CN109337845B - Acinetobacter Y-3L-asparaginase gene and expression and application thereof - Google Patents

Acinetobacter Y-3L-asparaginase gene and expression and application thereof Download PDF

Info

Publication number
CN109337845B
CN109337845B CN201811384145.0A CN201811384145A CN109337845B CN 109337845 B CN109337845 B CN 109337845B CN 201811384145 A CN201811384145 A CN 201811384145A CN 109337845 B CN109337845 B CN 109337845B
Authority
CN
China
Prior art keywords
asparaginase
acinetobacter
gene
asaase
pcr amplification
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.)
Active
Application number
CN201811384145.0A
Other languages
Chinese (zh)
Other versions
CN109337845A (en
Inventor
吕凤霞
焦琳舒
陆兆新
张充
别小妹
赵海珍
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Agricultural University
Original Assignee
Nanjing Agricultural University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Nanjing Agricultural University filed Critical Nanjing Agricultural University
Publication of CN109337845A publication Critical patent/CN109337845A/en
Application granted granted Critical
Publication of CN109337845B publication Critical patent/CN109337845B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/82—Asparaginase (3.5.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01001—Asparaginase (3.5.1.1)

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Polymers & Plastics (AREA)
  • Food Science & Technology (AREA)
  • Nutrition Science (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention relates to an acinetobacter Y-3L-asparaginase gene and expression and application thereof. The L-asparaginase gene disclosed by the invention is derived from a strain of acinetobacter which is screened from soilAcinetobacter soli) Y-3 (seed deposit number: CGMCC NO. 14831), the nucleotide sequence of which is shown as SEQ ID NO. 1. The L-asparaginase has good catalytic activity, effectively inhibits the generation of acrylamide in fried foods, can radically control the generation of potential carcinogen acrylamide in the high-temperature processing of starch-containing foods, and has wide application prospect in the field of food processing.

