgcatcatta aaggtgaaga gctatcaaat 540
cgcttagtcg gtgctcaaac gccatggact gttgctgcat ttgaagaaga ttcaggctct 600
gttgatcctg aaacaggcac acctgcactc gctcgttatg ccaaacaaat cggtgtgaaa 660
atttatacca actgtgcagt aagaggtatt gaaactgcgg gtggtaaaat ctctgatgtg 720
gtgagtgaga aaggggcgat taaaacgtct caagttgtac tcgctggggg tatctggtcg 780
cgtttattta tgggcaatat gggtattgat atcccaacgc tcaatgtata tctatcacaa 840
caacgtgtct caggggttcc tggtgcacca cgtggtaatg tgcatttacc taatggtatt 900
catttccgcg aacaagcgga tggtacttat gccgttgcac cacgtatctt tacgagttca 960
atagtcaaag atagcttcct gctagggcct aaatttatgc acttattagg tggcggagag 1020
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gcggcaggtt ttgatacacc attaaatact cgcatcatta aaggtgaaga gctatcaaat 540
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gtgagtgaga aaggggcgat taaaacgtct caagttgtac tcgctggggg tatctggtcg 780
cgtttattta tgggcaatat gggtattgat atcccaacgc tcaatgtata tctatcacaa 840
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ttaccgttgg aattctctat tggtgaagat ctatttaatt catttaaaat gccgacctct 1080
tggaatttag atgaaaaaac accattcgaa caattccgag ttgccacggc aacacaaaat 1140
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tcagaagttg ttgaacgttg gggtgccgtt gtgagtccaa catttgatga attacctatc 1260
atttctgagg tcaaagaata cccaggctta gtgattaaca cggcaacagt gtggggtatg 1320
acagaaggcc cggcagcggg tgaagtgacc gctgatattg tcatgggcaa gaaacctgtt 1380
attgatccaa cgccgtttag tttggatcgt tttaagaagt aa 1422
<210>4
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<213> Artificial sequence
<400>4
gattaacacg gcagcagtgt ggggtatg 28
<210>5
<211>28
<212>DNA
<213> Artificial sequence
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cataccccac actgctgccg tgttaatc 28
Claims (10)
1. An L-amino acid deaminase mutant characterized in that the amino acid sequence is translated from a nucleotide as shown in SEQ ID No. 2.
2. A gene encoding a mutant of the L-amino acid deaminase of claim 1.
3. A method for improving the catalytic efficiency of L-leucine by L-amino acid deaminase, characterized in that the amino acid threonine at position 436 of the L-amino acid deaminase encoded by SEQ ID NO.3 derived from Proteus mirabilis is mutated to alanine.
4. A method for constructing the mutant of claim 1, wherein the mutation site is determined by structural analysis based on the L-amino acid deaminase homology modeling encoded by Proteus mirabilis SEQ ID NO. 3; designing a site-directed mutagenesis primer, constructing a mutagenesis plasmid T436A-pET20b by using a pET20b vector carrying an L-amino acid deaminase gene coded by SEQ ID NO.3 as a template through site-directed mutagenesis, transforming escherichia coli BL21(DE3) cells by the mutated plasmid, and selecting and verifying to obtain a positive monoclonal mutagenesis mutant T436A.
5. A genetically engineered bacterium expressing the L-amino acid deaminase mutant according to claim 1.
6. A method for producing alpha-ketoisocaproic acid, characterized in that the genetically engineered bacterium of claim 5 is used as a whole-cell catalyst to convert a substrate containing L-leucine to obtain alpha-ketoisocaproic acid.
7. The method of claim 6, wherein the reaction of a system containing 20-25 g/L whole cells and 100-120 g/LL-leucine at 30-35 ℃ for 18-22 h produces α -ketoisocaproic acid.
8. The method of claim 7, wherein the whole cells are harvested as follows: inoculating the seed solution of the genetically engineered bacterium of claim 5 into a fermentation medium according to an inoculation amount of 1-3%, and culturing at 35-37 ℃ to OD6000.6-0.8, adding IPTG with the final concentration of 0.4-0.6 mM, inducing for 10-12 h at 25-28 ℃, and collecting cells.
9. The method according to claim 8, wherein the fermentation medium used for the fermentation contains per liter: peptone 12g, yeast extract 24g, glycerin 4mL, potassium dihydrogen phosphate 2.31g, dipotassium hydrogen phosphate 16.42 g.
10. Use of a mutant of an L-amino acid deaminase according to claim 1 for the fermentative production of α -ketoisocaproic acid.
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| CN108841844B (en) * | 2018-06-26 | 2022-09-06 | 江南大学 | Method for efficiently producing phenylpyruvic acid |
| CN109097383A (en) * | 2018-08-10 | 2018-12-28 | 浙江正硕生物科技有限公司 | A kind of method of high flux screening l-amino acid deamination enzyme mutant recombinant bacterial strain |
| CN109897837A (en) * | 2019-03-21 | 2019-06-18 | 江南大学 | A kind of l-amino acid deaminase mutant and its application |
| CN110643585B (en) * | 2019-11-08 | 2021-09-03 | 江南大学 | Method for producing alpha-ketone-beta-methyl n-pentanoic acid by using amino acid deaminase |
| CN111269900B (en) * | 2020-03-17 | 2021-09-28 | 江南大学 | Preparation and application of L-amino acid deaminase mutant |
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| WO2002033110A3 (en) * | 2000-10-17 | 2003-02-13 | Pcbu Services Inc | PRODUCTION OF α-HYDROXY-CARBOXYLIC ACIDS USING A COUPLED ENZYME SYSTEM |
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