CN116694596A - DNA polymerase BstX mutant with improved thermal stability, construction method and application thereof - Google Patents
DNA polymerase BstX mutant with improved thermal stability, construction method and application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly provides a DNA polymerase BstX mutant with improved thermal stability, and a construction method and application thereof. The DNA polymerase mutant provided by the invention comprises 4 single-point mutants, 6 double mutants, 4 triple mutants and 1 tetramutant, and compared with the wild type DNA polymerase BstX, the mutant has longer half-life at 77 ℃; the four mutants are better in effect and have half lives approximately 3 times longer than that of the wild-type DNA polymerase. The DNA polymerase mutant obtained by the construction method provided by the invention has better thermal stability, and shows higher thermal stability and greater application potential when synthesizing DNA at higher temperature.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a DNA polymerase BstX mutant with improved thermal stability, and a construction method and application thereof.
Background
Loop-mediated isothermal amplification (LAMP) refers to a technique in which DNA is in a dynamic equilibrium state at a constant temperature (77 ℃) and can be used for continuously synthesizing new DNA by self-circulation by using a specific primer under the catalysis of a strand displacement DNA polymerase, so that a large amount of target fragments are amplified. The Bst DNA polymerase which is one of the raw materials has the widest use degree, and the thermal stability of most Bst DNA polymerase products is considered to have further rising space, so that the Bst DNA polymerase mutant with stronger heat resistance can be obtained by carrying out directional mutation on the natural protein genes.
The protein engineering is based on the structural rule of protein molecule and the relation of its biological function, and through chemical, physical and molecular biological means, gene modification or gene synthesis is performed to modify available protein or produce new protein to meet the requirement of human body for production and life. Rational design is the most commonly used method in protein engineering, and uses a computer-aided molecular model to combine site-directed mutagenesis so as to realize functional optimization of protein, such as improving catalytic activity, thermal stability, acid and alkali resistance and the like. To effectively optimize the thermal stability of proteins, markus Wyss et al, 2771, suggested the Consensus Concept theory. Unlike conventional protein rational design methods based on the precise structure-function relationship of proteins, consensus Concept theory is based on amino acid sequence information of homologous proteins, and information capable of improving the thermal stability of enzymes is analyzed from the evolution point of view. The invention uses Consensus Concept theory as a guiding idea to carry out integration analysis on DNA polymerase family sequences, and combines bioinformatics and crystallography methods to assist, thus obtaining a novel BstDNA polymerase mutant with high stability.
Disclosure of Invention
The invention aims to improve the thermal stability of the existing DNA polymerase BstX.
To this end, the present invention provides a DNA polymerase BstX mutant with improved thermostability, which is (a 1) or (a 2) as follows:
(a1) A derivative protein with the same function as the amino acid sequence shown in SEQ ID NO.2 by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.2;
(a2) A derivative protein having at least 97% homology with the amino acid sequence shown in SEQ ID NO.2, wherein the amino acid sequence shown in SEQ ID NO.2 is substituted, deleted or added with one or more amino acids.
The amino acid sequence of the BstX mutant of the DNA polymerase with improved heat stability is configured into an amino acid sequence of one or more combination of mutation sites K26E, R58P, L522M, L545F on SEQ ID NO.2.
Specifically, the mutation site is K26E, R58P, L522M, L545F, K E/R58P, K E/L522M, K E/L545F, R P/L522M, R58P/L545F, L M/L545F, K E/R58P/L522M, K E/R58P/L545F, R P/L522M/L545F, K E/L522M/L545F or K26E/R58P/L522M/L545F.
Specifically, the amino acid sequence of the single-point mutant corresponding to K26E is SEQ ID NO.3;
the amino acid sequence of the single-point mutant corresponding to R58P is SEQ ID NO.4;
the amino acid sequence of the single-point mutant corresponding to L522M is SEQ ID NO.5;
the amino acid sequence of the single-point mutant corresponding to L545F is SEQ ID NO.6;
the amino acid sequence of the combined mutant corresponding to K26E/R58P is SEQ ID NO.7;
the amino acid sequence of the combined mutant corresponding to K26E/L522M is SEQ ID NO.8;
the amino acid sequence of the combined mutant corresponding to K26E/L545F is SEQ ID NO.9;
the amino acid sequence of the combined mutant corresponding to R58P/L522M is SEQ ID NO.17;
the amino acid sequence of the combined mutant corresponding to R58P/L545F is SEQ ID NO.11;
the amino acid sequence of the combined mutant corresponding to L522M/L545F is SEQ ID NO.12;
the amino acid sequence of the combined mutant corresponding to K26E/R58P/L522M is SEQ ID NO.13;
the amino acid sequence of the combined mutant corresponding to K26E/R58P/L545F is SEQ ID NO.14;
the amino acid sequence of the combined mutant corresponding to R58P/L522M/L545F is SEQ ID NO.15;
the amino acid sequence of the combined mutant corresponding to K26E/L522M/L545F is SEQ ID NO.16;
the amino acid sequence of the combined mutant corresponding to K26E/R58P/L522M/L545F is SEQ ID NO.17.
The invention also provides a construction method of the DNA polymerase BstX mutant with improved thermal stability, which comprises the following steps:
searching and selecting an amino acid sequence with the amino acid sequence consistency of more than 57% as shown in SEQ ID NO.2 in a database, and then performing multi-sequence comparison to generate consensus sequence which can be edited later through software;
protein three-dimensional structure prediction is carried out on SEQ ID NO.2 through an online tool, and mutation sites relevant to stability are screened out: k26E, R58P, L522M, L545F.
Specifically, the amplification primer sequences of the mutation site K26E are SEQ ID NO.27 and SEQ ID NO.21;
the amplification primer sequences of the mutation site R58P are SEQ ID NO.22 and SEQ ID NO.23;
the amplification primer sequences of the mutation site L522M are SEQ ID NO.24 and SEQ ID NO.25;
the amplified primer sequences of the mutation site L545F are SEQ ID NO.26 and SEQ ID NO.27.
The invention also provides a gene for encoding the BstX mutant of the DNA polymerase.
The invention also provides a recombinant plasmid containing the gene.
The invention also provides a soluble protein, immobilized enzyme or engineering bacteria containing the DNA polymerase BstX mutant.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the DNA polymerase BstX mutant with improved thermal stability comprises a single-point mutant and a combined mutant, and compared with the wild DNA polymerase BstX, the single-point mutant and the combined mutant have longer half lives at 77 ℃; in particular, the combination mutant shows a superposition effect of the thermal stability of the single point mutation, and the half life of the combination mutant is about 3 times that of the wild type. The mutant has excellent catalytic activity and good application prospect.