Description

Acinetobacter Y-3L-asparaginase gene and expression and application thereof
Technical Field
The invention relates to an acinetobacter Y-3L-asparaginase gene and expression and application thereof, belonging to the technical field of biology.
Background
L-asparaginase (EC3.5.1.1), which is a threonine amino hydrolase, specifically catalyzes the formation of L-aspartic acid and ammonia from L-asparagine. The L-asparaginase can hydrolyze asparagine to prevent Maillard reaction of asparagine and reducing sugar to generate acrylamide, so that the formation of acrylamide in the high-heat processed food is controlled radically, and the production process, the appearance, the flavor and the nutrition of the product are not changed.
The use of L-asparaginase to remove asparagine from the starting material is currently the most effective method for controlling the acrylamide level in high-heat processed foods. By adding asparaginase prior to baking or frying the food, the L-asparagine in the food material is hydrolyzed to L-aspartic acid and ammonia such that the L-asparagine cannot continue to participate in the Maillard reaction, thereby significantly reducing the amount of acrylamide that is produced during the thermal processing of the food.
The L-asparaginase has wide sources, and microorganisms such as plants, animals, bacteria, fungi, yeasts, actinomycetes and the like can produce the asparaginase, and the asparaginase from different sources has different biological characteristics. The extraction process of the L-asparaginase from animal and plant sources is complex, and the yield is low, so that the L-asparaginase from microorganism sources which is easy to culture and purify has great advantages, but the yield of the L-asparaginase from a wild strain is low, and the separation and purification method is complicated.
Disclosure of Invention
The invention aims to clone a gene fragment capable of coding L-asparaginase from genome DNA of Acinetobacter Y-3 by using a genetic engineering technology means, realize the efficient expression of the gene fragment in prokaryotic cells, and apply the gene fragment to fried foods, effectively inhibit the generation of acrylamide in the fried foods and radically control the generation of potential carcinogen acrylamide in the high-temperature processing of starch-containing foods.
The invention clones the L-asparaginase gene by a genetic engineering technology, constructs genetic engineering bacteria, obtains a novel recombinant L-asparaginase, realizes the efficient expression of the L-asparaginase gene, effectively controls the generation of potential cancerogenic substance acrylamide in the high-temperature processing of starch-containing foods, and has great application prospect in the field of food processing.
The invention separates an acinetobacter Y-3 from soil beside Yongxing town hot spring in Haikou city of Hainan province, and the classification is named acinetobacterAcinetobacter soli) The strain has been deposited in China general microbiological culture Collection center, with the address: the collection number of the strain is CGMCC NO 14831 in the national institute of microbiology, national institute of sciences, no.1, no. 3, beijing, chaoyang district, north Chen, west road.
An L-asparaginase gene AsAase derived from Acinetobacter as described aboveAcinetobacter soli) Y-3 has a nucleotide sequence shown in SEQ ID NO. 1.
The L-asparaginase gene AsAase encodes an L-asparaginase.
A method for preparing L-asparaginase, comprising the steps of:
(1) Performing PCR amplification of the L-asparaginase gene AsAase described above;
(2) Constructing an L-asparaginase gene AsAase prokaryotic expression vector;
(3) Recombinant L-asparaginase is expressed in prokaryotic cells.
In the preparation method of the L-asparaginase, the following primers are adopted for PCR amplification in the step (1):
F1-SacI：CGAGCTCATGAATAAAATTGCCTTAATTT；
R1-XhoI：CCGCTCGAGTTAATCCTCAGCCTTAATGGTGG；
F2-BglII：TGCAAAAGCCGCAGCAGATCTATGAATAAAATTGCCTT；
R2-KpnI：GAATTCGAGCTCCCGGGTACCTTAATCCTCAGCCTTA。
the preparation method of the L-asparaginase comprises the step (1)The PCR amplification system is as follows: 2 XTaq Master Mix 25. Mu.L, DNA template 2. Mu.L, dd H 2 O19. Mu.L, 2. Mu.L each of the upstream and downstream primers; PCR amplification conditions: pre-denaturation at 94℃for 3 min; denaturation at 94℃for 50s, annealing at 50℃for 50s, elongation at 72℃for 60s,30 cycles; extending at 72℃for 10min.
In the preparation method of the L-asparaginase, the expression vector used in the step (2) is any one of pET-30a, pET-32a, PCBS221 or PCBS 345.
In the preparation method of the L-asparaginase, the prokaryotic expression host used in the step (3) is escherichia coli or bacillus subtilis.
The application of the L-asparaginase in inhibiting the production of acrylamide in foods.
In the application, the raw materials are soaked in an L-asparaginase solution with the concentration of 20-30IU/mL, the material ratio is 1:2-1:3, the soaking temperature is 35-40 ℃, the time is 0.5-1.5h, and the acrylamide content can be reduced by 40-60%.
