CN109897837A - A kind of l-amino acid deaminase mutant and its application - Google Patents
A kind of l-amino acid deaminase mutant and its application Download PDFInfo
- Publication number
- CN109897837A CN109897837A CN201910216813.7A CN201910216813A CN109897837A CN 109897837 A CN109897837 A CN 109897837A CN 201910216813 A CN201910216813 A CN 201910216813A CN 109897837 A CN109897837 A CN 109897837A
- Authority
- CN
- China
- Prior art keywords
- amino acid
- gly
- mutant
- ala
- val
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a kind of l-amino acid deaminase mutant and its applications, belong to genetic engineering field.On the basis of present invention l-amino acid deaminase amino acid sequence shown in SEQ ID NO.1, after being mutated to one or more in G206, P272, V276, V283, E340, a variety of l-amino acid deaminase mutants have been obtained；The yield that wherein mutant G206R/P272F/V276C/V283I/E340S (its amino acid sequence is as shown in SEQ ID NO.3) is catalyzed sodium glutamate production α-KG is up to 100.96gL‑1.L-amino acid deaminase mutant provided by the invention will promote the industrialized production of α-KG to a certain extent.
Description
Technical field
The present invention relates to a kind of l-amino acid deaminase mutant and its applications, belong to genetic engineering field.
Background technique
α-ketoglutaric acid (α-ketoglutarate, abbreviation α-KG) is widely used in food, medicine, chemical industry and cosmetics
In equal industrial circles.In field of medicaments, α-KG can mitigate the kidney burden of nephrotic, can also control the intracorporal blood lipid of people
It is horizontal；In field of food, α-KG energy active balance body nitrogen metabolism, while also increasing food nutrition as additive；Changing
Cosmetic field, α-KG, which can remove free radical in conjunction with intracellular excessive nitrogen, improves skin quality etc.；In industrial circle,
α-KG can be used for animal feed additive, promote animal skeleton growth, anti-stress ability etc..
L-amino acid deaminase (PM1) from proteus mirabilis (Proteus mirabilis) can be by glutamic acid
Sodium is converted into α-ketoglutaric acid (α-KG).Gazi Sakir Hossain et al. (Bioconversion of l-glutamic
acid to α-ketoglutaric acid by an immobilized whole-cell biocatalyst
Expressing l-amino acid deaminase from Proteus mirabilis) proteus mirabilis will be come from
The l-amino acid deaminase of (Proteus mirabilis) heterogenous expression in bacillus subtilis and Escherichia coli, last benefit
Pidolidone is biologically converted into α-ketoglutaric acid with immobilized whole-cell biocatalyst, using Pidolidone as substrate, from withered
α-KG generation the level separated in careless bacillus and Escherichia coli be 55.3 ± 1.73 and 21.7 ± 0.39g/mg protein/point
Clock, the maximum conversion rate that Pidolidone is converted into α-KG is 31% (w/w).
In order to improve substrate specificity and reduce product consumption, Gazi Sakir Hossain et al. (Improved
production of α-ketoglutaric acid(α-KG)by a Bacillus subtilis whole-cell
biocatalyst via engineering of l-amino acid deaminase and deletion of theα-KG
Utilization pathway) 3 wheel fallibilities are further carried out by the l-amino acid deaminase to proteus mirabilis source
The gene knockout of α-KG Consumption in PCR and bacillus subtilis, it is determined that 6 critical sites F110, A255, E349,
R228, T249, I352 have simultaneously carried out fixed point saturation mutation, wherein mutant F110I/A255T/E349D/R228C/T249S/
I352A makes the biological transformation ratio of Pidolidone production α-KG by 31.5% to 83.25%, α-KG output increased to 10.08g/
L.SucA gene is knocked out, yield is further increased to 12.21g/L.
Although utilizing the L- amino for deriving from proteus mirabilis (Proteus mirabilis) by the research of forefathers
The sour full cell production α-KG of deaminase has obtained remarkable progress, but to further increase α-KG yield, l-amino acid deaminase
There are still very big progress spaces for molecular modification.
Summary of the invention
In order to solve the above-mentioned technical problem, the present invention is residual by the partial amino-acid of molecular modification l-amino acid deaminase
Base, so that product α-ketoglutaric acid yield significantly improves.
The first purpose of the invention is to provide a kind of l-amino acid deaminase mutant, the mutant includes: in SEQ
On the basis of amino acid sequence shown in ID NO.1, to one or more position in G206, P272, V276, V283, E340
The amino acid sequence that point obtains after being mutated.