2. The construction method of the DNA polymerase BstX mutant with improved thermal stability provided by the invention is different from the rational design based on the precise structure-function relationship of protein, and is characterized in that Consensus Concept theory is taken as a guiding thought, information capable of improving the thermal stability of the enzyme is analyzed from the evolution angle, the DNA polymerase BstX family sequence is subjected to integrated analysis, and the bioinformatics and crystallography methods are combined for assistance, so that the novel DNA polymerase BstX mutant with high stability is obtained.
The present invention will be described in further detail with reference to the accompanying drawings.
Drawings
FIG. 1 is a schematic diagram of the BstDNA polymerase protein provided in example 2 of the present invention simulating crystal structure and the distribution of mutation sites on the crystal structure.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in the following examples, and it is obvious that the described examples are only some examples of the present invention, but not all examples. Although representative embodiments of the present invention have been described in detail, those skilled in the art to which the invention pertains will appreciate that various modifications and changes can be made without departing from the scope of the invention. Accordingly, the scope of the invention should not be limited to the embodiments, but should be defined by the appended claims and equivalents thereof.
The invention provides a DNA polymerase BstX mutant with improved thermal stability, which is shown in the following (a 1) or (a 2):
(a1) A derivative protein with the same function as the amino acid sequence shown in SEQ ID NO.2 by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.2;
(a2) A derivative protein having at least 97% homology with the amino acid sequence shown in SEQ ID NO.2, wherein the amino acid sequence shown in SEQ ID NO.2 is substituted, deleted or added with one or more amino acids.
The amino acid sequence of the DNA polymerase BstX mutant with improved thermostability is configured as the amino acid sequence after mutation of mutation site K26E, R58P, L522M, L545F, K E/R58P, K26E/L522M, K E/L545F, R P/L522M, R P/L545F, L522M/L545F, K E/R58P/L522M, K E/R58P/L545F, R P/L522M/L545F, K E/L522M/L545F or K26E/R58P/L522M/L545F on SEQ ID NO.2.
Wherein the amino acid sequence of the single-point mutant corresponding to K26E is SEQ ID NO.3;
the amino acid sequence of the single-point mutant corresponding to R58P is SEQ ID NO.4;
the amino acid sequence of the single-point mutant corresponding to L522M is SEQ ID NO.5;
the amino acid sequence of the single-point mutant corresponding to L545F is SEQ ID NO.6;
the amino acid sequence of the combined mutant corresponding to K26E/R58P is SEQ ID NO.7;
the amino acid sequence of the combined mutant corresponding to K26E/L522M is SEQ ID NO.8;
the amino acid sequence of the combined mutant corresponding to K26E/L545F is SEQ ID NO.9;
the amino acid sequence of the combined mutant corresponding to R58P/L522M is SEQ ID NO.17;
the amino acid sequence of the combined mutant corresponding to R58P/L545F is SEQ ID NO.11;
the amino acid sequence of the combined mutant corresponding to L522M/L545F is SEQ ID NO.12;
the amino acid sequence of the combined mutant corresponding to K26E/R58P/L522M is SEQ ID NO.13;
the amino acid sequence of the combined mutant corresponding to K26E/R58P/L545F is SEQ ID NO.14;
the amino acid sequence of the combined mutant corresponding to R58P/L522M/L545F is SEQ ID NO.15;
the amino acid sequence of the combined mutant corresponding to K26E/L522M/L545F is SEQ ID NO.16;
the amino acid sequence of the combined mutant corresponding to K26E/R58P/L522M/L545F is SEQ ID NO.17.
The invention also provides a construction method of the DNA polymerase BstX mutant with improved thermal stability, which comprises the following steps:
searching the amino acid sequences shown in SEQ ID NO.2 in a Pfam database and an NCBI database, removing repeated identical sequences, selecting an amino acid sequence with the amino acid sequence shown in SEQ ID NO.2 being more than 37%, performing multi-sequence comparison through Clustalx1.83 software, finishing the residual amino acid sequence into fasta files, uploading the fasta files to a Consensu Maker v2.7.7 server, and generating Consensus sequence which can be edited later by the online software after setting parameters are modified according to requirements;
predicting the three-dimensional structure of the obtained protein shown in SEQ ID NO.2 by using a Swissmul online tool, observing the crystal structure of the protein shown in SEQ ID NO.2 by using a PyMOL, and screening out mutation sites related to heat stability as follows: k26E, R58P, L522M, L545F.
The amplification primer sequences of the mutation site K26E are SEQ ID NO.27 and SEQ ID NO.21;
the amplification primer sequences of the mutation site R58P are SEQ ID NO.22 and SEQ ID NO.23;
the amplification primer sequences of the mutation site L522M are SEQ ID NO.24 and SEQ ID NO.25;
the amplified primer sequences of the mutation site L545F are SEQ ID NO.26 and SEQ ID NO.27.
The effect of the DNA polymerase BstX mutant having improved thermostability of the present invention is examined by the following specific examples.
Example 1:
this example provides a mutant of BstX DNA polymerase with improved thermostability, wherein BstX DNA polymerase is wild-type BstX DNA polymerase from Bacillus stearothermophilus, named protein BstX DNA polymerase, the nucleic acid sequence encoding BstX DNA polymerase protein is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO.2.
SEQ ID NO.1
GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGATGTGATCACCGAAGAAATGCTGGCAGATAAAGCAGCACTGGTTGTTGAAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCTGGTTAATGAACATGGTCGCTTTTTTATGCGTCCGGAAACCGCACTGGCCGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAAGCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTATTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCTGAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGAAACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTGTTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTTCGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGATCTGCGCAATAATGAACAGGATCAGCTGCTGACCAAACTGGAACAGCCGCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGATACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGCGTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACATCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAGCTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGATGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCTGCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCTGCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAATCAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCTGCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGCCTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAGCCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTGATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATGGATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGTCAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATGGTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTATCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAAATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGCATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCGTAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGCAGCCGATATTATCAAAAAAGCAATGATTGATCTGGCAGCCCGTCTGAAAGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAACTGATTCTGGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTGGTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAAAGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG
SEQ ID NO.2
AEGEKPLEEMEFAIVDVITEEMLADKAALVVEVMEENYHDAPIVGIALVNEHGRFFMRPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEEQLQARLLLQVHDELILEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
The DNA polymerase BstX mutant with improved thermostability provided in this example includes: the amino acid sequence shown in SEQ ID NO.2 is substituted, deleted or added with one or more amino acids to form a derivative protein with the same function as the amino acid sequence shown in SEQ ID NO.2 (namely BstX DNA polymerase protein), or the amino acid sequence shown in SEQ ID NO.2 is substituted, deleted or added with one or more amino acids to form a derivative protein with at least 97% homology with the amino acid sequence shown in SEQ ID NO.2 (namely BstX DNA polymerase protein).