1. The invention digs Acinetobacter @Acinetobacter soli) The L-asparaginase gene derived from Y-3 and the expression thereof in prokaryotic cells are realized, and a novel genetic engineering strain for high-yield L-asparaginase is constructed.
2. The recombinant L-asparaginase AsAase is applied to fried foods, and the generation of acrylamide in the fried foods is effectively inhibited after the L-asparaginase is treated.
Drawings
FIG. 1 shows agarose gel electrophoresis patterns of an Acinetobacter Y-3L-asparaginase gene fragment
M, DNAMmarker: DS2000;1: acinetobacter Y-3L-asparaginase gene fragment AsAase
FIG. 2 is a schematic diagram showing electrophoresis of purified protein expressed by recombinant L-asparaginase
M: protein marker 26610;1: acinetobacter Y-3L-asparaginase purified protein
FIG. 3 is an acrylamide solution quality test chart of potato chips before and after treatment with L-asparaginase
A: diluting acrylamide extract of potato chips which are not subjected to enzyme treatment, and detecting a map of the liquid; b: enzyme-treated potato chips fried acrylamide extract liquid quality detection map
Detailed Description
Example 1
Acinetobacter @Acinetobacter soli) Cloning of Y-3L-asparaginase Gene
(1) Acinetobacter @Acinetobacter soli) Y-3 whole genome extraction: acinetobacter Y-3 thalli are collected by centrifugation, and the whole genome of the bacteria is extracted by adopting a Bacterial DNA kit kit of OMGA company.
(2) L-asparaginase primer design: according to NCBI databaseAcinetobacter soliThe AsAase gene sequence in the whole genome nucleic acid sequence is designed, and PCR upstream and downstream primers F1, R1, F2 and R2 of the L-asparaginase gene are designed:
F1：5’ CGAGCTCATGAATAAAATTGCCTTAATTT 3’(SacI)
R1：5’ CCGCTCGAGTTAATCCTCAGCCTTAATGGTGG 3’(XhoI)
F2：5’TGCAAAAGCCGCAGCAGATCTATGAATAAAATTGCCTT 3’(BglII)
R2：5’ GAATTCGAGCTCCCGGGTACCTTAATCCTCAGCCTTA 3’(KpnI)
(3) Cloning of the L-asparaginase gene: the whole genome of the acinetobacter Y-3 is used as a template, the primers designed above are used for PCR amplification, and a PCR amplification system is adopted: 2 XTaq Master Mix 25. Mu.L, DNA template 2. Mu.L, dd H 2 O19. Mu.L, 2. Mu.L each of the upstream and downstream primers; PCR amplification conditions: pre-denaturation at 94℃for 3 min; denaturation at 94℃for 50s, annealing at 50℃for 50s, elongation at 72℃for 60s,30 cycles; extending at 72℃for 10min. Gel recovery and purification are carried out by adopting a gel recovery kit, the purified product is connected with a pMD19-T carrier at 16 ℃ overnight, and the purified product is transformed into competent cells by a heat shock methodE.coliDH 5. Alpha. Was plated on ampicillin (Amp) resistant plates, positive transformants were picked up for sterilization (as shown in FIG. 1) and sent to the golden St. Nanj company for sequencing.
The sequencing result is analyzed by computer software to obtain a 981bp fragment (shown as SEQ ID NO. 1), namely the Acinetobacter L-asparaginase gene AsAase, which codes for a protein consisting of 326 amino acids.
Example 2
Expression and purification of recombinant L-asparaginase in E.coli
(1) Construction of an expression vector: the pMD19-T-AsAase and vector pET30a (+) were usedSac I andXhoi, double enzyme digestion is carried out at 37 ℃ for 9h. Recovery of target fragment, T, using gel recovery kit 4 The enzyme was ligated overnight at 16℃and transformed into competent cellsE.coliDH 5. Alpha. Was plated on kanamycin (Kana) resistant plates, positive transformants were picked up for enrichment, and the extracted plasmid was designated pET30a (+) -AsAase. Transformation of plasmid pET30a (+) -AsAase into competent cellsE.coliBL21 (DE 3), plated on Kana resistant plates, positive transformants were picked and sequenced.
(2) Expression of recombinant L-asparaginase: the recombinant bacteria were inoculated into LB liquid medium containing 100. Mu.g/mL Kana, cultured overnight at 37℃and 180rpm, and seed liquid was prepared. Then, the strain was transferred to 100ml of LB liquid medium containing the corresponding antibiotic at an inoculum size of 1%, between 37℃and OD 0.6-0.8, 100. Mu.l of IPTG (100 mg/ml) was added, and expression was induced overnight at 20 ℃.
(3) Purification of recombinant L-asparaginase: and (3) centrifugally collecting thalli at a low temperature, suspending thalli in a buffer solution, performing ultrasonic crushing, and centrifugally taking the supernatant again to obtain a crude enzyme solution. Since the target protein is fused with a histidine tag during plasmid construction, ni can be used 2+ -NTA affinity column purification of recombinant L-asparaginase. The method comprises the following steps:
loading: the final concentration of the sample is adjusted to 5mM imidazole to enhance adsorptivity, and the sample is subjected to membrane filtration treatment; ni before loading 2+ The NTA affinity column was equilibrated with 10 column volumes of 5mM imidazole buffer; sample is circularly loaded and adsorbed for 3 times; eluting the unadsorbed hetero protein with 5mM imidazole buffer solution after loading, and eluting the hetero protein with 50mM imidazole buffer solution;