In one embodiment of the invention, the mutant is V276C/V283I/E340G, P272F/V283I/
E340S or P272F/V283I/E340G.
In one embodiment of the invention, the mutant is V276C/E340S, P272F/V276C/V283I/
E340S or G206R/P272F/V283I/E340G.
In one embodiment of the invention, the mutant is G206R/P272F/V276C/V283I/E340S,
Amino acid sequence is as shown in SEQ ID NO.3.
A second object of the present invention is to provide the genes for encoding the mutant.
Third object of the present invention is to provide the carriers or cell that carry the gene.
Fourth object of the present invention is to provide the genetic engineering bacterium for expressing the l-amino acid deaminase mutant.
In one embodiment of the invention, the genetic engineering bacterium is with Escherichia coli for host.
In one embodiment of the invention, the genetic engineering bacterium is using pET20b as carrier.
Fifth object of the present invention is to provide a kind of method for preparing α-ketoglutaric acid, the l-amino acid deaminase is prominent
Variant or full cell containing the l-amino acid deaminase mutant are catalyst, are substrate preparation α -one penta using sodium glutamate
Diacid.
In one embodiment of the invention, the whole-cell catalyst concentration is 20 ± 5g/L.
In one embodiment of the invention, the substrate glutamic acid na concn is 200 ± 10g/L.
In one embodiment of the invention, the condition of the conversion are as follows: 30 ± 5 DEG C of temperature, transformation time 60 ±
10h。
Beneficial effects of the present invention:
(1) having determined influences in the l-amino acid deaminase of proteus mirabilis (Proteus mirabilis)
Catalytic activity and Binding Capacity and coenzyme effect 5 key amino acids G206, P272, V276, V283, E340, and to its into
Row unit point and multidigit point combinatorial mutagenesis, have obtained the more plants of l-amino acid deaminase mutants that can make α-KG output increased；
(2) wherein mutant V276C/E340S, P272F/V276C/V283I/E340S, G206R/P272F/V283I/
E340G, α-KG output increased 42.2%, 53.6%, 49.2%, α-KG yield is made to be respectively 86.38gL respectively-1、
93.29g·L-1、90.57g·L-1；
(3) α-KG yield highest of mutant G206R/P272F/V276C/V283I/E340S, amino acid sequence is such as
Shown in SEQ ID NO.3, α-KG yield is up to 100.96gL-1, with amino acid sequence L- amino as shown in SEQ ID NO.1
For sour deaminase compared to improving 66.3%, the molar yield of sodium glutamate conversion production α-KG is 64.7%.
Detailed description of the invention
Fig. 1 is l-amino acid deaminase single-point mutants yield testing result.
Fig. 2 is l-amino acid deaminase complex mutation body yield testing result.
Specific embodiment
In the different l-amino acid deaminase mutants in description the application, it is all made of following nomenclature:
(1) when including " alphanumeric ", for example, referring to when G206 to the amino acid sequence as shown in SEQ ID NO.1
In the 206th glycine (G) be mutated；
(2) when for " alphanumeric letter ", for example, referring to when G206R by the amino acid as shown in SEQ ID NO.1
The 206th glycine (G) in sequence is mutated into arginine (R)；
(3) letter is the designatory letter of orresponding amino acid.
Seed culture medium (LB culture medium): peptone 10g, yeast powder 5g, sodium chloride 10g, distilled water constant volume.
Fermentation medium (TB culture medium): peptone 12g, yeast powder 24g, glycerol 5g, dipotassium hydrogen phosphate trihydrate
16.37g, acid dihydride potassium 2.31g.
Sample preparation: it is centrifuged after extracting reaction solution 50 μ l dilution certain multiple, the membrane filtration in 0.22 μm of aperture of supernatant, filter
Liquid is sample to be tested.
The content detection of α-ketoglutaric acid: 1260 type high performance liquid chromatograph of Agilent, Bole Aminex HPX-87H
Chromatographic column (300x 7.8mm, 9 μm), column temperature are 40 DEG C, and sample volume is 10 μ 1, and mobile phase is 5mmo1/L dilute sulfuric acid, mobile phase warp
The membrane filtration in 0.22 μm of aperture, ultrasonic 30min degassing, flow velocity 0.6ml/min, ultraviolet detection wavelength are 210nm, when sample introduction
Between be 15min.