Specifically, a certain site is selected to carry out single-point mutation on the amino acid sequence shown in SEQ ID NO.2, and 4 single-point mutants of DNA polymerase BstX are respectively obtained, wherein the mutation sites are as follows: the activity of the 4 DNA polymerase BstX single-point mutants is determined by K26E, R58P, L522M, L545F, and the amino acid sequences of the 4 DNA polymerase BstX single-point mutants are respectively SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.
SEQ ID NO.3
AEGEKPLEEMEFAIVDVITEEMLADEAALVVEVMEENYHDAPIVGIALVNEHGRFFMRPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEEQLQARLLLQVHDELILEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
SEQ ID NO.4
AEGEKPLEEMEFAIVDVITEEMLADKAALVVEVMEENYHDAPIVGIALVNEHGRFFMPPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEEQLQARLLLQVHDELILEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
SEQ ID NO.5
AEGEKPLEEMEFAIVDVITEEMLADKAALVVEVMEENYHDAPIVGIALVNEHGRFFMRPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDMAARLKEEQLQARLLLQVHDELILEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
SEQ ID NO.6
AEGEKPLEEMEFAIVDVITEEMLADKAALVVEVMEENYHDAPIVGIALVNEHGRFFMRPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEEQLQARLLLQVHDELIFEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
Or selecting a plurality of mutation sites from the amino acid sequence shown in SEQ ID NO.2 for combination, for example, selecting 2 mutation sites from the 4 mutation sites for combination, and respectively obtaining the following 6 DNA polymerase BstX mutants with improved heat stability, wherein the combination mutation sites are as follows: K26E/R58P, K E/L522M, K26E/L545F, R P/L522M, R P/L545F, L M/L545F, and the amino acid sequences thereof are SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.17, SEQ ID NO.11, and SEQ ID NO.12, respectively.
SEQ ID NO.7
AEGEKPLEEMEFAIVDVITEEMLADEAALVVEVMEENYHDAPIVGIALVNEHGRFFMPPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEEQLQARLLLQVHDELILEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
SEQ ID NO.8
AEGEKPLEEMEFAIVDVITEEMLADEAALVVEVMEENYHDAPIVGIALVNEHGRFFMRPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDMAARLKEEQLQARLLLQVHDELILEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
SEQ ID NO.9
AEGEKPLEEMEFAIVDVITEEMLADEAALVVEVMEENYHDAPIVGIALVNEHGRFFMRPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEEQLQARLLLQVHDELIFEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
SEQ ID NO.17
AEGEKPLEEMEFAIVDVITEEMLADKAALVVEVMEENYHDAPIVGIALVNEHGRFFMPPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDMAARLKEEQLQARLLLQVHDELILEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
SEQ ID NO.11
AEGEKPLEEMEFAIVDVITEEMLADKAALVVEVMEENYHDAPIVGIALVNEHGRFFMPPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEEQLQARLLLQVHDELIFEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
SEQ ID NO.12
AEGEKPLEEMEFAIVDVITEEMLADKAALVVEVMEENYHDAPIVGIALVNEHGRFFMRPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDMAARLKEEQLQARLLLQVHDELIFEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
Or 3 mutation sites are selected from the 4 mutation sites to be combined to respectively obtain 4 DNA polymerase BstX mutants with improved thermal stability, wherein the combined mutation sites are as follows: the amino acid sequences of K26E/R58P/L522M, K E/R58P/L545F, K E/L522M/L545F, R P/L522M/L545F are SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16 respectively.
SEQ ID NO.13
AEGEKPLEEMEFAIVDVITEEMLADEAALVVEVMEENYHDAPIVGIALVNEHGRFFMPPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDMAARLKEEQLQARLLLQVHDELILEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
SEQ ID NO.14
AEGEKPLEEMEFAIVDVITEEMLADEAALVVEVMEENYHDAPIVGIALVNEHGRFFMPPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEEQLQARLLLQVHDELIFEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
SEQ ID NO.15
AEGEKPLEEMEFAIVDVITEEMLADKAALVVEVMEENYHDAPIVGIALVNEHGRFFMPPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDMAARLKEEQLQARLLLQVHDELIFEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
SEQ ID NO.16
AEGEKPLEEMEFAIVDVITEEMLADEAALVVEVMEENYHDAPIVGIALVNEHGRFFMRPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDMAARLKEEQLQARLLLQVHDELIFEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
Or 4 mutation sites are selected from the 4 mutation sites to be combined to obtain 1 DNA polymerase BstX mutant with improved thermal stability, wherein the combined mutation sites are as follows: the amino acid sequence of K26E/R58P/L522M/L545F is SEQ ID NO.17.
SEQ ID NO.17
AEGEKPLEEMEFAIVDVITEEMLADEAALVVEVMEENYHDAPIVGIALVNEHGRFFMPPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDMAARLKEEQLQARLLLQVHDELIFEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
Example 2:
the embodiment provides a construction method of a DNA polymerase BstX mutant with improved thermal stability, which comprises the following steps:
1. cloning of wild-type DNA polymerase BstX Gene
Performing codon optimization on a wild DNA polymerase BstX gene by taking escherichia coli as a host cell to obtain an optimized BstX DNA polymerase gene, wherein the nucleic acid sequence of the optimized BstX DNA polymerase gene is SEQ ID NO.1, and the expressed amino acid sequence of the optimized BstX DNA polymerase gene is SEQ ID NO.2; using SEQ ID NO.1 as a target gene, and adopting an upstream amplification primer SEQ ID NO.18 and a downstream amplification primer SEQ ID NO.19 to amplify the target gene;
the nucleic acid sequence of SEQ ID NO.18 is:
5’-ACTGCTCATATGGCGGAAGGCGAAAAACCGCT-3' (wherein the restriction endonuclease NdeI recognition site is underlined);
the nucleic acid sequence of SEQ ID NO.19 is:
5’-TCAGCTCTCGAGTTTCGCATCATACCAGGTCGGGC-3' (in which the restriction enzyme XhoI recognition site is underlined).
The amplification conditions were: amplification was carried out at 95℃for 2min, then at 56℃for 27sec, at 72℃for 97sec for 37 cycles, and finally at 72℃for 17min.
