CN107988131A - A kind of method of high yield α -one-γ-methylmercapto butyric acid - Google Patents
A kind of method of high yield α -one-γ-methylmercapto butyric acid Download PDFInfo
- Publication number
- CN107988131A CN107988131A CN201810035749.8A CN201810035749A CN107988131A CN 107988131 A CN107988131 A CN 107988131A CN 201810035749 A CN201810035749 A CN 201810035749A CN 107988131 A CN107988131 A CN 107988131A
- Authority
- CN
- China
- Prior art keywords
- butyric acid
- methylmercapto butyric
- coli
- genetic engineering
- amino acid
- 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.)
- Granted
Links
Classifications
-
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
- C12Y104/03002—L-Amino-acid oxidase (1.4.3.2)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of method of high yield α ketone γ methylmercapto butyric acids, belong to technical field of bioengineering.The gene that L amino acid oxidases are encoded from rhodococcus erythropolis is connected by the present invention by molecular biology method after artificial synthesized and codon optimization with expression vector.The expression plasmid built is imported into E.coli BL21 (DE3), the enzyme engineering bacteria of amino-acid oxidase containing L of the high copy restructuring of expression vector is obtained by kalamycin resistance plate screening.For converting L methionines production α ketone γ methylmercapto butyric acids.By controlling biomass, temperature and pH, the efficient production of α ketone γ methylmercapto butyric acids is realized.The wet thallus reaction 24h of 30g/L is put under conditions of 20 DEG C, pH7.5, α ketone γ methylmercapto butyric acids yield is up to 95.18g/L, conversion ratio 95.84%, space-time yield 3.97g/L/h.
Description
Technical field
The present invention relates to a kind of method of high yield α -one-γ-methylmercapto butyric acid, belong to technical field of bioengineering.
Background technology
L-methionine (L-methionine) can only be obtained as necessary amino acid by diet.For Ren Hejia
The health of fowl is important.Functioning also for cell, there play the role of to be important.Because l-methionine can quilt in body
It is rapid to decompose, therefore it is seldom to enter sanguimotor amount.α -one-γ-derivative of the methylmercapto butyric acid as l-methionine
Thing, can increase the utilization ratio of body.In addition, the safe and non-toxic life that can suppress tumour cell of α -one-γ-methylmercapto butyric acid
It is long, also it is used to treat uremic patient.
α -one-γ-methylmercapto butyric acid (α-Keto- γ-methylthiobutyric acid, abbreviation KMTB) is that one kind contains
There are the ketone acid of difunctional, molecular formula C5H8O3S.The production method of α -one-γ-methylmercapto butyric acid includes：Chemical synthesis and
Enzyme catalysis method.At present, industrially producing alpha -one-γ-methylmercapto butyric acid mainly uses organic synthesis method, is related to a series of complex
Chemical reaction process, so as to cause raw material sources, environmental pollution etc. a series of problems.Compared with chemical method, enzyme catalysis method
More convenient and easy to operate, its reaction condition is gentle, and accessory substance is few, has high selectivity and high efficiency, while also have
The characteristics of environmentally friendly, be more conducive to the large-scale production of industry.Enzyme catalysis method production α -one-γ-methylmercapto butyric acid, just
It is the enzyme using micro-organisms, conversion methionine produces α -one-γ-methylmercapto butyric acid.Using enzymatic conversion method methionine
α -one-γ-methylmercapto butyric acid is produced, is essentially all a step enzymatic reaction, therefore can be to avoid anti-in many metabolic pathways
Feedback is adjusted, so as to achieve the purpose that largely to accumulate α -one-γ-methylmercapto butyric acid.Therefore it is how extensive using biotechnology method
Safe α -one-γ-methylmercapto butyric acid is prepared, is domestic and international academia and industrial circle focal issue of interest.
Report for work and α -one-γ-methylmercapto butyric acid is produced as substrate with D-AAO using D-Met.But use this side
Method, can be accompanied by the generation of hydrogen peroxide.Hydrogen peroxide can cause the denaturation of protein, and then influence D-AAO
Availability.In order to eliminate the influence of hydrogen peroxide, it is necessary to add catalase, the extra cost for adding production.And with
L-methionine is more cheap for substrate, its conversion production α -one-γ-methylmercapto butyric acid is catalyzed more with L-amino acid oxidase
It is economical.