The determination in embodiment 1:L- amino acid deaminase saturation mutation site
With document (Hossain GS, Li J, Shin HD, et al.Bioconversion of l-glutamic acid
toα-ketoglutaric acid by an immobilized whole-cell biocatalyst expressing L-
amino acid deaminase from Proteus mirabilis.Journal of Biotechnology,2014,169
(Complete): 112-120. in), Hossain GS et al. is to from proteus mirabilis (Proteus mirabilis)
L-amino acid deaminase (PM1) be transformed after obtain amino acid sequence l-amino acid deamination as shown in SEQ ID NO.1
Based on enzyme mutant, the present embodiment on this basis, to amino acid sequence l-amino acid deamination as shown in SEQ ID NO.1
Enzyme mutant is further transformed.5 crucial amino for influencing catalytic activity and Binding Capacity and coenzyme effect have been determined
Sour G206, P272, V276, V283, E340.
Foundation, mutant screening and the determination of yield in embodiment 2:L- amino acid deaminase single-point mutants library
(1) by amino acid sequence as shown in SEQ ID NO.1 (nucleotide sequence is as shown in SEQ ID NO.2) L- amino
Sour deaminase gene is connected on carrier pET20b, obtains recombinant vector pET20b-PM1, building process is referring to document
(Hossain GS,Li J,Shin HD,et al.Bioconversion of l-glutamic acid toα-
ketoglutaric acid by an immobilized whole-cell biocatalyst expressing L-amino
acid deaminase from Proteus mirabilis.Journal of Biotechnology,2014,169
(Complete): 112-120. the trifle of page 114 the 2.2nd).
(2) acquisition of single-point saturation mutation l-amino acid deaminase
Using the recombinant vector pET20b-PM1 in step (1) as template, upstream and downstream primer containing mutation sites (is shown in Table
1) after, carrying out full plasmid PCR respectively, phosphorylation and company are carried out using Blunting Kination Ligation (BKL) Kit method
It connects, obtains the single-point saturation mutation l-amino acid deaminase of G206, P272, V276, V283, E340.The condition of full plasmid PCR
Are as follows: 98 DEG C of 3min；Then 98 DEG C of 10s, 55 DEG C of 15s, 72 DEG C of 2min30s amount to 25 circulations；Last 72 DEG C of 10min.
Connection product is transferred to e. coli bl21 (DE3) respectively, and the Escherichia coli after conversion are coated on containing 100
On the LB plate of the ammonia benzyl antibiotic of μ g/mL, 37 DEG C of culture 12h obtain the l-amino acid deaminase containing single-point saturation mutation
Recombination bacillus coli.
1 rite-directed mutagenesis primer of table
(3) screening of mutant
High flux screening: in the mutation library that step (2) is obtained each 100 monoclonals of random picking into 96 orifice plates, with
Contain the recombination bacillus coli of amino acid sequence l-amino acid deaminase as shown in SEQ ID NO.1 as control, 37 DEG C of trainings
Support 10h.It is gone in 48 orifice plates by 50% inoculum concentration, while adding the IPTG of final concentration of 0.06mmol/L-, carried out in 37 DEG C
Fiber differentiation is centrifuged (4200 × g, 5min, 4 DEG C) after 4h, abandons supernatant.Substrate glutamic acid sodium is dissolved in the PB of 0.2M, and (pH is
6.0) in buffer, make the final concentration of 50g/L of sodium glutamate.600 μ l substrate glutamic acid sodium are added to containing recombination large intestine bar
Catalysis reaction is carried out in 48 orifice plates of bacterium, 37 DEG C, centrifugation stops reaction after 1h, measures product α-KG in supernatant by development process
Content.
Develop the color measurement method are as follows: takes 50 μ l supernatants and 100 μ l 0.1%2, the mixing of 4- dinitrophenylhydrazine, room temperature puts 5min
1ml 1.5M sodium hydroxide solution is added afterwards, 200 μ l liquid are drawn after colour stable in 96 shallow bore hole plates, microplate reader detects it
Absorption value at 520nm.
Shaking flask secondary screening: the forward mutation assay body obtained after high flux screening is inoculated in LB culture medium, 37 DEG C, 220rpm mistake
Night culture.Later by 2% switching in TB culture medium, 37 DEG C, 220rpm is cultivated to OD600Final concentration is added when=2.0
The IPTG of 0.06mmol/L is induced.20 DEG C of inducing temperature, induction time 12h.It is centrifuged under 4 DEG C, 5095 × g after induction
10min collects thallus, and thallus is washed twice with 0.2M PB buffer again, suspends spare.
