CN105062997A - L-asparaginase mutant with improved enzyme activity and construction method thereof - Google Patents

L-asparaginase mutant with improved enzyme activity and construction method thereof Download PDF

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CN105062997A
CN105062997A CN201510526470.6A CN201510526470A CN105062997A CN 105062997 A CN105062997 A CN 105062997A CN 201510526470 A CN201510526470 A CN 201510526470A CN 105062997 A CN105062997 A CN 105062997A
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mutant
enzyme
seqidno
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饶志明
龙水清
张显
杨套伟
徐美娟
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Jiangnan University
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Institute Of Food Biotechnology Jiangnan University (rugao)
Rugao Jiangda Food Biotechnology Research Institute Co Ltd
Jiangnan University
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Priority to PCT/CN2015/094554 priority patent/WO2017031839A1/en
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    • 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
    • A23L5/10—General methods of cooking foods, e.g. by roasting or frying
    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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Abstract

The invention discloses an L-asparaginase mutant with the improved enzyme activity and a construction method thereof, and belongs to the field of gene engineering. According to the L-asparaginase mutant, on the basis of amino acid shown in the SEQ ID NO.2, the 107th glycine is mutated into aspartic acid. The obtained mutant is expressed in bacillus subtilis, fermentation is performed in a shake flask for 24 h and then the enzyme activity is 961 U/mL; the enzyme activity of the mutant is improved by 80%, the appetency of a substrate is decreased by 50% compared with protoenzyme, the catalytic efficiency is improved by 84%, and meanwhile the specific enzyme activity is improved by 83%. According to the L-asparaginase mutant, it is shown that the 107th amino acid residue has a great influence on the enzyme catalytic action, a certain foundation is provided for research on the enzyme catalytic mechanism, and the enzyme industrial application potential is improved.