The content of the invention
The present invention first purpose be to provide a kind of recombinant bacterium of high yield L-amino acid oxidase, be using Escherichia coli as
Host, using the plasmid of pET series as carrier, expresses L-amino acid oxidase, the L-amino acid oxidase contains such as SEQ ID
Amino acid sequence shown in NO.1.
In one embodiment of the invention, the carrier is pET serial carriers.
In one embodiment of the invention, the carrier is pET28a.
In one embodiment of the invention, the Escherichia coli include E.coli BL21, E.coli JM109,
E.coli DH5 α or E.coli TOP10.
Second object of the present invention is to provide a kind of construction method of the recombinant bacterium of high yield L-amino acid oxidase, described
Method is to be connected carrier with the gene of encoded L-amino acids oxidizing ferment, is converted into host cell；The gene contains such as SEQ
Nucleotide sequence shown in ID NO.2.
In one embodiment of the invention, the gene of the encoded L-amino acids oxidizing ferment such as SEQ ID NO.1 institutes
Show, the Escherichia coli are E.coli BL21 (DE3).
In one embodiment of the invention, the host cell includes but not limited to Escherichia coli.
In one embodiment of the invention, it is described structure high yield L-amino acid oxidase recombinant bacterium specifically include as
Lower step：Using pET28a as carrier, gene shown in SEQ ID NO.2 is connected with carrier, in E. coli BL21
(DE3) L-amino acid oxidase shown in recombination expression SEQ ID NO.1 in.
Third object of the present invention is to provide the method using recombinant bacterium production L-amino acid oxidase, the side
Method is by the recombinant bacterium culture to OD600For 0.6-0.8, the expression of IPTG induction L-amino acid oxidases is added.
In one embodiment of the invention, the method is by the recombinant bacterium culture to OD600For 0.6-0.8, add
After entering final concentration of 0.4mM IPTG, induction 12h is carried out at 25 DEG C.
Fourth object of the present invention is to provide the method using recombinant bacterium production α -one-γ-methylmercapto butyric acid, its
It is characterized in that, the method is using l-methionine as substrate, using recombinant bacterium conversion of substrate production α -one-γ-first sulphur
Base butyric acid.
In one embodiment of the invention, the conversion condition is：PH 7~9, conversion temperature are 15~37 DEG C, are turned
Change 20~24h of the time.
In one embodiment of the invention, the conversion condition is pH7~8,20 DEG C of conversion temperature, transformation time
24h。
In one embodiment of the invention, the conversion carries out in shaking table, shaking speed 200rpm.
In one embodiment of the invention, converted with the wet cell of the recombinant bacterium, wet cell additive amount is
25-35g/L。
In one embodiment of the invention, the transformation system is to containing final concentration of 80-200g/L L- first sulphur ammonia
The wet cell that the recombinant bacterium is added in the reaction system of acid carries out resting cell.
In one embodiment of the invention, described convert adds into the l-methionine solution that concentration is 100g/L
The full cell of 30g/L, 24h is converted at 20 DEG C.
In one embodiment of the invention, the transformation system is the L- amino using l-methionine as substrate
The additive amount of acid oxidase whole-cell catalyst is 30g/L.
The present invention also provides the recombinant bacterium food, medicine, chemical field application.
Beneficial effect：1st, the present invention is the L-amino acid oxidase conversion l-methionine used from rhodococcus erythropolis
α -one-γ-methylmercapto butyric acid is produced, which shows high vigor after e. coli expression, can preferably meet work
The demand of industry large-scale production.2nd, 20 DEG C, the yield of α -one-γ-methylmercapto butyric acid reaches as high as under conditions of pH7.5
95.18g/L, transformation period need 24h, and production efficiency is greatly improved.