Resting cell system: whole-cell catalyst concentration 20g/L, substrate glutamic acid na concn 200g/L.Full cell turns
Change condition: 30 DEG C of temperature, revolving speed 220rpm, transformation time 60h.Product α-KG yield is measured, yield is higher than to the bacterial strain of control
Carry out the sequencing of l-amino acid deaminase.
Determination of yield: after sequencing carry out result sequence alignment, by amino acid as the result is shown mutate bacterial strain again into
Row fermented and cultured and resting cell, method and condition are finally produced using high performance liquid chromatography measurement α-KG with " shaking flask secondary screening "
Amount.Wherein mutant G206R, P272F, V276C, V283I, E340S, E340G yield improve 12.5% compared with control respectively,
18.2%, 14.8%, 4.5%, 27.6%, 17.0% (being shown in Table 2).
2 α-KG yield of table
Embodiment 3:L- amino acid deaminase multiple spot complex mutation and determination of yield
By the mutational site G206R, P272F of simple point mutation output increased determined in embodiment 2, V276C, V283I,
E340S, E340G are combined mutation, according to the method in embodiment 2, measure P272F/V276C in complex mutation body,
V276C/V283I、V276C/E340S、V276C/E340G、P272F/V283I、P272F/E340S、P272F/E340G、
V283I/E340S、P272F/V276C/V283I、P272F/V276C/E340G、V276C/V283I/E340G、P272F/
V283I/E340S、272L/V283I/E340G、P272F/V276C/V283I/E340S、G206R/P272F/V283I/E340G、
G206R/P272F/V276C/V283I/E340S yield is above control.Compared with the control group yield be respectively increased 27.3%,
28.5%, 42.2%, 25.8%, 5.4%, 23.7%, 22.1%, 16.5%, 16.1%, 28.6%, 34.7%, 35.1%,
30.3%, 53.6%, 49.2%, 66.3% (being shown in Table 3).Wherein, mutant G206R/P272F/V276C/V283I/E340S
α-KG yield highest, for amino acid sequence as shown in SEQ ID NO.3, α-KG yield is up to 100.96gL-1, sodium glutamate
The molar yield of conversion production α-KG is 64.7%, this mutant strain is named as E.coli BL21-pET-20b (+)-
pm1152。
The molar yield calculation formula of sodium glutamate conversion production α-KG:
Molar yield=(product α-KG concentration/α-KG relative molecular mass) ÷ (substrate glutamic acid sodium total concentration/paddy ammonia
Sour sodium relative molecular mass)
3 α-KG yield of table
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of l-amino acid deaminase mutant and its application
<160> 13
<170> PatentIn version 3.3
<210> 1
<211> 473
<212> PRT
<213>artificial synthesized
<400> 1
Met Asn Ile Ser Arg Arg Lys Leu Leu Leu Gly Val Gly Ala Ala Gly
1 5 10 15
Val Leu Ala Gly Gly Ala Ala Leu Val Pro Met Val Arg Arg Asp Gly
20 25 30
Lys Phe Val Glu Ala Lys Ser Arg Ala Ser Phe Val Glu Gly Thr Gln
35 40 45
Gly Ala Leu Pro Lys Glu Ala Asp Val Val Ile Ile Gly Ala Gly Ile
50 55 60
Gln Gly Ile Met Thr Ala Ile Asn Leu Ala Glu Arg Gly Met Ser Val
65 70 75 80
Thr Ile Leu Glu Lys Gly Gln Ile Ala Gly Glu Gln Ser Gly Arg Ala
85 90 95
Tyr Ser Gln Ile Ile Ser Tyr Gln Thr Ser Pro Glu Ile Phe Pro Leu
100 105 110
His His Tyr Gly Lys Ile Leu Trp Arg Gly Met Asn Glu Lys Ile Gly
115 120 125
Ala Asp Thr Ser Tyr Arg Thr Gln Gly Arg Val Glu Ala Leu Ala Asp
130 135 140
Glu Lys Ala Leu Asp Lys Ala Gln Ala Trp Ile Lys Thr Ala Lys Glu
145 150 155 160
Ala Ala Gly Phe Asp Thr Pro Leu Asn Thr Arg Ile Ile Lys Gly Glu
165 170 175
Glu Leu Ser Asn Arg Leu Val Gly Ala Gln Thr Pro Trp Thr Val Ala