eluting: eluting the hetero protein, eluting with 10 times of medium volume of 200mM imidazole buffer solution, collecting effluent liquid to obtain purified sample, and dialyzing to remove imidazole to obtain purified protein. Purified proteins were subjected to SDS-PAGE electrophoresis (as shown in FIG. 2).
Example 3
Expression and purification of recombinant L-asparaginase in bacillus subtilis
(1) Construction of an expression vector: the pMD19-T-AsAase and the vector PCBS345 were usedBglII and IIKpnI, double enzyme digestion is carried out at 37 ℃ for 9h. Recovery of target fragment, T, using gel recovery kit 4 The enzyme was ligated overnight at 16℃and transformed into competent cellsE.coliDH5 alpha is coated on a resistance plate, positive transformant is picked up for enrichment, and the extracted plasmid is named PCBS345-AsAase. Transformation of plasmid PCBS345-AsAase into competent cellsB. subtilis 001, picking positive transformants for sequencing.
(2) Expression of recombinant L-asparaginase: the recombinant bacteria are inoculated in a fermentation culture medium and cultured for 24-48h at 37 ℃ and 180 rpm.
(3) Purification of recombinant L-asparaginase: and (5) centrifugally collecting thalli at a low temperature, wherein the supernatant is crude enzyme liquid. The crude enzyme solution purification method is as follows:
ammonium sulfate fractional precipitation: adding ground (NH) to the supernatant at low temperature 4 ) 2 SO 4 Standing the powder at 0deg.C until the powder has different saturation, centrifuging to collect supernatant, and precipitating with (NH) 4 ) 2 SO 4 Is dissolved in Tris-HCl buffer.
Hydrophobic chromatography: and (3) after the redissolved precipitate passes through a membrane, loading the precipitate to a hydrophobic chromatography column, and collecting target proteins by gradient elution and removing salt ions by dialysis.
Example 4: application of L-asparaginase in food
The use of L-asparaginase in food has been supported by many prior art examples, which use potato chips as an example for application tests, as follows:
1. test procedure
(1) Potato chip manufacturing: cleaning potato, peeling, cutting into 2mm slices, and washing with ultrapure water to clean starch granules adsorbed on the surface of potato. Soaking potato chips in 20-30IU/mL pure enzyme solution at 35-40deg.C for 0.5-1 hr. Drying in an oven, frying, and cooling to room temperature.
(2) Separation and purification of acrylamide in a sample: 2g of the crushed sample is mixed with 10mL of deionized water, vortexed, subjected to ultrasonic treatment for 30min, centrifuged at 8000rpm for 10min, the supernatant is added with n-hexane in a ratio of 1:1 for degreasing, centrifuged at 8000rpm for 10min, and the aqueous phase is degreased once again. 5mL of methanol and 5mL of water are used for activating an ACA solid phase extraction column, a water phase is sampled, 5mL of water is used for leaching, a small column is dried by pumping, 5mL of methanol is used for eluting, nitrogen is blown to dryness, 0.5mL of ultrapure water is used for redissolving, and the acrylamide content of the liquid is measured after passing through a 0.22 mu m filter membrane.
2. Effect of L-asparaginase in inhibiting acrylamide formation in potato chips
The concentration of acrylamide extracted from potato chips after L-asparaginase treatment is reduced by 40-60% (as shown in FIG. 3) compared to that before treatment, which is effective in inhibiting the production of acrylamide in fried foods.
Sequence listing
<110> Nanjing agricultural university
<120> an Acinetobacter Y-3L-asparaginase gene, expression and use thereof
<140> 201811384145 .0
<141> 2018-11-20
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 980
<212> DNA
<213> acinetobacter soli
<400> 1
atgaataaaa ttgccttaat ttgtatgggt ggaacctttg gttgtattgg tgaaccgctt 60
gcaccaatgc cctatgatgc ctttcaaccc caattagaac gcattcttcc accacaatac 120
gcagtcgatt gttttaaagc cccttcaatt aaagatagta gcgcctgcac cgccgcagac 180
tggttgttac tggtacaaca gattaagggg ttgcagcttg agggctatac acactttgtg 240
gtgattcacg gtaccgatac catgagttat gcgtctgcaa ctttggcacg ctttttggga 300
caaacctgtc acgtggtgat cacgggtagt cagtatccgc tgcttaacac tgcgggcgac 360
gatacacgtg agtttaccga tgcgaacgag aatttatact ttgcattaga gagcgttcaa 420
cgtttagcaa gcggtaccta tcaggctttt catcatcaaa tttttcacgg tcaaaccgtt 480
cttaaaaccc atacaactga gctggatgca ttttccggtg tctcggccga tgcaccttgt 540
gatacggtca caaatgcgat ggctgtagat gctgccatgg ttgcacgtgc cgatgaactg 600
gccatagtga atctgatgca tcaacctgtg cgtccagcgc acttggcaca gcaacttgaa 660
aatattgcac agcgaccacc gcacttttta attttacaag gattcggaac cggtaatatt 720
gcggtcaatg acgccattct ggcgcagttt caacaattac gagcccaagg ctgtctggtg 780
gtgttaacta cacaagtcac cttcggcaaa attgatcagc gctatgccat tagtgactgg 840
gtacattgtg caggcattgc caccaatacc acgcatggtc atgccgatct gtatgccaag 900
ctgctacaac tttacttaga gtatgatgat gctgaaggtt ggtatcgaca ctgggatacc 960
accattaagg ctgaggatta 980
<210> 2
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 2
cgagctcatg aataaaattg ccttaattt 29
<210> 3
<211> 32
<212> DNA
<213> Artificial Sequence
<400> 3
ccgctcgagt taatcctcag ccttaatggt gg 32
<210> 4
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 4
tgcaaaagcc gcagcagatc tatgaataaa attgcctt 38
<210> 5
<211> 37
<212> DNA
<213> Artificial Sequence
<400> 5
gaattcgagc tcccgggtac cttaatcctc agcctta 37