Brief description of the drawings
Fig. 1：The HPLC analysis collection of illustrative plates of converted product；(A)：α -one-γ-methylmercapto butyric acid sodium standard items；((B)：Reaction
The conversion fluid of 24h；
Fig. 2：Influence of the different biomasses to α -one-γ-methylmercapto butyric acid yield；
Fig. 3：Influence of the conversion to α -one-γ-methylmercapto butyric acid yield under condition of different temperatures；
Fig. 4：Influence of the conversion to α -one-γ-methylmercapto butyric acid yield under condition of different pH；
Fig. 5：Influence of the L-amino acid oxidase in other sources to α -one-γ-methylmercapto butyric acid yield.
Embodiment
Sample to be tested pre-processes：Take conversion fluid 10000rpm to centrifuge 2min, collect supernatant, and with α -one-γ-methyl mercapto
Butyric acid sodium salt is as standard items, preparing standard solution.Supernatant and standard solution after appropriateness is diluted is micro- through 0.22 μm respectively
After the membrane filtration of hole, with high effective liquid chromatography for measuring α -one-γ-methylmercapto butyric acid and the content of remaining l-methionine.
The measure of α -one-γ-methylmercapto butyric acid content：Using high performance liquid chromatography.Chromatographic column：C18ODS
HYPERSIL (250mm × 4.6mm, 5 μm)；Mobile phase：, 0.0275% dilute sulfuric acid, with 0.22 μm of membrane filtration；Column temperature：40
℃；Detection wavelength：225nm；Sample size：10μL；Flow velocity:1.0mL/min.
The calculating of space-time yield：Space-time yield (g/L/h)=KMTB yield (g/L)/transformation time (h)
Embodiment 1：The structure of the genetic engineering bacterium containing L-amino acid oxidase
(1) L-AAO deaminase gene of the artificial synthesized codon optimization containing BamHI and XhoI restriction enzyme sites,
As shown in SEQ ID NO.2.
(2) restriction enzyme BamHI and XhoI is used by 37 DEG C of digestion 2h of target gene and expression vector pET28a；
(3) with T4 ligases respectively by 16 DEG C of connection 10h of the target gene after digestion and glue reclaim and plasmid pET28a；
(4) expression plasmid built is imported into E.coli BL21 (DE3), is trained in the LB tablets for receiving mycin containing card
Support 12h；
(5) bacterium colony grown in tablet is subjected to PCR and digestion verification, the plasmid containing target gene is subjected to sequencing and is tested
Card, selects the right-on bacterial strain of target gene, as expresses L-amino acid oxidase genetically engineered E.coli BL21-L-
AAO。
Embodiment 2：The induced expression of genetic engineering bacterium
(1) by the genetically engineered E.coli BL21-L-AAO access LB slant medium cultures 12h of structure；
(2) a ring inclined-plane seed is connect in LB culture mediums, cultivates 6h；
(3) E.coli BL21-L-AAO seed liquors are accessed in TB fermentation mediums, cultivated to OD600For 0.6, add eventually
Concentration is that the IPTG of 0.4mM is induced, and thalline, sterile saline washing thalline are collected after inducing 12h in 25 DEG C.
Embodiment 3：Influence of the different biomasses to production α -one-γ-methylmercapto butyric acid
The wet thallus obtained in embodiment 2 is taken, is collected by centrifugation after thalline and carries out resting cell as catalyst.Control
PH is 8.0,30 DEG C of temperature, adjusts the additive amount (30g/L, 40g/L, 50g/L, 60g/L) of thalline.Reaction solution includes final concentration of
The l-methionine of 100g/L, 20mM Tris-HCL.After converting 24h, supernatant after being reacted using high effective liquid chromatography for measuring
The situation (as shown in Figure 2) of the concentration of middle α -one-γ-methylmercapto butyric acid, as the final concentration of 30g/L of biomass, α -one-γ-
The concentration of methylmercapto butyric acid is 77.45g/L；When biomass is 40g/L, the concentration of α -one-γ-methylmercapto butyric acid is
94.46g/L；As the final concentration of 50g/L of biomass, the concentration of α -one-γ-methylmercapto butyric acid is 96.84g/L；Work as biomass
During final concentration of 60g/L, the concentration of α -one-γ-methylmercapto butyric acid is 97.59g/L..