Description

The altheine enzyme mutant of a kind of enzyme raising alive and construction process thereof
Technical field
The present invention relates to altheine enzyme mutant and the construction process thereof of the raising alive of a kind of enzyme, belong to gene engineering technology field.
Background technology
Altheine hydrolytic deaminization base can be formed L-Aspartic acid and ammonia by L-ASP (L-asparaginaseamidohydrolase, E.C.3.5.1.1).L-ASP has anti-tumor activity, and be applied to treatment acute lymphoblastic leukemia and Huo Jinsen disease etc. at present, Recent study finds that L-ASP can also reduce the generation of acrylamide in fried food product.L-ASP size, structure and character are different because originating difference.Altheine enzyme source is relatively more extensive, all finds containing L-ASP in guinea pig serum, plant and microorganism.
Heterogenous expression L-ASP distinct issues are, expressing quantity is low, L-ASP enzyme is lived low.Therefore, Fixedpoint mutation modified L-ASP, improves enzymatic activities, significant for raising L-ASP industrial applications prospect.
Summary of the invention
The present invention provide firstly a kind of enzyme altheine enzyme mutant improved alive, and its aminoacid sequence is the sequence shown in SEQIDNO.1.
The nucleotide sequence of described mutant of encoding is the sequence shown in SEQIDNO.3.
Described mutant is on the amino acid whose basis of amino acid as shown in sequence SEQIDNO.2, and 107 amino acids are become aspartic acid by glycine mutation.
Present invention also offers a kind of genetic engineering bacterium of expressing described altheine enzyme mutant.
The preparation method of described genetic engineering bacterium, on the basis of nucleotide sequence shown in SEQIDNO.4, the codon mutation of the glycine of coding the 107th has been become the codon of codes for aspartate, obtain recombination, recombination is connected to expression vector and obtains recombinant plasmid, in recombinant plasmid transformed to subtilis Host Strains, namely obtain Bacillus subtilis genes engineering bacteria.
In one embodiment of the invention, described expression vector is pMA5.
In one embodiment of the invention, described preparation method, specifically:
(1) arrange as template with nucleotides sequence shown in SEQIDNO.4, Flprimer (sequence is as shown in SEQIDNO.5), Rlprimer (sequence is as shown in SEQIDNO.6) is primer, carries out PCR and namely obtains the recombination G107D shown in SEQIDNO.3.
(2) by recombination sequence obtained in the previous step; be connected in pMA5 expression vector, obtain recombinant plasmid pMA5-G107D, recombinant plasmidization transforms B.subtilis168; obtain recombined bacillus subtilis engineering strain, called after pMA5-G107D/B.subtilis168.
The present invention is on the basis of natural L-ASP, and by rite-directed mutagenesis biotechnology transformation altheine enzyme molecular structure, the pure enzyme liquor ratio enzyme of mutant enzyme improves 83% before living and comparatively suddenling change.The substrate avidity K of mutant enzyme G107D mcomparatively reduce by 50% before sudden change, and catalytic efficiency improves (k catwith K mratio) 84%.The present invention shows that 107 katalysis of amino acids residue to enzyme have considerable influence, provides certain basis, and improve the industrial application potentiality of this enzyme to the research of the catalytic mechanism of this enzyme.Gained of the present invention can be used for the medicine preparing treatment acute lymphoblastic leukemia and Huo Jinsen disease, also can be used for the generation reducing acrylamide in fried food product.
Embodiment
Embodiment 1 is containing the structure of the recombinant vectors of altheine enzyme mutant
(1) acquisition of G107D mutant: arrange as template with nucleotides sequence shown in SEQIDNO.4, Fprimer (sequence is as shown in SEQIDNO.5), Rprimer (sequence is as shown in SEQIDNO.6) are primer, carry out PCR and namely obtain the recombination shown in SEQIDNO.3.
(2) recombination and pMA5 are used BamHI, MluI double digestion respectively, spending the night with T4DNA ligase enzyme 16 DEG C after purifying connects.Connect product chemistry method and transform JM109 competent cell.Conversion fluid coating is dull and stereotyped containing kantlex (50mg/L) LB, extracts plasmid, the recombinant plasmid that double digestion checking builds, called after pMA5-G107D.Examining order is completed by the raw work in Shanghai.
Embodiment 2 is produced L-ASP subtilis engineering bacteria and is built
Recombinant plasmid pMA5-G107D chemical method embodiment 1 obtained is transformed into B.subtilis168 competent cell, and concrete grammar is as follows:
(1) solution needed for transformation experiment following (g/L):
Sp-A:(NH 4) 2sO 44, K 2hPO 428, Trisodium Citrate 12Sp-B:MgSO 47H 2o0.4
100 × CAYE:Casaminoacid20, yeast powder 100SpI substratum: Sp-A49%, Sp-B49%, 50% glucose 2%, 100 × CAYE2%SpII substratum: SpI substratum 98%, 50mmol/LCaCl 21%, 250mmol/LMgCl 21%.115 DEG C of moist heat sterilizations.
(2) by single colony inoculation of B.Subtilis168 to (50mL centrifuge tube) in 2mLSpI substratum, 37 DEG C, 200r/min overnight incubation;
(3) 100 μ L nutrient solutions are got in 5mLSpI substratum, 37 DEG C, 200r/min is cultured to logarithmic phase (OD600 value is about 1), about 4 ~ 5h;
(4) 200 μ L nutrient solutions are got in 2mLSpII substratum, 37 DEG C, 200r/min cultivates 90min, 20 μ L10mmol/LEGTA are added after taking-up, in 37 DEG C, 200r/min continuation cultivation 10min, then be distributed into 500 μ L often to manage, add 5 μ L recombinant plasmid pMA5-G107D, mixing, 37 DEG C, 200r/min cultivates 90min, gets bacterium liquid coating resistant panel.Cultivate 12h for 37 DEG C, picking positive transformant is verified.Obtain recombinant bacterium pMA5-G107D/B.subtilis168.
Embodiment 3 recombinant bacterium pMA5-G107D/B.subtilis168L-asparagus fern phthalein amine enzyme high expression and enzyme activity determination.
(1) recombinant bacterium pMA5-G107D/B.subtilis168 embodiment 2 built and the control strain pMA5-ansz/B.subtilis168 expressing the enzyme do not suddenlyd change are inoculated in l0mL respectively containing in the LB substratum of kantlex, 37 DEG C of shaking culture are spent the night, next day by 4% inoculum size transfer in bacillus subtilis fermention medium, cultivate 24h for 37 DEG C, get fermented liquid in 4 DEG C, the centrifugal l0min of 10000r/min, supernatant is the outer crude enzyme liquids of born of the same parents, cytoclasis supernatant liquor is crude enzyme liquid in born of the same parents, for the mensuration of enzyme activity.
(2) bacillus subtilis fermention medium: soy peptone 10g/L, K 2hPO 42.3g/L, KH 2pO 41.7g/L, corn steep liquor 15g/L, urea 3g/L, glucose 40g/L, MgSO 40.75g/L, NaCl5g/L.Regulate pH6.8-7.0.
(3) enzyme is lived and is defined: under 40 DEG C of reaction conditionss, can be converted into 1 μm of olNH by catalysis L-asparagus fern phthalein amine in per minute 3required enzyme amount is a Ge Meihuo unit.
(4) L-ASP enzyme activity determination method: take altheine as substrate, by being determined at the NH discharged in catalyzed reaction 3amount measure enzyme live.Reaction mixture (1mL) consists of: 400 μ L25mML-l-asparagines (being dissolved in 50mMpH7.5Tris-HCl); 400 μ L50mMpH7.5Tris-HCl; The enzyme solution of 100 μ L proper concns.Reaction mixture, at 40 DEG C, under pH7.5 condition, after reaction 15min, adds 100 μ L15% (W/V%) solution of trichloroacetic acid termination reactions.The reaction solution of trichoroacetic acid(TCA) termination reaction is added as blank before enzyme reaction.Reaction mixture is centrifugal 10min under 20000g condition, gets 200uL supernatant liquor and joins in the deionized water of 4.8mL.In above-mentioned system, add the Nessler's reagent of 200 μ L, under being determined at 450nm wavelength, survey absorbancy, the NH discharged by color reaction survey enzyme reaction 3amount.
(3) result shows that total enzyme of the L-ASP that recombinant bacterium pMA5-G107D/B.subtilis168 expresses (with the summation of enzymatic activities in born of the same parents) alive is 961U/mL, raising 80% more alive than control strain pMA5-ansz/.subtilis168 (534.2U/mL) L-ASP enzyme.
(4) the outer crude enzyme liquid of the born of the same parents that obtain of step (1) purified after obtain L-ASP G107D ansz, analyze the recombinant L-asparaginase G107D after purifying anszzymologic property, as table 1, substrate avidity K mcomparatively reduce by 50%, catalytic efficiency k before sudden change cat/ K mimprove 84%, simultaneously raising 83% more alive than enzyme.Due to the raising of catalytic efficiency, add G107D anszratio enzyme live.
Table 1G107D anszreactive kinetics parameters
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.