Claims (10)

1. Acinetobacter from soilAcinetobacter soli) Y-3 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO:14831.
2. An L-asparaginase gene AsAase derived from Acinetobacter as set forth in claim 1Acinetobacter soli) Y-3 has a nucleotide sequence shown in SEQ ID NO. 1.
3. The L-asparaginase encoded by the L-asparaginase gene AsAase of claim 2.
4. A method for preparing L-asparaginase, comprising the steps of:
(1) Performing PCR amplification of the L-asparaginase gene AsAase of claim 2;
(2) Constructing an L-asparaginase gene AsAase prokaryotic expression vector;
(3) Recombinant L-asparaginase is expressed in prokaryotic cells.
5. The method of claim 4, wherein the PCR amplification in step (1) uses the following primers:
F1-SacI：CGAGCTCATGAATAAAATTGCCTTAATTT；
R1- XhoI：CCGCTCGAGTTAATCCTCAGCCTTAATGGTGG；
F2- BglII：TGCAAAAGCCGCAGCAGATCTATGAATAAAATTGCCTT；
R2- KpnI：GAATTCGAGCTCCCGGGTACCTTAATCCTCAGCCTTA。
6. the method of claim 4, wherein the PCR amplification system in step (1) is: 2 XTaq Master Mix 25. Mu.L, DNA template 2. Mu.L, dd H 2 O19. Mu.L, 2. Mu.L each of the upstream and downstream primers; PCR amplification conditions: pre-denaturation at 94℃for 3 min; denaturation at 94℃for 50s, annealing at 50℃for 50s, elongation at 72℃for 60s,30 cycles; extending at 72℃for 10min.
7. The method according to claim 4, wherein the expression vector used in the step (2) is any one of pET-30a, pET-32a, PCBS221 or PCBS 345.
8. The method according to claim 4, wherein the prokaryotic cell used in the step (3) is Escherichia coli or Bacillus subtilis.
9. Use of the L-asparaginase according to claim 3 in the inhibition of acrylamide formation in food products.
10. The use according to claim 9, wherein the raw material is soaked in an L-asparaginase solution having a concentration of 20-30IU/mL, the material to L-asparaginase solution ratio is 1:2-1:3, the soaking temperature is 35-40 ℃, and the time is 0.5-1.5h.
CN201811384145.0A 2017-11-29 2018-11-20 Acinetobacter Y-3L-asparaginase gene and expression and application thereof Active CN109337845B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201711228833.3A CN108728371A (en) 2017-11-29 2017-11-29 The expression and application of a kind of acinetobacter calcoaceticus Y-3 and its altheine enzyme gene
CN2017112288333 2017-11-29