Embodiment 4：Conversion temperature compares the influence of production α -one-γ-methylmercapto butyric acid
The wet thallus obtained in embodiment 2 is taken, is collected by centrifugation after thalline and carries out resting cell as catalyst.Control
PH is 8.0, and it is 15~37 DEG C to adjust reaction temperature, measures its optimum temperature.Reaction solution includes the L- first of final concentration of 100g/L
Methyllanthionine, the wet thallus of 20mM Tris-HCL, 30g/L.After converting 24h, supernatant after being reacted using high effective liquid chromatography for measuring
The situation (as shown in Figure 3) of the concentration of α -one-γ-methylmercapto butyric acid in liquid, the results show that when temperature is 15 DEG C, α -one-
γ-methylmercapto butyric acid concentration is 80.29g/L；When temperature is 20 DEG C, the concentration of α -one-γ-methylmercapto butyric acid is 87.35g/
L；When temperature is 25 DEG C, the concentration of α -one-γ-methylmercapto butyric acid is 83.10g/L；When temperature is 30 DEG C, α -one-γ-first
The concentration of sulfenyl butyric acid is 77.34g/L；When temperature is 37 DEG C, the concentration of α -one-γ-methylmercapto butyric acid is 75.08g/L.When
Temperature concentration of α -one-γ-methylmercapto butyric acid at 20 DEG C reaches highest 87.35g/L, conversion ratio 88.06%, space-time yield
For 3.64g/L/h；When temperature is higher than 20 DEG C, with the rise of temperature, the yield of α -one-γ-methylmercapto butyric acid is under gradually
Drop.
Embodiment 5：Convert influences of the pH to production α -one-γ-methylmercapto butyric acid
The wet thallus obtained in embodiment 2 is taken, is collected by centrifugation after thalline and carries out resting cell as catalyst.Control
Temperature is 20 DEG C, and it is 7.0~9.0 to adjust reaction pH.Reaction solution includes the l-methionine of final concentration of 100g/L, 20mM
The wet thallus of Tris-HCL, 30g/L.After converting 24h, α -one-γ in supernatant after being reacted using high effective liquid chromatography for measuring-
The situation (as shown in Figure 4) of the concentration of methylmercapto butyric acid, the results show that when pH be 7.0 when, α -one-γ-methylmercapto butyric acid it is dense
Spend for 88.90g/L；When pH is 7.5, the concentration of α -one-γ-methylmercapto butyric acid is 95.18g/L；When pH is 8.0, α -one-
The concentration of γ-methylmercapto butyric acid is 87.87g/L；When pH is 8.5, the concentration of α -one-γ-methylmercapto butyric acid is 71.50g/L；
When pH is 9.0, the concentration of α -one-γ-methylmercapto butyric acid is 62.55g/L.When pH is 7.5, α -one-γ-methylmercapto butyric acid
Concentration reaches highest 95.18g/L, conversion ratio 95.84%, space-time yield 3.97g/L/h；When pH is higher than 7.5, with pH
Rise, the yield of α -one-γ-methylmercapto butyric acid is being gradually reduced.
Embodiment 6
Take the wet thallus obtained in embodiment 2, as whole-cell catalyst be used to converting l-methionine production α -one-
γ-methylmercapto butyric acid.Prepare the reaction system of 20mL substrates and L-amino acid oxidase.Reaction mixture is by final concentration of
The full cell of the l-methionine of 100g/L, 20mMTris-HCL and 30g/L.After reacting 24h under 20 DEG C of reaction condition, lead to
Cross 0.22 μm of filter to be filtered, and analysis result is carried out with high performance liquid chromatography.At this time, KMTB yield reaches
95.18g/L, conversion ratio 95.84%, space-time yield 3.97g/L/h.α -one-γ-methylmercapto butyric acid is realized efficiently to produce.