Claims (8)

1. an altheine enzyme mutant, is characterized in that, the aminoacid sequence of described mutant is as shown in SEQIDNO.1.
2. the gene of mutant described in coding claim 1.
3. the recombinant expression vector containing gene described in claim 2.
4. express the genetic engineering bacterium of altheine enzyme mutant described in claim 1 for one kind.
5. prepare the method for genetic engineering bacterium described in claim 4 for one kind, it is characterized in that, on the basis of sequence shown in SEQIDNO.4,107th glycine mutation is become aspartic acid, obtain recombination, recombination is linked expression vector and obtains recombinant plasmid, in recombinant plasmid transformed to subtilis Host Strains, namely obtain Bacillus subtilis genes engineering bacteria.
6. preparation method according to claim 5, it is characterized in that, described method is specifically: (1) with nucleotide sequence shown in SEQIDNO.4 for template, with the primer of sequence as shown in SEQIDNO.5, SEQIDNO.6, carry out PCR, 107 amino acids namely obtaining encoding are become the G107D mutant gene sequence of aspartic acid by glycine mutation; (2) by recombination sequence obtained in the previous step, be connected in pMA5 expression vector, obtain recombinant plasmid pMA5-G107D, recombinant plasmidization transforms B.Subtilis, obtains recombined bacillus subtilis genetic engineering bacterium.
7. the application of altheine enzyme mutant described in claim 1 in the medicine preparing treatment acute lymphoblastic leukemia and Huo Jinsen disease.
8. altheine enzyme mutant described in claim 1 is reducing the application in fried food product in acrylamide generation.
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106434612A (en) * 2016-10-21 2017-02-22 江南大学 Asparaginase mutant and application thereof
CN107828768A (en) * 2017-12-13 2018-03-23 江南大学 A kind of L asparagines enzyme mutant and its construction method
CN107988194A (en) * 2017-12-15 2018-05-04 江南大学 L-Aspartic acid α-decarboxylation the enzyme variants and its construction method that a kind of enzyme activity improves
CN108070581A (en) * 2017-12-15 2018-05-25 江南大学 L-Aspartic acid β-decarboxylation the enzyme mutant and its application that a kind of enzyme activity improves
CN108094976A (en) * 2017-12-15 2018-06-01 江南大学 Application of one plant of thermophilic L-ASP in high temperature frying food
CN108559734A (en) * 2018-01-15 2018-09-21 江南大学 The l-lactate dehydrogenase mutant and its application that a kind of catalytic efficiency improves
CN109266635A (en) * 2018-11-20 2019-01-25 江南大学 A kind of altheine enzyme mutant and its construction method that enzyme activity improves
WO2019113965A1 (en) * 2017-12-15 2019-06-20 江南大学 Thermophilic l-asparaginase mutant and screening and fermentation method therefor
CN112941059A (en) * 2021-02-23 2021-06-11 江南大学 L-asparaginase mutant and expression thereof in bacillus subtilis