Publications (2)

Publication Number Publication Date
CN109337845A CN109337845A (en) 2019-02-15
CN109337845B true CN109337845B (en) 2023-11-07

Family

ID=63940326

Family Applications (2)

Application Number Title Priority Date Filing Date
CN201711228833.3A Withdrawn CN108728371A (en) 2017-11-29 2017-11-29 The expression and application of a kind of acinetobacter calcoaceticus Y-3 and its altheine enzyme gene
CN201811384145.0A Active CN109337845B (en) 2017-11-29 2018-11-20 Acinetobacter Y-3L-asparaginase gene and expression and application thereof

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN201711228833.3A Withdrawn CN108728371A (en) 2017-11-29 2017-11-29 The expression and application of a kind of acinetobacter calcoaceticus Y-3 and its altheine enzyme gene

Country Status (1)

Country Link
CN (2) CN108728371A (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110256535B (en) * 2019-06-14 2021-01-01 中国石油大学(华东) L-asparaginase XiDL and coding gene and application thereof
CN112725318A (en) * 2020-12-31 2021-04-30 南京农业大学 Preparation method and application of mycobacterium gordonii L-asparaginase

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
asparaginase [Acinetobacter soli];NCBI Reference Sequence: WP_025095353.1;《GenBank》;20170816;第1页 *
陈辉.降低丙烯酰胺危害的措施.《食品安全概论》.中国轻工业出版社,2011,(第1版),第129-130页. *

Also Published As

Publication number Publication date
CN109337845A (en) 2019-02-15
CN108728371A (en) 2018-11-02

Similar Documents

Publication Publication Date Title
US7695751B2 (en) Detoxifizyme with activity of transforming aflatoxin and the gene encodes thereof
CN110527674A (en) Polypeptide, its polynucleotides of separation, and the additive comprising polypeptide, its purposes and method
CN112961846B (en) Zearalenone degrading enzyme mutant with improved enzyme activity and coding gene and application thereof
CN110669745B (en) Zearalenone degrading enzyme mutant with improved thermal stability and application thereof
CN109337845B (en) Acinetobacter Y-3L-asparaginase gene and expression and application thereof
CN112760299B (en) Glucose oxidase mutant with improved thermal stability as well as coding gene and application thereof
CN107760621A (en) Iprodione degradation bacteria, digestive enzyme IpaH and its encoding gene ipaH and its application
CN111394326B (en) Vomitoxin degrading enzyme DDH and application thereof in detoxification of trichothecene toxins
JP4663631B2 (en) AMP deaminase derived from actinomycetes and use thereof
CN104212757A (en) Method for high-efficiently producing L-theanine through production of coli [gamma]-glutamylmethylamine synthetase with escherichia coli
CN117264975A (en) Cloning, expression and application of vomitoxin detoxification enzyme gene
CN109097316B (en) Method for improving yield of antifungal peptide bacillus D by over-expressing degU gene
CN109022474B (en) Method for improving yield of antifungal peptide bacillus D by over-expressing spo0A gene
CN107022536B (en) Cellulase mutant with high catalytic efficiency, and gene and application thereof
CN108410843B (en) New pullulanase, and coding gene and application thereof
CN108102934B (en) Aspergillus niger strain capable of producing pectin lyase at high yield
CN109593661B (en) Gene engineering bacterium for producing echinocandin B and construction method and application thereof
CN113355334B (en) Corn salt-tolerant gene and application thereof
CN113174352B (en) Bacillus subtilis HF1 mutant and construction method and application thereof
CN110117583A (en) Thermostabilization and the ratio phytase ECAPPA mutant living improved and its gene and application
CN114958804A (en) Neutral phytase mutant
CN111549007B (en) Transaminase TSTA, preparation method and application
Tork et al. New tannase-producing Lactobacillus Sp. Nrc10: Gene cloning, enzyme purification, and characterization
CN107058267B (en) Modified lignocellulose and application thereof
CN112410319B (en) Nuclease At _ NrnCM and coding gene 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