180 185 190
Ala Phe Glu Glu Asp Ser Gly Ser Val Asp Pro Glu Thr Gly Thr Pro
195 200 205
Ala Leu Ala Arg Tyr Ala Lys Gln Ile Gly Val Lys Ile Tyr Thr Asn
210 215 220
Cys Ala Val Arg Gly Ile Glu Thr Ala Gly Gly Lys Ile Ser Asp Val
225 230 235 240
Val Ser Glu Lys Gly Ala Ile Lys Thr Ser Gln Val Val Leu Ala Gly
245 250 255
Gly Ile Trp Ser Arg Leu Phe Met Gly Asn Met Gly Ile Asp Ile Pro
260 265 270
Thr Leu Asn Val Tyr Leu Ser Gln Gln Arg Val Ser Gly Val Pro Gly
275 280 285
Ala Pro Arg Gly Asn Val His Leu Pro Asn Gly Ile His Phe Arg Glu
290 295 300
Gln Ala Asp Gly Thr Tyr Ala Val Ala Pro Arg Ile Phe Thr Ser Ser
305 310 315 320
Ile Val Lys Asp Ser Phe Leu Leu Gly Pro Lys Phe Met His Leu Leu
325 330 335
Gly Gly Gly Glu Leu Pro Leu Glu Phe Ser Ile Gly Glu Asp Leu Phe
340 345 350
Asn Ser Phe Lys Met Pro Thr Ser Trp Asn Leu Asp Glu Lys Thr Pro
355 360 365
Phe Glu Gln Phe Arg Val Ala Thr Ala Thr Gln Asn Thr Gln His Leu
370 375 380
Asp Ala Val Phe Gln Arg Met Lys Thr Glu Phe Pro Val Phe Glu Lys
385 390 395 400
Ser Glu Val Val Glu Arg Trp Gly Ala Val Val Ser Pro Thr Phe Asp
405 410 415
Glu Leu Pro Ile Ile Ser Glu Val Lys Glu Tyr Pro Gly Leu Val Ile
420 425 430
Asn Thr Ala Thr Val Trp Gly Met Thr Glu Gly Pro Ala Ala Gly Glu
435 440 445
Val Thr Ala Asp Ile Val Met Gly Lys Lys Pro Val Ile Asp Pro Thr
450 455 460
Pro Phe Ser Leu Asp Arg Phe Lys Lys
465 470
<210> 2
<211> 1422
<212> DNA
<213>artificial synthesized
<400> 2
atgaacattt caaggagaaa gctactttta ggtgttggtg ctgcgggcgt tttagcaggt 60
ggtgcggctt tagttccaat ggttcgccgt gacggcaaat ttgtggaagc taaatctaga 120
gcatcatttg ttgaaggtac gcaaggggct cttcctaaag aagcagatgt agtgattatt 180
ggtgccggta ttcaggggat catgaccgct attaaccttg ctgaacgtgg tatgagtgtc 240
actatcttag aaaagggtca gattgccggt gagcaatcag gccgtgcata cagccaaatt 300
attagttacc aaacatcacc agaaatcttc ccattacacc attatgggaa aatattatgg 360
cgtggcatga atgagaaaat tggtgcagat 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 taaaacttct caagttgtac tcgccggggg tatctggtca 780
cgtttattta tgggcaatat gggtattgat atcccaacgc tcaatgtata tctatcacaa 840
caacgtgtct caggggttcc tggtgcacca cgtggtaatg tgcatttacc taatggtatt 900
catttccgcg agcaagcgga tggtacttat gccgttgcac cacgtatctt tacgagttca 960
atagtcaaag atagcttcct gctagggcct aaatttatgc acttattagg tggcggagag 1020
ttaccgttgg aattctctat tggtgaagat ctatttaatt catttaaaat gccgacctct 1080
tggaatttag atgaaaaaac accattcgaa caattccgag ttgccacggc aacacaaaat 1140
acgcaacact tagatgctgt tttccaaaga atgaaaacag aattcccagt atttgaaaaa 1200
tcagaagttg ttgaacgttg gggtgccgtt gtgagtccaa catttgatga attacctatc 1260
atttctgagg tcaaagaata cccaggctta gtgattaaca cggcaacagt gtggggtatg 1320
accgaaggcc cagcagcggg tgaagtgacc gctgatattg tcatgggcaa gaaacctgtt 1380
attgatccaa cgccgtttag tttggatcgt tttaagaagt aa 1422
<210> 3
<211> 473
<212> PRT
<213>artificial synthesized
<400> 3
Met Asn Ile Ser Arg Arg Lys Leu Leu Leu Gly Val Gly Ala Ala Gly
1 5 10 15
Val Leu Ala Gly Gly Ala Ala Leu Val Pro Met Val Arg Arg Asp Gly
20 25 30
Lys Phe Val Glu Ala Lys Ser Arg Ala Ser Phe Val Glu Gly Thr Gln
35 40 45
Gly Ala Leu Pro Lys Glu Ala Asp Val Val Ile Ile Gly Ala Gly Ile
50 55 60
Gln Gly Ile Met Thr Ala Ile Asn Leu Ala Glu Arg Gly Met Ser Val
65 70 75 80
Thr Ile Leu Glu Lys Gly Gln Ile Ala Gly Glu Gln Ser Gly Arg Ala
85 90 95
Tyr Ser Gln Ile Ile Ser Tyr Gln Thr Ser Pro Glu Ile Phe Pro Leu
100 105 110