Reference examples 1
According to the identical strategy of embodiment 1~2, in expression in escherichia coli Corynebacterium glutamicum, [Genbank is logged in respectively
Number：AJE67504], bacillus subtilis [Genbank accession number：AKN12962], bacillus thuringiensis [Genbank log in
Number AEA14048], bacillus megaterium [Genbank accession number WP_026679766], [Genbank is logged in Bacillus cercus
Number AQQ62701] source L-amino acid oxidase, compare α -one-γ-methylmercapto butyric acid under the same conditions in embodiment 6
Yield, the results are shown in Figure 5, under the conditions of same conversion, when expression derives from the L-amino acid oxidase in Corynebacterium glutamicum,
The yield of α -one-γ-methylmercapto butyric acid is 49.12g/L；When expression derives from the L-amino acid oxidase in bacillus subtilis
When, the yield of α -one-γ-methylmercapto butyric acid is 57.96g/L；When expression derives from the l-amino acid in bacillus thuringiensis
During oxidizing ferment, the yield of α -one-γ-methylmercapto butyric acid is 18.40g/L；When expression derives from the L- ammonia in bacillus megaterium
During base acid oxidase, the yield of α -one-γ-methylmercapto butyric acid is 29.53g/L；Derived from when expressing in Bacillus cercus
During L-amino acid oxidase, the yield of α -one-γ-methylmercapto butyric acid is 48.20g/L.
Although the present invention is disclosed as above with preferred embodiment, 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 LISTING
<110>Southern Yangtze University
<120>A kind of method of high yield α -one-γ-methylmercapto butyric acid
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 534
<212> PRT
<213>Artificial sequence
<400> 1
Met Pro Tyr Thr Arg Arg Ser Phe Met Arg Ser Leu Gly Ile Thr Gly
1 5 10 15
Gly Ala Gly Leu Ala Leu Gly Ala Met Ser Ser Ile Gly Leu Ala Pro
20 25 30
Ala Val Ala Ser Thr Pro Arg Arg Phe Gln Ala Pro Ala Ala Gly Asp
35 40 45
Leu Ile Gly Lys Val Lys Gly Asn His Ser Val Val Ile Leu Gly Gly
50 55 60
Gly Pro Ser Gly Leu Cys Ser Ala Tyr Glu Leu Gln Lys Ala Gly Tyr
65 70 75 80
Lys Val Thr Val Leu Glu Ala Arg Asn Arg Pro Gly Gly Arg Val Trp
85 90 95
Ser Ile Arg Asn Gly Thr Glu Glu Thr Asp Leu Asn Gly Glu Thr Gln
100 105 110
Thr Cys Thr Phe Ser Glu Gly His Phe Tyr Asn Leu Gly Ala Thr Arg
115 120 125
Ile Pro Gln Asn His Asn Thr Ile Asp Tyr Cys Arg Glu Leu Gly Val
130 135 140
Glu Leu Gln Met Phe Gly Asn Gln Asn Ala Asn Thr Phe Val Asn Tyr
145 150 155 160
Thr Gly Asn Lys Pro Leu Ala Asn Gln Ser Ile Thr Tyr Arg Ala Ala
165 170 175
Lys Ala Asp Thr Tyr Gly Tyr Met Ser Glu Leu Leu Gln Lys Ala Thr
180 185 190
Asn Gln Gly Ala Leu Asp Gln Val Leu Ser Lys Glu Asp Lys Glu Ala
195 200 205
Leu Ser Ser Phe Leu Thr Asp Phe Gly Asp Leu Ser Ser Asp Gly Arg
210 215 220
Tyr Val Gly Ser Ser Arg Arg Gly His Ser Ala Glu Pro Gly Ala Gly
