CN106520814A - Thermo-stable nuclease and preparation method thereof - Google Patents
Thermo-stable nuclease and preparation method thereof Download PDFInfo
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- CN106520814A CN106520814A CN201611236993.8A CN201611236993A CN106520814A CN 106520814 A CN106520814 A CN 106520814A CN 201611236993 A CN201611236993 A CN 201611236993A CN 106520814 A CN106520814 A CN 106520814A
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- nucleic acid
- thermostable nucleic
- acid enzyme
- nuclease
- enzyme
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- 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
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- 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)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
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- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
Abstract
The invention discloses thermo-stable nuclease and a preparation method thereof. The nuclease prepared by the invention is high-temperature nuclease, and the nuclease, within a temperature range from 50 degrees to 70 degrees, achieves the maximum activity. Meanwhile, the nuclease, as an important enzymatic preparation capable of eliminating nucleic acid pollution in a biological production, can simultaneously hydrolyze DNA and RNA. The thermo-stable nuclease provided by the invention has the beneficial effects that the nuclease provided by the invention is thermo-stable protein, which is convenient for preservation and transportation at normal temperature and is capable of reducing storage cost.
Description
Technical field
The present invention relates to belong to gene engineering technology field, more particularly, to a kind of thermostable nucleic acid enzyme and its preparation side
Method.
Background technology
Nuclease is the enzyme that a class can hydrolyze the nucleic acid molecules such as DNA and RNA.According to substrate preference difference, nuclease point
For RNA nucleases and DNA nucleases.Nuclease has in the food industry and is widely used, for producing mononucleotide product, with
And the nucleic acid impurities gone in the biological products such as removing protein, sugar.Most nucleases are normal temperature nuclease, are easily lost in high temperature
It is living, inconvenience is brought to production and transport, be especially unsuitable for high temperature production technique.
The content of the invention
In order to improve the heat endurance of nuclease, its enzymatic activity and stability at high temperature is improved, the present invention is developed
A kind of thermostable nucleic acid enzyme, while the nuclease can digest RNA and DNA molecular simultaneously, is very suitable in food industry
Simultaneously eliminate RNA and two kinds of impurity of DNA, and Combination mononucleotide product cannot production technology.
Technical scheme is specific as follows.
The present invention provides a kind of preparation method of thermostable nucleic acid enzyme, comprises the following steps that：
（1）Build the recombinant expression plasmid of thermostable nucleic acid enzyme
Based on thermophilic microorganism genomic information, PCR expands the thermostable nucleic acid enzyme gene of its coding, obtains heat endurance core
Phytase gene, and by gene cloning to prokaryotic expression carrier pET28 etc., build thermostable nucleic acid enzyme recombinant expression plasmid；
（2）Recombinant expressed thermostable nucleic acid enzyme
By the thermostable nucleic acid enzyme recombinant expression plasmid conversion Bacillus coli expression host BL21 for building（DE3）Deng obtaining heat
Stability nuclease recombinant strains；Which is carried out into thermostable nucleic acid enzyme induction expression with derivant IPTG again；
（3）The thermostable nucleic acid enzyme of affinity purification expression
The Escherichia coli after abduction delivering thermostable nucleic acid enzyme are collected by centrifugation, thalline are resuspended in into non-denatured protein lysates,
Ultrasonic disruption thalline, 65 degree of heating, 30 minutes inactivation Escherichia coli impurity proteins, is collected by centrifugation containing thermostable nucleic acid enzyme
Escherichia coli cracking supernatant；Recycle immobilization nickel ion affinity purification resin that thermostable nucleic acid is purified from supernatant
Enzyme；
（4）The enzymatic activity of the thermostable nucleic acid enzyme of detection purifying, the especially enzymatic activity under high temperature, obtain thermostable nucleic acid
Enzyme.
（5）Enzyme assay will be carried out by thermostable nucleic acid enzyme after purification, and therefrom filter out
Thermostable nucleic acid enzyme.
In the present invention, step（1）In, the thermophilic microorganism includes Pyrococcus furiosus etc..
In the present invention, step（2）It is middle Fiber differentiation is carried out with derivant IPTG actual conditions be：First by heat endurance core
To OD600=0.4-1.0, adding 0.5mM derivants IPTG, that 3 are cultivated at a temperature of 37 DEG C is little for sour enzyme recombinant strains culture
Shi Jinhang abduction deliverings.
In the present invention, step（3）In, the composition of the non-denatured protein lysates is as follows：20 mM pH value are 8.0
Tris-HCl, 300 mM NaCl, 0.5mM DTT, 10vol% glycerine.
In the present invention, the amino acid sequence of the thermostable nucleic acid enzyme of the thermophilic microorganism Pyrococcus furiosus
Row are respectively such as SEQ ID NO:Shown in 1.