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103060299A (en) * 2011-10-19 2013-04-24 北京安百胜生物科技有限公司 L-asparaginase variant with increased activity
WO2014170811A2 (en) * 2013-04-16 2014-10-23 Sonawane Avinash Novel mutant l-asparaginases
CN104371993A (en) * 2014-10-22 2015-02-25 江南大学 Asparaginase mutant with enhanced enzyme activity

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102864163A (en) * 2012-09-20 2013-01-09 江南大学 Method for efficiently expressing bacillus subtilis L-asparaginase
CN103243063A (en) * 2013-05-24 2013-08-14 江南大学 High-efficiency expression of bacillus subtilis L-asparaginase (ansZ) in escherichia coli
CN104611317B (en) * 2015-03-09 2018-07-06 江南大学 A kind of method for improving L-ASP secreting, expressing

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103060299A (en) * 2011-10-19 2013-04-24 北京安百胜生物科技有限公司 L-asparaginase variant with increased activity
WO2014170811A2 (en) * 2013-04-16 2014-10-23 Sonawane Avinash Novel mutant l-asparaginases
CN104371993A (en) * 2014-10-22 2015-02-25 江南大学 Asparaginase mutant with enhanced enzyme activity

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SHUIQING LONG ET AL.: "Amino acid residues adjacent to the catalytic cavity of tetramer l-asparaginase II contribute significantly to its catalytic efficiency and thermostability", 《ENZYME AND MICROBIAL TECHNOLOGY》 *
陈建华等: "E.coli L-天冬酰胺酶B细胞抗原表位氨基酸残基Lys196对其抗原性的影响", 《中国药科大学学报》 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106434612A (en) * 2016-10-21 2017-02-22 江南大学 Asparaginase mutant and application thereof
CN107828768A (en) * 2017-12-13 2018-03-23 江南大学 A kind of L asparagines enzyme mutant and its construction method
CN107828768B (en) * 2017-12-13 2020-10-09 江南大学 L-asparaginase mutant and construction method thereof
CN108094976A (en) * 2017-12-15 2018-06-01 江南大学 Application of one plant of thermophilic L-ASP in high temperature frying food
CN108070581A (en) * 2017-12-15 2018-05-25 江南大学 L-Aspartic acid β-decarboxylation the enzyme mutant and its application that a kind of enzyme activity improves
WO2019113965A1 (en) * 2017-12-15 2019-06-20 江南大学 Thermophilic l-asparaginase mutant and screening and fermentation method therefor
CN108094976B (en) * 2017-12-15 2020-03-06 江南大学 Application of thermophilic L-asparaginase in high-temperature fried food
CN107988194B (en) * 2017-12-15 2020-08-04 江南大学 L-aspartic acid α -decarboxylase variant with improved enzyme activity and construction method thereof
CN108070581B (en) * 2017-12-15 2020-09-04 江南大学 L-aspartate beta-decarboxylase mutant with improved enzyme activity and application thereof
CN107988194A (en) * 2017-12-15 2018-05-04 江南大学 L-Aspartic acid α-decarboxylation the enzyme variants and its construction method that a kind of enzyme activity improves
US11001825B2 (en) 2017-12-15 2021-05-11 Jiangnan University Thermophilic L-asparaginase mutant and screening and fermentation methods thereof
CN108559734A (en) * 2018-01-15 2018-09-21 江南大学 The l-lactate dehydrogenase mutant and its application that a kind of catalytic efficiency improves
CN108559734B (en) * 2018-01-15 2020-09-04 江南大学 L-lactate dehydrogenase mutant with improved catalytic efficiency and application thereof
CN109266635A (en) * 2018-11-20 2019-01-25 江南大学 A kind of altheine enzyme mutant and its construction method that enzyme activity improves
CN109266635B (en) * 2018-11-20 2020-12-01 江南大学 L-asparaginase mutant with improved enzyme activity and construction method thereof
CN112941059A (en) * 2021-02-23 2021-06-11 江南大学 L-asparaginase mutant and expression thereof in bacillus subtilis

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Applicant before: Jiangnan University

Applicant before: Institute of food biotechnology, Jiangnan University (Rugao)

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Application publication date: 20151118

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