After the reaction was completed, the PCR amplification product was detected by 1.5% agarose gel electrophoresis to obtain a 1.7kb band having a length corresponding to the expected result. Recovering and purifying the target fragment according to the standard operation of a kit, carrying out double enzyme digestion on the target fragment and pET28a plasmid by using restriction endonucleases XhoI and NdeI, then adopting T4 DNA ligase to carry out ligation, converting the obtained ligation product into competent cells of escherichia coli BL21 (DE 3), coating the transformed cells on an LB plate containing 57 mug/ml kanamycin, extracting positive cloning plasmids, sequencing, and obtaining recombinant plasmids pET28a-Bst by correctly accessing pET28a plasmids as a result of the accurate sequence of cloned DNA polymerase BstX;
wherein, the wild type DNA polymerase BstX is derived from Bacillus stearothermophilus;
BstX DNA polymerase gene is supplied by Jin Weizhi Biotechnology Co., ltd;
the PCR amplification enzyme is KOD high-fidelity polymerase provided by Toyobo.
Expression and purification of BstX DNA polymerase protein
Inoculating engineering bacteria in an glycerol pipe into a 4mL LB culture medium test tube containing 177 mug/mL Kan according to the volume ratio of 1%, and culturing for 12h at 37 ℃ and 227 rpm; 4mL of the bacterial liquid was transferred to a shaking flask of 1L LB medium containing 57. Mu.g/mL Kan, and cultured at 37℃and 227rpm for 2.5 hours, so that OD677 reached about 7.9, and then 7.1mM IPTG inducer was added to induce culture at 277rpm and 25℃for 14 hours. And (3) ultrasonically crushing the escherichia coli bacterial suspension obtained after fermentation, and performing one-step Ni-NTA affinity chromatography treatment to obtain BstX DNA polymerase protein with the purity of more than 95 percent, wherein the amino acid sequence is SEQ ID NO.2.
Multiple sequence alignment of BstX DNA polymerase homologous proteins and Consensu analysis
3.1. Entering a Pfam database homepage (http:// Pfam. Xfam. Org /), inputting an amino acid SEQUENCE of BstX DNA polymerase in a SEQUENCE SEARCH tool for searching, directly feeding back an alignment result of the amino acid SEQUENCE of the whole family of the protein by a server, displaying various amino acid abundances of each mutation site in a columnar graph, and automatically generating consensus SEQUENCE of the protein family by the website;
3.2. inputting the amino acid sequences shown in SEQ ID NO.2 into NCBI protein database and Pfam database, finding out all protein sequences with the consistency of more than 37% with the amino acid sequence (SEQ ID NO. 2) of BstX DNA polymerase protein by using Blast tool, deleting the repeated identical sequences, arranging the residual amino acid sequences into fasta format, inputting Clustalx1.83 software for multi-sequence comparison, and outputting the comparison results in the formats of aln, dnd and fasta, wherein the dnd file is a constructed evolution tree file, and the aln and fasta files are different forms of sequence files;
the fasta files are uploaded to a Consensu Maker v2.7.7 (http:// www.hiv.lanl.gov/content/sequence/CONSENSUS/content. Html) server, and after the parameters are modified as needed, the online software will generate Consensus sequence that can be edited later.
3.3. The amino acid sequence of BstX DNA polymerase protein (SEQ ID NO. 2) was compared to the amino acid abundance map of each position of the family consensus sequence.
Simulation of BstX DNA polymerase protein three-dimensional Structure and selection of mutant Hot spots
4.1. Three-dimensional structure prediction of BstX DNA polymerase protein (amino acid sequence SEQ ID NO. 2) is obtained through Swissmul online tool pair;
4.2. the crystal structure of BstX DNA polymerase protein (amino acid sequence SEQ ID NO. 2) is observed by using PyMOL, the mutation sites and the mutation forms to be selected are rechecked according to structural information, and the mutant sites most likely to improve the thermal stability of the BstX DNA polymerase protein are screened out under the following screening conditions:
(1) The criteria for judging a certain site as a candidate site are:
(1) most proteins of this family have a high overall height of amino acid abundance at this site;
(2) the amino acid of the site is conserved;
(3) the amino acid with higher frequency of occurrence of the locus has larger physical and chemical property difference, such as charge difference, polarity strength, steric hindrance and the like, with the amino acid of the BstX DNA polymerase protein at the locus.
(2) Removing the vicinity of the active center, i.e. from the catalytic residueAmino acid residues within the scope are removed from the amino acid residues in the entrapped or semi-entrapped state.
After the two-step screening, a total of 17 different sites are left, and most of the sites are positioned on the surface of BstX DNA polymerase protein molecules, as shown in figure 1, and the arrows indicate mutation sites.
(3) The 17 mutant forms are analyzed in detail one by one according to the crystal structure of the BstX DNA polymerase protein, and mutants which can improve the thermal stability of the BstX DNA polymerase protein are screened.
The main judgment criteria are as follows: (1) the mutation should eliminate the original acting force forms which are unfavorable for heat stabilization, such as electrostatic repulsion, charge aggregation and the like; (2) mutations should not disrupt the existing force patterns and stable protein structures that facilitate thermostability; (3) mutations should introduce new forms of forces that favor thermal stabilization, such as hydrogen bonding, salt bridging, hydrophobic interactions, etc.
The number of the co-designed single-point mutants is 4, and mutation sites of the single-point mutants are respectively as follows: k26E, R58P, L522M, L545F;
the activity of the 4 DNA polymerase BstX single-point mutants is measured, 4 DNA polymerase BstX mutants with improved thermal stability are screened, and mutation sites are as follows: the amino acid sequences of the corresponding single-point mutants of K26E, R58P, L522M, L545F are SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.
5. Construction, expression and purification of mutants
Construction of BstX DNA polymerase protein Single Point mutant
Taking the recombinant plasmid pET28a-Bst in the step 1 as a template, taking a pair of complementary oligonucleotides with mutation sites as amplification primers, and carrying out full plasmid PCR (polymerase chain reaction) amplification by using KOD high-fidelity enzyme to obtain the recombinant plasmid with the specific mutation sites;
the amplification primer pairs used were:
(1) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.27 and the downstream amplification primer SEQ ID NO.21 of the mutation site K26E are as follows, respectively:
SEQ ID NO.27：
5'-CAGATGAAGCAGCACTGGTTGTTG-3'；
SEQ ID NO.21：
5'-TTCAACAACCAGTGCTGCTTCAT-3'；
(2) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.22 and the downstream amplification primer SEQ ID NO.23 of the mutation site R58P are as follows:
SEQ ID NO.22：
5'-TTTTTTATGCCGCCGGAAACC-3'；
SEQ ID NO.23：
5'-TTTCCGGCGGCATAAAAAAGCGA-3'；
(3) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.24 and the downstream amplification primer SEQ ID NO.25 of the mutation site L522M are as follows, respectively:
SEQ ID NO.24：
5'-AATGATTGATCTGGCAGCCCG-3'；
SEQ ID NO.25：
5'-GGGCTGCCAGATCAATCATTGC-3'；
(4) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.26 and the downstream amplification primer SEQ ID NO.27 of the mutation site L545F are as follows, respectively:
SEQ ID NO.26：
5'-AACTGATTTTTGAAGCACCGAAAG-3'；
SEQ ID NO.27：
5'-TTTCGGTGCTTCAAAAATCAGTTC-3'；
the amplification conditions were: amplifying for 2min at 95 ℃, then amplifying for 27sec at 56 ℃ and 97sec at 72 ℃ for 37 cycles, and finally amplifying for 17min at 72 ℃; the PCR amplification product is recovered by gel, the product is recovered by digestion of the gel with DpnI enzyme at 37 ℃ for 2 hours, and the initial template is degraded; transferring the digested product into competent cells of escherichia coli BL21 (DE 3), coating the competent cells on an LB agar plate containing 57 mug/mL kanamycin, culturing overnight at 37 ℃, screening positive clones, and sequencing and verifying to obtain recombinant bacteria containing a single-point mutant of DNA polymerase BstX;
wherein the KOD high-fidelity enzyme is provided by TakaRa;
the DpnI enzyme is supplied by Fermentas.
Construction of BstX DNA polymerase protein combinatorial mutants
By using a construction method similar to that of single-point mutants, accumulating and combining the single-point mutants with improved stability, selecting a plurality of mutation sites from the amino acid sequence shown in SEQ ID NO.2 for combining, for example, selecting 2-4 mutation sites from the 4 mutation sites for combining, and respectively obtaining different DNA polymerase BstX combined mutants:
(1) 2 mutation sites are selected for combination, 6 DNA polymerase BstX mutant DNA polymerase BstX combined mutants with improved thermal stability can be constructed, and the combined mutation sites are respectively: the amino acid sequences of the 6 DNA polymerase BstX combined mutants with improved thermal stability are respectively SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.17, SEQ ID NO.11 and SEQ ID NO.12;
(2) 3 mutation sites are selected for combination, 4 DNA polymerase BstX combination mutants with improved thermal stability can be constructed, and the combination mutation sites are respectively: the amino acid sequences of the 4 DNA polymerase BstX combined mutants with improved thermal stability are SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16 respectively, wherein the amino acid sequences of the K26E/R58P/L522M, K26E/R58P/L545F, R58P/L522M/L545F and K26E/L522M/L545F;
(3) And 4 mutation sites are selected for combination, 1 DNA polymerase BstX combination mutant with improved heat stability can be constructed, and the combination mutation sites are respectively: the amino acid sequence of the combined mutant of the 1 DNA polymerase BstX with improved heat stability is SEQ ID NO.17.
Example 3:
this example provides a gene encoding a DNA polymerase BstX mutant with improved thermostability as described in example 1:
(1) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of K26E is SEQ ID NO.28;
SEQ ID NO.28
GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGA
TGTGATCACCGAAGAAATGCTGGCAGATAAAGCAGCACTGGTTGTTG
AAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCT
GGTTAATGAACATGGTCGCTTTTTTATGCGTCCGGAAACCGCACTGGC
CGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAA
GCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTA
TTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCT
GAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGA
AACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTG
TTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTT
CGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGAT
CTGCGCAATAATGAACAGGATCAGCTGCTGACCAAACTGGAACAGCC
GCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGA
TACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGC
GTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACA
TCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAG
CTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGA
TGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCT
GCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCT
GCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAAT
CAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCT
GCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGC
CTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAG
CCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTG
ATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATG
GATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGT
CAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATG
GTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTA
TCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAA
ATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGC
ATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCG
TAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGC
AGCCGATATTATCAAAAAAGCAATGATTGATCTGGCAGCCCGTCTGAA
AGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAAC
TGATTCTGGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTG
GTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAA
AGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG
(2) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of R58P is SEQ ID NO.29;
SEQ ID NO.29 (1770 bases for the remaining sequences, but 1769 bases for 29 only: GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGATGTGATCACCGAAGAAATGCTGGCAGATAAAGCAGCACTGGTTGTTGAAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCTGGTTAATGAACATGGTCGCTTTTTTATGCCCCGGAAACCGCACTGGCCGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAAGCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTATTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCTGAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGAAACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTGTTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTTCGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGATCTGCGCAATAATGAACAGGATCAGCTGCTGACCAAACTGGAACAGCCGCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGATACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGCGTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACATCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAGCTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGATGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCTGCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCTGCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAATCAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCTGCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGCCTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAGCCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTGATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATGGATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGTCAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATGGTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTATCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAAATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGCATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCGTAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGCAGCCGATATTATCAAAAAAGCAATGATTGATCTGGCAGCCCGTCTGAAAGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAACTGATTCTGGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTGGTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAAAGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG)
(3) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of L522M is SEQ ID NO.37;
SEQ ID NO.37
GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGA
TGTGATCACCGAAGAAATGCTGGCAGATAAAGCAGCACTGGTTGTTG
AAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCT
GGTTAATGAACATGGTCGCTTTTTTATGCGTCCGGAAACCGCACTGGC
CGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAA
GCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTA
TTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCT
GAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGA
AACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTG
TTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTT
CGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGAT
CTGCGCAATAATGAACAGGATCAGCTGCTGACCAAACTGGAACAGCC
GCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGA
TACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGC
GTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACA
TCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAG
CTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGA
TGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCT
GCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCT
GCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAAT
CAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCT
GCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGC
CTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAG
CCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTG
ATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATG
GATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGT
CAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATG
GTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTA
TCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAA
ATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGC
ATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCG
TAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGC
AGCCGATATTATCAAAAAAGCAATGATTGATATGGCAGCCCGTCTGAA
AGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAAC
TGATTCTGGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTG
GTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAA
AGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG
(4) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site L545F is SEQ ID NO.31;
SEQ ID NO.31
GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGA
TGTGATCACCGAAGAAATGCTGGCAGATAAAGCAGCACTGGTTGTTG
AAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCT
GGTTAATGAACATGGTCGCTTTTTTATGCGTCCGGAAACCGCACTGGC
CGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAA
GCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTA
TTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCT
GAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGA
AACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTG
TTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTT
CGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGAT
CTGCGCAATAATGAACAGGATCAGCTGCTGACCAAACTGGAACAGCC
GCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGA
TACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGC
GTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACA
TCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAG
CTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGA
TGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCT
GCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCT
GCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAAT
CAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCT
GCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGC
CTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAG
CCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTG
ATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATG
GATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGT
CAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATG
GTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTA
TCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAA
ATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGC
ATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCG
TAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGC
AGCCGATATTATCAAAAAAGCAATGATTGATCTGGCAGCCCGTCTGAA
AGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAAC
TGATTTTTGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTG
GTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAA
AGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG
(5) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of K26E/R58P is SEQ ID NO.32;
SEQ ID NO.32
GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGA
TGTGATCACCGAAGAAATGCTGGCAGATGAAGCAGCACTGGTTGTTG
AAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCT
GGTTAATGAACATGGTCGCTTTTTTATGCCGCCGGAAACCGCACTGGC
CGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAA
GCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTA
TTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCT
GAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGA
AACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTG
TTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTT
CGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGAT
CTGCGCAATAATGAACAGGATCAGCTGCTGACCAAACTGGAACAGCC
GCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGA
TACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGC
GTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACA
TCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAG
CTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGA
TGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCT
GCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCT
GCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAAT
CAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCT
GCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGC
CTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAG
CCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTG
ATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATG
GATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGT
CAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATG
GTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTA
TCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAA
ATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGC
ATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCG
TAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGC
AGCCGATATTATCAAAAAAGCAATGATTGATCTGGCAGCCCGTCTGAA
AGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAAC
TGATTCTGGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTG
GTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAA
AGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG
(6) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of K26E/L522M is SEQ ID NO.33;
SEQ ID NO.33
GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGA
TGTGATCACCGAAGAAATGCTGGCAGATGAAGCAGCACTGGTTGTTG
AAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCT
GGTTAATGAACATGGTCGCTTTTTTATGCGTCCGGAAACCGCACTGGC
CGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAA
GCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTA
TTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCT
GAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGA
AACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTG
TTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTT
CGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGAT
CTGCGCAATAATGAACAGGATCAGCTGCTGACCAAACTGGAACAGCC
GCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGA
TACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGC
GTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACA
TCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAG
CTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGA
TGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCT
GCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCT
GCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAAT
CAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCT
GCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGC
CTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAG
CCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTG
ATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATG
GATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGT
CAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATG
GTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTA
TCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAA
ATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGC
ATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCG
TAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGC
AGCCGATATTATCAAAAAAGCAATGATTGATATGGCAGCCCGTCTGAA
AGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAAC
TGATTCTGGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTG
GTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAA
AGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG
(7) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of K26E/L545F is SEQ ID NO.34;
CTCGAGSEQ ID NO.34
GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGA
TGTGATCACCGAAGAAATGCTGGCAGATGAAGCAGCACTGGTTGTTG
AAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCT
GGTTAATGAACATGGTCGCTTTTTTATGCGTCCGGAAACCGCACTGGC
CGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAA
GCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTA
TTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCT
GAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGA
AACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTG
TTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTT
CGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGAT
CTGCGCAATAATGAACAGGATCAGCTGCTGACCAAACTGGAACAGCC
GCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGA
TACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGC
GTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACA
TCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAG
CTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGA
TGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCT
GCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCT
GCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAAT
CAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCT
GCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGC
CTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAG
CCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTG
ATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATG
GATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGT
CAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATG
GTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTA
TCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAA
ATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGC
ATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCG
TAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGC
AGCCGATATTATCAAAAAAGCAATGATTGATCTGGCAGCCCGTCTGAA
AGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAAC
TGATTTTTGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTG
GTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAA
AGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG
(8) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of R58P/L522M is SEQ ID NO.35;
SEQ ID NO.35
GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGA
TGTGATCACCGAAGAAATGCTGGCAGATAAAGCAGCACTGGTTGTTG
AAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCT
GGTTAATGAACATGGTCGCTTTTTTATGCCGCCGGAAACCGCACTGGC
CGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAA
GCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTA
TTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCT
GAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGA
AACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTG
TTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTT
CGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGAT
CTGCGCAATAATGAACAGGATCAGCTGCTGACCAAACTGGAACAGCC
GCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGA
TACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGC
GTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACA
TCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAG
CTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGA
TGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCT
GCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCT
GCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAAT
CAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCT
GCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGC
CTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAG
CCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTG
ATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATG
GATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGT
CAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATG
GTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTA
TCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAA
ATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGC
ATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCG
TAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGC
AGCCGATATTATCAAAAAAGCAATGATTGATATGGCAGCCCGTCTGAA
AGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAAC
TGATTCTGGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTG
GTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAA
AGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG
(9) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of R58P/L545F is SEQ ID NO.36;
SEQ ID NO.36
GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGA
TGTGATCACCGAAGAAATGCTGGCAGATAAAGCAGCACTGGTTGTTG
AAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCT
GGTTAATGAACATGGTCGCTTTTTTATGCCGCCGGAAACCGCACTGGC
CGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAA
GCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTA
TTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCT
GAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGA
AACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTG
TTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTT
CGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGAT
CTGCGCAATAATGAACAGGATCAGCTGCTGACCAAACTGGAACAGCC
GCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGA
TACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGC
GTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACA
TCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAG
CTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGA
TGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCT
GCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCT
GCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAAT
CAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCT
GCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGC
CTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAG
CCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTG
ATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATG
GATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGT
CAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATG
GTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTA
TCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAA
ATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGC
ATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCG
TAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGC
AGCCGATATTATCAAAAAAGCAATGATTGATCTGGCAGCCCGTCTGAA
AGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAAC
TGATTTTTGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTG
GTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAA
AGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG
(17) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of L522M/L545F is SEQ ID NO.37;
SEQ ID NO.37
GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGA
TGTGATCACCGAAGAAATGCTGGCAGATAAAGCAGCACTGGTTGTTG
AAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCT
GGTTAATGAACATGGTCGCTTTTTTATGCGTCCGGAAACCGCACTGGC
CGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAA
GCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTA
TTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCT
GAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGA
AACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTG
TTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTT
CGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGAT
CTGCGCAATAATGAACAGGATCAGCTGCTGACCAAACTGGAACAGCC
GCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGA
TACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGC
GTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACA
TCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAG
CTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGA
TGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCT
GCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCT
GCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAAT
CAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCT
GCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGC
CTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAG
CCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTG
ATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATG
GATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGT
CAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATG
GTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTA
TCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAA
ATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGC
ATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCG
TAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGC
AGCCGATATTATCAAAAAAGCAATGATTGATATGGCAGCCCGTCTGAA
AGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAAC
TGATTTTTGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTG
GTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAA
AGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG
(11) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of K26E/R58P/L522M is SEQ ID NO.38;
SEQ ID NO.38
GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGA
TGTGATCACCGAAGAAATGCTGGCAGATGAAGCAGCACTGGTTGTTG
AAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCT
GGTTAATGAACATGGTCGCTTTTTTATGCCGCCGGAAACCGCACTGGC
CGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAA
GCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTA
TTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCT
GAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGA
AACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTG
TTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTT
CGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGAT
CTGCGCAATAATGAACAGGATCAGCTGATGACCAAACTGGAACAGCC
GCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGA
TACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGC
GTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACA
TCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAG
CTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGA
TGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCT
GCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCT
GCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAAT
CAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCT
GCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGC
CTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAG
CCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTG
ATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATG
GATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGT
CAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATG
GTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTA
TCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAA
ATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGC
ATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCG
TAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGC
AGCCGATATTATCAAAAAAGCAATGATTGATCTGGCAGCCCGTCTGAA
AGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAAC
TGATTCTGGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTG
GTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAA
AGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG
(12) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of K26E/R58P/L545F is SEQ ID NO.39;
SEQ ID NO.39
GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGA
TGTGATCACCGAAGAAATGCTGGCAGATGAAGCAGCACTGGTTGTTG
AAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCT
GGTTAATGAACATGGTCGCTTTTTTATGCCGCCGGAAACCGCACTGGC
CGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAA
GCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTA
TTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCT
GAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGA
AACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTG
TTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTT
CGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGAT
CTGCGCAATAATGAACAGGATCAGCTGCTGACCAAACTGGAACAGCC
GCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGA
TACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGC
GTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACA
TCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAG
CTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGA
TGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCT
GCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCT
GCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAAT
CAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCT
GCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGC
CTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAG
CCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTG
ATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATG
GATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGT
CAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATG
GTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTA
TCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAA
ATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGC
ATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCG
TAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGC
AGCCGATATTATCAAAAAAGCAATGATTGATCTGGCAGCCCGTCTGAA
AGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAAC
TGATTTTTGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTG
GTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAA
AGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG
(13) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of R58P/L522M/L545F is SEQ ID NO.47;
SEQ ID NO.47
GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGA
TGTGATCACCGAAGAAATGCTGGCAGATGAAGCAGCACTGGTTGTTG
AAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCT
GGTTAATGAACATGGTCGCTTTTTTATGCCGCCGGAAACCGCACTGGC
CGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAA
GCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTA
TTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCT
GAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGA
AACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTG
TTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTT
CGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGAT
CTGCGCAATAATGAACAGGATCAGCTGCTGACCAAACTGGAACAGCC
GCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGA
TACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGC
GTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACA
TCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAG
CTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGA
TGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCT
GCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCT
GCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAAT
CAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCT
GCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGC
CTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAG
CCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTG
ATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATG
GATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGT
CAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATG
GTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTA
TCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAA
ATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGC
ATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCG
TAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGC
AGCCGATATTATCAAAAAAGCAATGATTGATCTGGCAGCCCGTCTGAA
AGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAAC
TGATTTTTGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTG
GTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAA
AGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG
(14) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of R58P/L522M/L545F is SEQ ID NO.41;
SEQ ID NO.41
GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGA
TGTGATCACCGAAGAAATGCTGGCAGATGAAGCAGCACTGGTTGTTG
AAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCT
GGTTAATGAACATGGTCGCTTTTTTATGCGTCCGGAAACCGCACTGGC
CGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAA
GCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTA
TTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCT
GAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGA
AACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTG
TTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTT
CGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGAT
CTGCGCAATAATGAACAGGATCAGCTGCTGACCAAACTGGAACAGCC
GCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGA
TACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGC
GTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACA
TCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAG
CTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGA
TGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCT
GCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCT
GCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAAT
CAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCT
GCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGC
CTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAG
CCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTG
ATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATG
GATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGT
CAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATG
GTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTA
TCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAA
ATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGC
ATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCG
TAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGC
AGCCGATATTATCAAAAAAGCAATGATTGATATGGCAGCCCGTCTGAA
AGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAAC
TGATTTTTGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTG
GTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAA
AGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG
(15) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of K26E/R58P/L522M/L545F is shown as SEQ ID NO.42.
SEQ ID NO.42
GCCGAAGGTGAAAAACCGCTGGAAGAAATGGAATTTGCCATTGTTGA
TGTGATCACCGAAGAAATGCTGGCAGATGAAGCAGCACTGGTTGTTG
AAGTTATGGAAGAGAATTATCATGATGCACCGATTGTTGGTATTGCCCT
GGTTAATGAACATGGTCGCTTTTTTATGCCGCCGGAAACCGCACTGGC
CGATAGCCAGTTTCTGGCATGGCTGGCCGATGAAACCAAAAAGAAAA
GCATGTTTGATGCCAAACGTGCAGTTGTTGCACTGAAATGGAAAGGTA
TTGAACTGCGTGGTGTTGCATTTGATCTGCTGCTGGCAGCATATCTGCT
GAATCCGGCACAGGATGCCGGTGATATTGCAGCAGTTGCAAAAATGA
AACAGTATGAAGCAGTGCGTAGTGATGAAGCCGTTTATGGTAAAGGTG
TTAAACGTAGCCTGCCGGATGAACAGACCCTGGCAGAACATCTGGTT
CGTAAAGCAGCCGCAATTTGGGCATTAGAACAGCCTTTTATGGATGAT
CTGCGCAATAATGAACAGGATCAGCTGCTGACCAAACTGGAACAGCC
GCTGGCAGCCATTCTGGCCGAGATGGAATTTACCGGTGTTAATGTGGA
TACCAAACGTCTGGAACAAATGGGTAGCGAACTGGCCGAACAGCTGC
GTGCAATTGAACAGCGTATTTATGAACTGGCAGGCCAAGAATTTAACA
TCAATAGCCCGAAACAGCTGGGTGTGATTCTGTTTGAAAAACTGCAG
CTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGA
TGTGCTGGAAAAACTGGCACCGCATCATGAAATCGTTGAAAACATTCT
GCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCT
GCTGAAAGTTGTTCGTCCGGATACCGGTAAAGTTCATACCATGTTTAAT
CAGGCACTGACCCAGACCGGTCGTCTGAGCAGCGCAGAACCGAATCT
GCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGC
CTTTGTTCCGAGCGAACCGGATTGGCTGATTTTTGCAGCAGATTATAG
CCAGATCGAACTGCGCGTTCTGGCACATATTGCAGATGATGATAATCTG
ATTGAAGCCTTTCAGCGCGATCTGGATATTCATACCAAAACAGCCATG
GATATCTTTCACGTTAGCGAAGAAGAGGTTACCGCAAATATGCGTCGT
CAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGATTATG
GTCTGGCACAGAATCTGAATATTACCCGTAAAGAAGCAGCCGAGTTTA
TCGAACGTTATTTTGCAAGCTTTCCGGGTGTCAAACAGTACATGGAAA
ATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGC
ATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCG
TAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGC
AGCCGATATTATCAAAAAAGCAATGATTGATATGGCAGCCCGTCTGAA
AGAAGAACAGTTACAGGCACGCCTGCTGCTGCAGGTTCATGATGAAC
TGATTTTTGAAGCACCGAAAGAAGAGATTGAACGTCTTTGTGAACTG
GTTCCGGAAGTGATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAA
AGTGGATTATCATTATGGTCCGACCTGGTATGATGCGAAATAACTCGAG example 4:
this example investigated the enzymatic characterization of the BstX mutant of DNA polymerase.
The wild type DNA polymerase BstX and the various DNA polymerase BstX mutants provided in example 2 were subjected to a thermal stability test according to the conventional method for measuring the activity of the DNA polymerase BstX, specifically:
the enzyme solution is incubated at a certain temperature, samples are taken at different treatment times, the residual activity percentage of the DNA polymerase BstX or the DNA polymerase BstX mutant is measured, the ln value of the residual activity percentage is plotted against the time t (min), the slope of the straight line is the inactivation constant kinact, and the half-life of the wild type DNA polymerase BstX or the DNA polymerase BstX mutant at the temperature is obtained from t1/2 = ln 2/kinact.
The experimental results show that the thermal stability of 4 single-point mutants and 11 combined mutants in the various DNA polymerase BstX mutants is obviously improved, as shown in the table 1:
TABLE 1 characterization of enzymatic Properties of wild-type DNA polymerase BstX and mutants
As can be seen from Table 1, the DNA polymerase BstX mutant provided by the invention comprises a single-point mutant and a combined mutant, and compared with the wild DNA polymerase BstX, the single-point mutant and the combined mutant have longer half lives at 77 ℃; in particular, the combination mutant shows a superposition effect of the thermal stability of the single point mutation, and the half life of the combination mutant is about 3 times that of the wild type.
The foregoing examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention, and all designs that are the same or similar to the present invention are within the scope of the present invention.
Claims (9)
1. A DNA polymerase BstX mutant with improved thermostability, characterized in that: the DNA polymerase BstX mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.2, and the mutation site is selected from one or more than one combination of K26E, R58P, L522M, L545F.
2. The thermostable enhanced DNA polymerase BstX mutant of claim 1, wherein: the mutation site is K26E, R58P, L522M, L F, K E/R58P, K E/L522M, K E/L545F, R P/L522M, R P/L545F, L M/L545F, K E/R58P/L522M, K E/R58P/L545F, R P/L522M/L545F, K E/L522M/L545F or K26E/R58P/L522M/L545F.
3. The thermostable enhanced DNA polymerase BstX mutant of claim 1, wherein:
the amino acid sequence of the single-point mutant corresponding to K26E is SEQ ID NO.3;
the amino acid sequence of the single-point mutant corresponding to R58P is SEQ ID NO.4;
the amino acid sequence of the single-point mutant corresponding to L522M is SEQ ID NO.5;
the amino acid sequence of the single-point mutant corresponding to L545F is SEQ ID NO.6;
the amino acid sequence of the combined mutant corresponding to K26E/R58P is SEQ ID NO.7;
the amino acid sequence of the combined mutant corresponding to K26E/L522M is SEQ ID NO.8;
the amino acid sequence of the combined mutant corresponding to K26E/L545F is SEQ ID NO.9;
the amino acid sequence of the combined mutant corresponding to R58P/L522M is SEQ ID NO.17;
the amino acid sequence of the combined mutant corresponding to R58P/L545F is SEQ ID NO.11;
the amino acid sequence of the combined mutant corresponding to L522M/L545F is SEQ ID NO.12;
the amino acid sequence of the combined mutant corresponding to K26E/R58P/L522M is SEQ ID NO.13;
the amino acid sequence of the combined mutant corresponding to K26E/R58P/L545F is SEQ ID NO.14;
the amino acid sequence of the combined mutant corresponding to R58P/L522M/L545F is SEQ ID NO.15;
the amino acid sequence of the combined mutant corresponding to K26E/L522M/L545F is SEQ ID NO.16;
the amino acid sequence of the combined mutant corresponding to K26E/R58P/L522M/L545F is SEQ ID NO.17.
4. A method for constructing a DNA polymerase BstX mutant with improved thermostability according to any one of claims 1 to 3, comprising the steps of:
searching and selecting an amino acid sequence with the amino acid sequence consistency of more than 57% as shown in SEQ ID NO.2 in a database, and then performing multi-sequence comparison to generate consensus sequence which can be edited later through software;
protein three-dimensional structure prediction is carried out on SEQ ID NO.2 through an online tool, and mutation sites relevant to stability are screened out: k26E, R58P, L522M, L545F.
5. The method for constructing a BstX mutant of DNA polymerase with improved thermostability according to claim 4, wherein:
the amplification primer sequences of the mutation site K26E are SEQ ID NO.27 and SEQ ID NO.21;
the amplification primer sequences of the mutation site R58P are SEQ ID NO.22 and SEQ ID NO.23;
the amplification primer sequences of the mutation site L522M are SEQ ID NO.24 and SEQ ID NO.25;
the amplified primer sequences of the mutation site L545F are SEQ ID NO.26 and SEQ ID NO.27.
6. A gene encoding the DNA polymerase BstX mutant with improved thermostability according to any one of claims 1 to 3.
7. A recombinant plasmid comprising the gene according to claim 6.
8. A soluble protein, immobilized enzyme or engineered bacterium comprising the thermostable enhanced DNA polymerase BstX mutant of any one of claims 1-3.
9. Use of a DNA polymerase BstX mutant with improved thermostability according to any of claims 1-3 for catalyzing DNA synthesis.
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