Compared with existing nuclease, the present invention has the marked improvement in terms of 2, specific as follows：
（1）Good heat endurance.Nuclease derives from high temperature microbe, and the growth of microorganism temperature is 100 degree, therefore the core
Sour enzyme it is thermally-stabilised very good, be incubated 2 hours at 80 degree, activity is substantially reduced, and is highly convenient for storage transport.
（2）Extensive substrate spectrum.The nuclease can hydrolyze DNA and RNA simultaneously, can be removed in biological products simultaneously
Two kinds of nucleic acid impurities.For producing during mononucleotide product, a kind of nuclease can just prepare Combination mononucleotide product.
Description of the drawings
Temperature-independents of the Fig. 1 for the thermostable nucleic acid enzymatic activity of thermophilic microorganism Pyrococcus furiosus
Linearity curve.
Specific embodiment
With reference to the accompanying drawings and examples, the specific embodiment of the present invention is further described.Following examples are only
For clearly illustrating technical scheme, and can not be limited the scope of the invention with this.
Embodiment：
The preparation of thermophilic microorganism Pyrococcus furiosus thermostable nucleic acid enzymes
The first step, design synthesis thermophilic microorganism Pyrococcus furiosus thermostable nucleic acid enzyme genes, and insert
PET28 expression vectors, build the recombinant expression plasmid of thermostable nucleic acid enzyme.Restructuring thermophilic microorganism Pyrococcus
Furiosus thermostable nucleic acid enzymes N-terminal is used for 6 continuous histidine affinity purification labels from pET28 carriers
Immobilization nickel ion affinity chromatograph is purified.
Second step, recombinant expressed thermophilic microorganism Pyrococcus furiosus thermostable nucleic acid enzymes.By thermophilic micro- life
Thing Pyrococcus furiosus thermostable nucleic acid enzymes recombinant expression plasmid converts Bacillus coli expression host BL21
（DE3）, obtain thermophilic microorganism Pyrococcus furiosus thermostable nucleic acid enzyme recombinant strains.Bacterial strain will be expressed
Cultivate to OD600=0.6, add 0.5mM derivant IPTG, cultivate 12 hours at a temperature of 16 DEG C, induce thermophilic microorganism
Pyrococcus furiosus thermostable nucleic acid expression of enzymes.
The amino acid sequence of thermophilic microorganism Pyrococcus furiosus thermostable nucleic acid enzyme recombinant proteins（Nitrogen end
→ carbon teminal）Such as SEQ IDNO:Shown in 1.
3rd step, thermophilic microorganism Pyrococcus furiosus thermostable nucleic acid enzyme affinity purifications.Step 2 is lured
After Escherichia coli after leading are collected by centrifugation, thalline is resuspended in protein lysate（20 mM Tris-HCl, pH 8.0, 300
MM NaCl, 0.5mM DTT, 10vol% glycerine）.Ultrasonic disruption thalline, is heated and inactivated Escherichia coli impurity protein, centrifugation
Collect the cellular lysate supernatant containing thermophilic microorganism Pyrococcus furiosus thermostable nucleic acid enzymes.Using fixation
Change nickel ion affinity purification purifying resin thermophilic microorganism Pyrococcus furiosus thermostable nucleic acid enzymes.
4th step, detects the enzymatic activity and enzyme activity of thermophilic microorganism Pyrococcus furiosus thermostable nucleic acid enzymes
Temperature dependency.DNA and RNA molecule by the use of 40 nucleotides of length adds thermophilic microorganism Pyrococcus as substrate
Furiosus thermostable nucleic acid enzymes, determine the enzymatic activity temperature dependency of thermostable nucleic acid enzyme.Measurement result shows thermophilic
The thermostable nucleic acid enzyme of microorganism Pyrococcus furiosus codings has highest activity in the range of 50-70 degree, together
When the albumen there is good heat endurance, concrete outcome is shown in Fig. 1.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, on the premise of without departing from the technology of the present invention principle, some improvements and modifications can also be made, these improvements and modifications
Also should be regarded as protection scope of the present invention.