225 230 235 240
Leu Asn Phe Gly Thr Glu Ile Glu Pro Phe Gly Met Ser Asp Val Ile
245 250 255
Gln Gly Gly Ile Gly Arg Ala Phe Ser Phe Glu Phe Gly Tyr Asp Gln
260 265 270
Ala Met Thr Met Met Thr Pro Val Gly Gly Met Asp Arg Ile Tyr Tyr
275 280 285
Lys Phe Gln Asp Ala Ile Gly Met Asp Asn Ile Glu Phe Gly Ala Glu
290 295 300
Val Ser Gly Met Lys Asn Val Pro Glu Gly Val Thr Val Asp Tyr Val
305 310 315 320
Val Asp Gly Lys Thr Lys Ser Ile Thr Ala Asp Tyr Ala Ile Cys Thr
325 330 335
Ile Pro Pro His Leu Ile Lys Arg Leu Asn Asn Asn Leu Pro Ser Asp
340 345 350
Ile Leu Leu Ala Leu Asp Ala Ala Lys Pro Ser Ser Ser Gly Lys Leu
355 360 365
Gly Ile Glu Tyr Ser Arg Arg Trp Trp Glu Thr Glu Asp Arg Ile Tyr
370 375 380
Gly Gly Ala Ser Asn Thr Asp Arg Asp Ile Ser Gln Ile Met Phe Pro
385 390 395 400
Tyr Asp His Tyr Asn Ser Asp Arg Gly Val Val Val Ala Tyr Tyr Ser
405 410 415
Ser Gly Lys Arg Gln Gln Ala Phe Glu Ser Leu Thr His Arg Gln Arg
420 425 430
Leu Ala Lys Ala Ile Ala Glu Gly Ala Glu Ile His Gly Asp Lys Tyr
435 440 445
Thr Arg Asp Ile Ser Ser Ser Phe Ser Gly Ser Trp Arg Arg Thr Lys
450 455 460
Tyr Ser Glu Ser Ala Trp Ala Ser Trp Ala Gly Ala Gly Asp Ser His
465 470 475 480
Gly Gly Met Ala Thr Pro Glu Tyr Thr Lys Leu Leu Glu Pro Val Asp
485 490 495
Arg Ile Tyr Phe Ala Gly Asp His Leu Ser Asn Ala Ile Ala Trp Gln
500 505 510
His Gly Ala Phe Thr Ser Ala Gln Asp Val Val Thr His Leu His Gln
515 520 525
Arg Val Ala Gln Thr Ala
530
<210> 2
<211> 1605
<212> DNA
<213>Artificial sequence
<400> 2
atgccgtaca cccgtcgttc tttcatgcgt tctctgggta tcaccggtgg tgctggtctg 60
gctctgggtg ctatgtcttc tatcggtctg gctccggctg ttgcttctac cccgcgtcgt 120
ttccaggctc cggctgctgg tgacctgatc ggtaaagtta aaggtaacca ctctgttgtt 180
atcctgggtg gtggtccgtc tggtctgtgc tctgcttacg aactgcagaa agctggttac 240
aaagttaccg ttctggaagc tcgtaaccgt ccgggtggtc gtgtttggtc tatccgtaac 300
ggtaccgaag aaaccgacct gaacggtgaa acccagacct gcaccttctc tgaaggtcac 360
ttctacaacc tgggtgctac ccgtatcccg cagaaccaca acaccatcga ctactgccgt 420
gaactgggtg ttgaactgca gatgttcggt aaccagaacg ctaacacctt cgttaactac 480
accggtaaca aaccgctggc taaccagtct atcacctacc gtgctgctaa agctgacacc 540
tacggttaca tgtctgaact gctgcagaaa gctaccaacc agggtgctct ggaccaggtt 600
ctgtctaaag aagacaaaga agctctgtct tctttcctga ccgacttcgg tgacctgtct 660
tctgacggtc gttacgttgg ttcttctcgt cgtggtcact ctgctgaacc gggtgctggt 720
ctgaacttcg gtaccgaaat cgaaccgttc ggtatgtctg acgttatcca gggtggtatc 780
ggtcgtgctt tctctttcga attcggttac gaccaggcta tgaccatgat gaccccggtt 840
ggtggtatgg accgtatcta ctacaaattc caggacgcta tcggtatgga caacatcgaa 900
ttcggtgctg aagtttctgg tatgaaaaac gttccggaag gtgttaccgt tgactacgtt 960
gttgacggta aaaccaaatc tatcaccgct gactacgcta tctgcaccat cccgccgcac 1020
ctgatcaaac gtctgaacaa caacctgccg tctgacatcc tgctggctct ggacgctgct 1080
aaaccgtctt cttctggtaa actgggtatc gaatactctc gtcgttggtg ggaaaccgaa 1140