His His Tyr Gly Lys Ile Leu Trp Arg Gly Met Asn Glu Lys Ile Gly
115 120 125
Ala Asp Thr Ser Tyr Arg Thr Gln Gly Arg Val Glu Ala Leu Ala Asp
130 135 140
Glu Lys Ala Leu Asp Lys Ala Gln Ala Trp Ile Lys Thr Ala Lys Glu
145 150 155 160
Ala Ala Gly Phe Asp Thr Pro Leu Asn Thr Arg Ile Ile Lys Gly Glu
165 170 175
Glu Leu Ser Asn Arg Leu Val Gly Ala Gln Thr Pro Trp Thr Val Ala
180 185 190
Ala Phe Glu Glu Asp Ser Gly Ser Val Asp Pro Glu Thr Arg Thr Pro
195 200 205
Ala Leu Ala Arg Tyr Ala Lys Gln Ile Gly Val Lys Ile Tyr Thr Asn
210 215 220
Cys Ala Val Arg Gly Ile Glu Thr Ala Gly Gly Lys Ile Ser Asp Val
225 230 235 240
Val Ser Glu Lys Gly Ala Ile Lys Thr Ser Gln Val Val Leu Ala Gly
245 250 255
Gly Ile Trp Ser Arg Leu Phe Met Gly Asn Met Gly Ile Asp Ile Phe
260 265 270
Thr Leu Asn Cys Tyr Leu Ser Gln Gln Arg Ile Ser Gly Val Pro Gly
275 280 285
Ala Pro Arg Gly Asn Val His Leu Pro Asn Gly Ile His Phe Arg Glu
290 295 300
Gln Ala Asp Gly Thr Tyr Ala Val Ala Pro Arg Ile Phe Thr Ser Ser
305 310 315 320
Ile Val Lys Asp Ser Phe Leu Leu Gly Pro Lys Phe Met His Leu Leu
325 330 335
Gly Gly Gly Ser Leu Pro Leu Glu Phe Ser Ile Gly Glu Asp Leu Phe
340 345 350
Asn Ser Phe Lys Met Pro Thr Ser Trp Asn Leu Asp Glu Lys Thr Pro
355 360 365
Phe Glu Gln Phe Arg Val Ala Thr Ala Thr Gln Asn Thr Gln His Leu
370 375 380
Asp Ala Val Phe Gln Arg Met Lys Thr Glu Phe Pro Val Phe Glu Lys
385 390 395 400
Ser Glu Val Val Glu Arg Trp Gly Ala Val Val Ser Pro Thr Phe Asp
405 410 415
Glu Leu Pro Ile Ile Ser Glu Val Lys Glu Tyr Pro Gly Leu Val Ile
420 425 430
Asn Thr Ala Thr Val Trp Gly Met Thr Glu Gly Pro Ala Ala Gly Glu
435 440 445
Val Thr Ala Asp Ile Val Met Gly Lys Lys Pro Val Ile Asp Pro Thr
450 455 460
Pro Phe Ser Leu Asp Arg Phe Lys Lys
465 470
<210> 4
<211> 25
<212> DNA
<213>artificial synthesized
<220>
<221> misc_feature
<222> (1)..(3)
<223> n is a, c, g, or t
<400> 4
nnnacacctg cactcgctcg ttatg 25
<210> 5
<211> 26
<212> DNA
<213>artificial synthesized
<400> 5
tgtttcagga tcaacagagc ctgaat 26
<210> 6
<211> 33
<212> DNA
<213>artificial synthesized
<220>
<221> misc_feature
<222> (1)..(3)
<223> n is a, c, g, or t
<400> 6
nnnacgctca atgtatatct atcacaacaa cgt 33
<210> 7
<211> 37
<212> DNA
<213>artificial synthesized
<400> 7
gatatcaata cccatattgc ccataaataa acgtgac 37
<210> 8
<211> 30
<212> DNA
<213>artificial synthesized
<220>
<221> misc_feature
<222> (1)..(3)
<223> n is a, c, g, or t
<400> 8
nnntatctat cacaacaacg tgtctcaggg 30
<210> 9
<211> 32
<212> DNA
<213>artificial synthesized
<400> 9
attgagcgtt gggatatcaa tacccatatt gc 32
<210> 10
<211> 25
<212> DNA
<213>artificial synthesized
<220>
<221> misc_feature
<222> (1)..(3)
<223> n is a, c, g, or t
<400> 10
nnntcagggg ttcctggtgc accac 25
<210> 11
<211> 32
<212> DNA
<213>artificial synthesized
<400> 11
acgttgttgt gatagatata cattgagcgt tg 32
<210> 12
<211> 28
<212> DNA
<213>artificial synthesized
<220>
<221> misc_feature
<222> (1)..(3)
<223> n is a, c, g, or t
<400> 12
nnnttaccgt tggaattctc tattggtg 28
<210> 13
<211> 30
<212> DNA
<213>artificial synthesized
<400> 13
tccgccacct aataagtgca taaatttagg 30
Claims (10)
1. a kind of l-amino acid deaminase mutant, which is characterized in that the amino acid sequence of the mutant includes: in SEQ ID
On the basis of amino acid sequence shown in NO.1, to one or more site in G206, P272, V276, V283, E340 into
The amino acid sequence obtained after row mutation.
2. mutant according to claim 1, which is characterized in that the mutant be V276C/V283I/E340G,
P272F/V283I/E340S or P272F/V283I/E340G.
3. mutant according to claim 1, which is characterized in that the mutant is V276C/E340S, P272F/
V276C/V283I/E340S or G206R/P272F/V283I/E340G.
4. mutant according to claim 1, which is characterized in that the mutant is G206R/P272F/V276C/
V283I/E340S, amino acid sequence is as shown in SEQ ID NO.3.
5. encoding the gene of mutant described in claim 1.
6. carrying the carrier or cell of gene described in claim 5.
7. expressing the genetic engineering bacterium of l-amino acid deaminase mutant described in claim 1.
8. genetic engineering bacterium according to claim 7, which is characterized in that with Escherichia coli be host.
9. genetic engineering bacterium according to claim 7, which is characterized in that using pET20b as expression vector.
10. a kind of method for preparing α-ketoglutaric acid, which is characterized in that with l-amino acid deamination enzyme mutant described in claim 1
Body or full cell containing the l-amino acid deaminase mutant are catalyst, are substrate preparation α -one penta 2 using sodium glutamate
Acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910216813.7A CN109897837A (en) | 2019-03-21 | 2019-03-21 | A kind of l-amino acid deaminase mutant and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910216813.7A CN109897837A (en) | 2019-03-21 | 2019-03-21 | A kind of l-amino acid deaminase mutant and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109897837A true CN109897837A (en) | 2019-06-18 |
Family
ID=66953708
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910216813.7A Pending CN109897837A (en) | 2019-03-21 | 2019-03-21 | A kind of l-amino acid deaminase mutant and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109897837A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841844A (en) * | 2018-06-26 | 2018-11-20 | 江南大学 | A kind of method of efficient production phenylpyruvic acid |
| CN110643585A (en) * | 2019-11-08 | 2020-01-03 | 江南大学 | Method for producing alpha-ketone-beta-methyl n-pentanoic acid by using amino acid deaminase |
| CN111269900A (en) * | 2020-03-17 | 2020-06-12 | 江南大学 | Preparation and application of L-amino acid deaminase mutant |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624576A (en) * | 2018-04-25 | 2018-10-09 | 江南大学 | A kind of mutant and the preparation method and application thereof of l-amino acid deaminase |
| CN109321541A (en) * | 2018-11-14 | 2019-02-12 | 江南大学 | A kind of mutant of L-amino acid oxidase |
-
2019
- 2019-03-21 CN CN201910216813.7A patent/CN109897837A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624576A (en) * | 2018-04-25 | 2018-10-09 | 江南大学 | A kind of mutant and the preparation method and application thereof of l-amino acid deaminase |
| CN109321541A (en) * | 2018-11-14 | 2019-02-12 | 江南大学 | A kind of mutant of L-amino acid oxidase |
Non-Patent Citations (3)
| Title |
|---|
| FLAVIA TODONE 等: "Active Site Plasticity in D-Amino Acid Oxidase: A Crystallographic Analysis", 《BIOCHEMISTRY》 * |
| MOBLEY,H.: "amino acid deaminase [Proteus mirabilis HI4320]", 《GENBANK DATABASE》 * |
| 王越 等: "L-氨基酸脱氨酶的分子改造及其用于全细胞催化法生产α-酮戊二酸条件的优化", 《生物工程杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841844A (en) * | 2018-06-26 | 2018-11-20 | 江南大学 | A kind of method of efficient production phenylpyruvic acid |