The amino acid sequence of thermostable nucleic acid enzyme：SEQ ID NO:1
1 MDKEGFLNKV REAVDVVKLH IELGHTIRII SHRDADGITS AAILAKALGR EGASFHISIV
61 KQVSEDLLRE LKDEDYKIFI FSDLGSGSLS LIKEYLKEKT VIILDHHPPE NVKLEEKHIL
121 VNPVQFGANS VRDLSGSGVT YFFARELNEK NRDLAYIAIV GAVGDMQEND GVFHGMNLDI
181 IEDGKSLGIL EVKKELRLFG RETRPLYQML AYATNPEIPE VTGDERKAIE WLKNKGFNPE
241 KKYWELSEEE KKKLHDFLII HMIKHGAGKE DIDRLIGDVV ISPLYPEGDP RHEAREFATL
301 LNATGRLNLG NLGVAVCLGD EEAFRKALKM VEDYKREQIE ARKWLLQNWN SEVWEGDHVY
361 VLYVGKSIRD TLVGIAASMA INAGLADPEK PVIVFADTDE DPNLLKGSAR TTERALAKGY
421 NLGEALRKAA ELVNGEGGGH AIAAGIRIPR ARLAEFRKLI DKILGEQVSK GGDKSES
1 MDKEGFLNKV REAVDVVKLH IELGHTIRII SHRDADGITS AAILAKALGR EGASFHISIV
61 KQVSEDLLRE LKDEDYKIFI FSDLGSGSLS LIKEYLKEKT VIILDHHPPE NVKLEEKHIL
121 VNPVQFGANS VRDLSGSGVT YFFARELNEK NRDLAYIAIV GAVGDMQEND GVFHGMNLDI
181 IEDGKSLGIL EVKKELRLFG RETRPLYQML AYATNPEIPE VTGDERKAIE WLKNKGFNPE
241 KKYWELSEEE KKKLHDFLII HMIKHGAGKE DIDRLIGDVV ISPLYPEGDP RHEAREFATL
301 LNATGRLNLG NLGVAVCLGD EEAFRKALKM VEDYKREQIE ARKWLLQNWN SEVWEGDHVY
361 VLYVGKSIRD TLVGIAASMA INAGLADPEK PVIVFADTDE DPNLLKGSAR TTERALAKGY
421 NLGEALRKAA ELVNGEGGGH AIAAGIRIPR ARLAEFRKLI DKILGEQVSK GGDKSES
Claims (5)
1. a kind of thermostable nucleic acid enzyme and preparation method thereof, it is characterised in that comprise the following steps that：
（1）Build the recombinant expression plasmid of thermostable nucleic acid enzyme
Based on thermophilic microorganism genomic information, PCR expands the thermostable nucleic acid enzyme of its coding, and nuclease gene is cloned
To prokaryotic expression carrier pET28, thermostable nucleic acid enzyme recombinant expression plasmid is built；
（2）Recombinant expressed thermostable nucleic acid enzyme
By the thermostable nucleic acid enzyme recombinant expression plasmid conversion Bacillus coli expression host BL21 for building（DE3）, obtain hot steady
Qualitative nuclease recombinant strains；Which is carried out into thermostable nucleic acid enzyme induction expression with derivant IPTG again；
（3）The thermostable nucleic acid enzyme of affinity purification expression
The Escherichia coli after abduction delivering thermostable nucleic acid enzyme are collected by centrifugation, thalline are resuspended in into non-denatured protein lysates,
Ultrasonic disruption thalline, in 65 degree of heating, 30 minutes inactivation overwhelming majority e. coli proteins, is collected by centrifugation large intestine bar after heating
Bacterium cracks supernatant；Recycle immobilization nickel ion affinity purification resin that thermostable nucleic acid enzyme is purified from supernatant；
（4）The enzymatic activity and peak optimization reaction temperature of detection thermostable nucleic acid enzyme, obtains highly active thermostable nucleic acid enzyme；
（5）The thermostable nucleic acid enzyme of purifying is carried out into enzymatic activity and peak optimization reaction temperature measuring, its enzymatic activity and enzyme activity is identified
Property temperature dependency.
2. preparation method according to claim 1, it is characterised in that：Step（1）In, the thermophilic microorganism includes
Pyrococcus furiosus。
3. preparation method according to claim 1, it is characterised in that：Step（2）Middle derivant IPTG carries out induction training
Foster actual conditions is：Thermostable nucleic acid enzyme recombinant strains culture is added into 0.5mM to OD600=0.4-1.0 first
Derivant IPTG is cultivated 3 hours at a temperature of 37 DEG C and is carried out abduction delivering.
4. the thermostable nucleic acid enzyme that the preparation method as described in one of claims 1 to 3 is obtained.
5. thermostable nucleic acid enzyme as claimed in claim 4, wherein its amino acid sequence such as SEQ ID NO:Shown in 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201611236993.8A CN106520814A (en) | 2016-12-28 | 2016-12-28 | Thermo-stable nuclease and preparation method thereof |
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| CN201611236993.8A CN106520814A (en) | 2016-12-28 | 2016-12-28 | Thermo-stable nuclease and preparation method thereof |
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| CN106520814A true CN106520814A (en) | 2017-03-22 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110959038A (en) * | 2017-05-15 | 2020-04-03 | C-乐克塔股份有限公司 | Enzyme products |
| CN112080485A (en) * | 2020-09-21 | 2020-12-15 | 北京格源天润生物技术有限公司 | Method for extracting ribonuclease from bovine pancreas |
-
2016
- 2016-12-28 CN CN201611236993.8A patent/CN106520814A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110959038A (en) * | 2017-05-15 | 2020-04-03 | C-乐克塔股份有限公司 | Enzyme products |
| CN112080485A (en) * | 2020-09-21 | 2020-12-15 | 北京格源天润生物技术有限公司 | Method for extracting ribonuclease from bovine pancreas |
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Application publication date: 20170322 |