gaccgtatct acggtggtgc ttctaacacc gaccgtgaca tctctcagat catgttcccg 1200
tacgaccact acaactctga ccgtggtgtt gttgttgctt actactcttc tggtaaacgt 1260
cagcaggctt tcgaatctct gacccaccgt cagcgtctgg ctaaagctat cgctgaaggt 1320
gctgaaatcc acggtgacaa atacacccgt gacatctctt cttctttctc tggttcttgg 1380
cgtcgtacca aatactctga atctgcttgg gcttcttggg ctggtgctgg tgactctcac 1440
ggtggtatgg ctaccccgga atacaccaaa ctgctggaac cggttgaccg tatctacttc 1500
gctggtgacc acctgtctaa cgctatcgct tggcagcacg gtgctttcac ctctgctcag 1560
gacgttgtta cccacctgca ccagcgtgtt gctcagaccg cttaa 1605
Claims (10)
1. a kind of genetic engineering bacterium of high yield L-amino acid oxidase, it is characterised in that using Escherichia coli as host, with pET systems
The plasmid of row is carrier, expresses L-amino acid oxidase；The L-amino acid oxidase contains the ammonia as shown in SEQ ID NO.1
Base acid sequence.
2. genetic engineering bacterium according to claim 1, it is characterised in that the carrier is pET28a；The Escherichia coli
Including E.coli BL21, E.coli JM109, E.coli DH5 α or E.coli TOP10.
A kind of 3. method for building genetic engineering bacterium described in claim 1, it is characterised in that by carrier and encoded L-amino acids oxygen
Change the gene connection of enzyme, convert into host cell；The gene contains the nucleotide sequence as shown in SEQ ID NO.2.
4. the according to the method described in claim 3, it is characterized in that, gene of the encoded L-amino acids oxidizing ferment such as SEQ ID
Shown in NO.1, the Escherichia coli are E.coli BL21 (DE3).
A kind of 5. method for producing L-amino acid oxidase, it is characterised in that using the genetic engineering described in claim 1 or 2
Bacterium.
6. according to the method described in claim 5, it is characterized in that, by the genetic engineering bacterium culture described in claim 1 or 2 extremely
OD600For 0.6-0.8, the expression of IPTG induction L-amino acid oxidases is added.
A kind of 7. method for producing α -one-γ-methylmercapto butyric acid, it is characterised in that the method is using l-methionine the bottom of as
Thing, using genetic engineering bacterium conversion of substrate production α -one-γ-methylmercapto butyric acid described in claim 1 or 2.
8. the method according to the description of claim 7 is characterized in that the conversion is in 7~9,15~37 DEG C of pH, conversion 20~
24h。
It is 9. the method according to the description of claim 7 is characterized in that wet thin with the genetic engineering bacterium described in claim 1 or 2
Born of the same parents are converted, and wet cell additive amount is 25-35g/L；The transformation system is to containing final concentration of 80-200g/L L- first sulphur
Resting cell is carried out in the reaction system of propylhomoserin.