| CN108841844B (en) * | 2018-06-26 | 2022-09-06 | 江南大学 | Method for efficiently producing phenylpyruvic acid |
| CN110643585A (en) * | 2019-11-08 | 2020-01-03 | 江南大学 | Method for producing alpha-ketone-beta-methyl n-pentanoic acid by using amino acid deaminase |
| CN111269900A (en) * | 2020-03-17 | 2020-06-12 | 江南大学 | Preparation and application of L-amino acid deaminase mutant |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shanmugam et al. | Biological nitrogen fixation | |
| CN109897837A (en) | A kind of l-amino acid deaminase mutant and its application | |
| AU756211B2 (en) | Transformed microorganisms with improved properties | |
| CN105255849B (en) | The glutamate decarboxylase mutation construction and its application that a kind of enzyme activity improves | |
| CN107937361B (en) | A kind of alanine dehydrogenase mutant and its application | |
| CN103249832B (en) | Utilize new the producing succinic acid mutant microorganism and utilize its method producing succinic acid of sucrose and glycerol simultaneously | |
| CN108795916A (en) | A kind of lysine decarboxylase mutant, its encoding gene and its expression and application | |
| CN106591330B (en) | Trans-L-1,2-proline-4- '-hydroxylase gene, enzyme, carrier, engineering bacteria and application | |
| CN110257315A (en) | A kind of bacillus subtilis and its construction method and application | |
| CN114634918B (en) | D-amino acid oxidase mutant, engineering bacteria and application | |
| CN109294966A (en) | A kind of the Corynebacterium glutamicum recombinant bacterium and its construction method of high yield L-Leu | |
| CN108949706A (en) | A kind of L-PROLINE -4- hydroxylase and its genetic engineering bacterium, construction method and application | |
| Luo et al. | Enhancement of pyruvic acid production in Candida glabrata by engineering hypoxia-inducible factor 1 | |
| Zhang et al. | Arginase from Bacillus thuringiensis SK 20.001: purification, characteristics, and implications for L-ornithine biosynthesis | |
| CN111690624A (en) | Method for synthesizing 2-O-alpha-D-glycerol glucoside by using microorganisms | |
| Jiang et al. | Enhanced low molecular weight poly-γ-glutamic acid production in recombinant Bacillus subtilis 1A751 with zinc ion | |
| CN105602880B (en) | The method of one plant of Corynebacterium glutamicum and its excessive synthetic phospholipid acyl serine | |
| CN105238797B (en) | A kind of mutator of gshF genes of Streptococcusagalactiae and its application | |
| CN109929822A (en) | A kind of Aspergillus oryzae lipase mutant and its application | |
| CN114350699A (en) | Strain for producing D-psicose 3-epimerase and application thereof | |
| CN111411066B (en) | Double-way composite neuraminic acid-producing bacillus subtilis and construction method thereof | |
| CN109576239A (en) | Heat-resisting phosphorylase and its application | |
| CN113444701A (en) | Saccharomyces cerevisiae endogenous squalene monooxygenase mutant and application thereof | |
| CN112442518B (en) | Method for producing spermidine by using cheap substrate and engineering bacteria | |
| CN105349557A (en) | Malic enzyme gene RKME2 and recombinant expression vector thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190618 |
|
| RJ01 | Rejection of invention patent application after publication |