10. genetic engineering bacterium described in claim 1 or 2 is in the application of food, medicine, chemical field.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810035749.8A CN107988131B (en) | 2018-01-15 | 2018-01-15 | Method for high-yield production of α -ketone-gamma-methylthiobutyric acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810035749.8A CN107988131B (en) | 2018-01-15 | 2018-01-15 | Method for high-yield production of α -ketone-gamma-methylthiobutyric acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107988131A true CN107988131A (en) | 2018-05-04 |
| CN107988131B CN107988131B (en) | 2020-06-09 |
Family
ID=62041093
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810035749.8A Active CN107988131B (en) | 2018-01-15 | 2018-01-15 | Method for high-yield production of α -ketone-gamma-methylthiobutyric acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107988131B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109321541A (en) * | 2018-11-14 | 2019-02-12 | 江南大学 | A kind of mutant of L-amino acid oxidase |
| CN109371070A (en) * | 2018-11-14 | 2019-02-22 | 江南大学 | A kind of method of high yield α-ketoisovaleric acid |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555642A (en) * | 2013-10-12 | 2014-02-05 | 江南大学 | Construction method and application of L-amino acid oxidase production recombinant strain |
-
2018
- 2018-01-15 CN CN201810035749.8A patent/CN107988131B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555642A (en) * | 2013-10-12 | 2014-02-05 | 江南大学 | Construction method and application of L-amino acid oxidase production recombinant strain |
Non-Patent Citations (1)
| Title |
|---|
| 毕春元等: "L-谷氨酸氧化酶的研究进展", 《生命科学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109321541A (en) * | 2018-11-14 | 2019-02-12 | 江南大学 | A kind of mutant of L-amino acid oxidase |
| CN109371070A (en) * | 2018-11-14 | 2019-02-22 | 江南大学 | A kind of method of high yield α-ketoisovaleric acid |
| CN109321541B (en) * | 2018-11-14 | 2020-12-01 | 江南大学 | Mutant of L-amino acid oxidase |
| CN109371070B (en) * | 2018-11-14 | 2024-05-28 | 江南大学 | Method for high yield of alpha-ketoisovaleric acid |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107988131B (en) | 2020-06-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR20090005052A (en) | Method for the enzymatic production of 2-hydroxy-2-methyl carboxylic acids | |
| CN105671010A (en) | Aldehyde ketone reductase mutant, gene, engineering bacterium and application of mutant | |
| CN104152505A (en) | Method for transforming and preparing 4-hydroxyl-L-isoleucine by using recombinant bacterial strains | |
| CN108467860A (en) | A kind of method of highly producing gamma-aminobutyric acid | |
| CN105026548A (en) | D-glucaric acid-producing bacterium, and method for manufacturing d-glucaric acid | |
| CN105695425A (en) | Cyclohexanone monooxygenase and application thereof in synthesis of esomeprazole | |
| CN109266595A (en) | A kind of building and application of the recombinant bacterium of conversion L-threonine production C4H9NO2 | |
| CN107937361A (en) | A kind of alanine dehydrogenase mutant and its application | |
| CN104130967B (en) | One plant of coexpression L lactic dehydrogenase and the Escherichia coli of hydrogenlyase and its construction method and application | |
| CN104131041A (en) | Production method for alpha-ketoglutaric acid | |
| CN104046586B (en) | One strain gene engineering bacterium and the application in producing (2R, 3R)-2,3-butanediol thereof | |
| CN107988131A (en) | A kind of method of high yield α -one-γ-methylmercapto butyric acid | |
| CN104673814B (en) | A kind of L threonine aldolases for coming from enterobacter cloacae and its application | |
| CN109593702B (en) | Method for synthesizing L-phenyllactic acid by whole cell transformation of genetic engineering strain | |
| CN109371070A (en) | A kind of method of high yield α-ketoisovaleric acid | |
| Li et al. | Genome shuffling of Aspergillus niger for improving transglycosylation activity | |
| CN104031892A (en) | Leucine dehydrogenase and gene for coding same | |
| CN114350630B (en) | L-pantolactone dehydrogenase, mutant and application thereof | |
| CN113174378B (en) | Glutamate dehydrogenase mutant, encoding gene thereof, genetically engineered bacterium and application thereof in preparation of L-2-aminobutyric acid | |
| CN105950595B (en) | (-)-gamma-lactam enzyme, gene, mutant, carrier and its preparation and application | |
| CN107177564A (en) | A kind of L lactic dehydrogenases in Lactobacillus casei source and its application | |
| CN114672525A (en) | Biosynthesis method and application of N-acetyl-5-methoxytryptamine | |
| CN113215120A (en) | Method for producing trans-4-hydroxy-L-proline by recombinant escherichia coli transformation | |
| CN108359666B (en) | A kind ofnudCGene and application thereof in preparation of nicotinic acid | |
| CN110804602A (en) | L-aspartic acid β -decarboxylase mutant and application 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |