CN116590254A

CN116590254A - DNA polymerase mutant and construction method and application thereof

Info

Publication number: CN116590254A
Application number: CN202310430779.XA
Authority: CN
Inventors: 熊艳; 秦朕龙; 田军龙
Original assignee: Suzhou Jinyizhen Biotechnology Co ltd
Current assignee: Suzhou Jinyizhen Biotechnology Co ltd
Priority date: 2023-04-20
Filing date: 2023-04-20
Publication date: 2023-08-15

Abstract

The invention belongs to the technical field of biology, and particularly provides a DNA polymerase mutant, 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

DNA polymerase mutant and construction method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a DNA polymerase mutant and a construction method and application thereof.

Background

BstDNA polymerase has the characteristics of high specificity, high sensitivity, simplicity, rapidness, low cost and the like in Lamp, and becomes a widely used commercial DNA multiple displacement amplification enzyme, most of products on the market have the optimal reaction temperature of 60 ℃, the reaction activity is affected to a certain extent at 70 ℃, and the reaction is basically not reacted at 80 ℃, so that the protein needs to be directionally modified to obtain the BstDNA polymerase with higher thermal stability.

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 thermostability of proteins, markus Wyss et al in 2001 suggested 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 mutant, the DNA polymerase BstX mutant being (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 90% 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 is configured into the amino acid sequence of one or more combination of mutation sites M34L, E169K, N463P, C556K on SEQ ID NO.2.

Specifically, the mutation site is M34L, E K, N463P, C556K, M L/E169 8238L/N463P, M L/C556K, E K/N463P, E K/C556 463K/C556K, M P/C556 463 34L/E169K/C556 463 34L/E169K/C556K, E K/N463P/C556K, M L/N463P/C556K or M34L/E169K/N463P/C556.

Specifically, the amino acid sequence of the single-point mutant corresponding to M34L is SEQ ID NO.3;

the amino acid sequence of the single-point mutant corresponding to E169K is SEQ ID NO.4;

the amino acid sequence of the single-point mutant corresponding to N463P is SEQ ID NO.5;

the amino acid sequence of the single-point mutant corresponding to C556K is SEQ ID NO.6;

the amino acid sequence of the combined mutant corresponding to M34L/E169K is SEQ ID NO.7;

the amino acid sequence of the combined mutant corresponding to M34L/N463P is SEQ ID NO.8;

the amino acid sequence of the combined mutant corresponding to M34L/C556K is SEQ ID NO.9;

the amino acid sequence of the combined mutant corresponding to E169K/N463P is SEQ ID NO.10;

the amino acid sequence of the combined mutant corresponding to E169K/C556K is SEQ ID NO.11;

the amino acid sequence of the combined mutant corresponding to the N463P/C556K is SEQ ID NO.12;

the amino acid sequence of the combined mutant corresponding to M34L/E169K/N463P is SEQ ID NO.13:

the amino acid sequence of the combined mutant corresponding to M34L/E169K/C556K is SEQ ID NO.14;

the amino acid sequence of the combined mutant corresponding to E169K/N463P/C556K is SEQ ID NO.15;

the amino acid sequence of the combined mutant corresponding to M34L/N463P/C556K is SEQ ID NO.16;

the amino acid sequence of the combined mutant corresponding to M34L/E169K/N463P/C556K is SEQ ID NO.17.

The invention also provides a construction method of the DNA polymerase mutant, which comprises the following steps:

searching and selecting an amino acid sequence with the amino acid sequence consistency of more than 50% 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: M34L, E169K, N463P, C556K.

Specifically, the amplification primer sequences of the mutation sites M34L are SEQ ID NO.20 and SEQ ID NO.21;

the amplification primer sequences of the mutation site E169K are SEQ ID NO.22 and SEQ ID NO.23;

the amplification primer sequences of the mutation site N463P are SEQ ID NO.24 and SEQ ID NO.25;

the amplification primer sequences of the mutation site C556K are SEQ ID NO.26 and SEQ ID NO.27.

The invention also provides a gene for encoding the DNA polymerase mutant.

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 mutant.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the invention provides that the DNA polymerase mutant comprises a single-point mutant and a combined mutant, and the half lives of the single-point mutant and the combined mutant are longer at 70 ℃ compared with the wild type DNA polymerase BstX and the wild type DNA polymerase BstX; 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 mutant provided by the invention is different from the rational design based on the precise structure-function relationship of protein, takes Consensus Concept theory as a guiding thought, analyzes information capable of improving enzyme thermal stability from an evolution angle, performs integrated analysis on a BstX family sequence of the DNA polymerase, and combines with the assistance of bioinformatics and crystallography methods to obtain a novel BstX mutant of the DNA polymerase with high stability.

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 mutant, which is (a 1) or (a 2) as follows:

The amino acid sequence of the BstX mutant of the DNA polymerase is configured as the amino acid sequence of the mutation site M34L, E169K, N463P, C556K, M L/E169K, M L/N463P, M L/C556K, E K/N463P, E K/C556K, N463P/C556K, M L/E169K/N463P, M L/E169K/C556K, E K/N463P/C556K, M L/N463P/C556K or M34L/E169K/N463P/C556 after mutation.

Wherein, the amino acid sequence of the single-point mutant corresponding to M34L is SEQ ID NO.3;

the amino acid sequence of the combined mutant corresponding to M34L/E169K/N463P is SEQ ID NO.13;

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 30%, 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 v2.0.0 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: M34L, E169K, N463P, C556K.

The amplification primer sequences of the mutation site M34L are SEQ ID NO.20 and SEQ ID NO.21;

the amplification primer sequences of the mutation sites E169K are SEQ ID NO.22 and SEQ ID NO.23;

the amplification primer sequence of the mutation site C556K is SEQ ID NO.26 and SEQ ID NO.27

The effect of the DNA polymerase mutant 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

SEQ ID NO.2

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 90% 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: M34L, E169K, N463P, C556K, and the activity of the 4 DNA polymerase BstX single-point mutants is determined, wherein the amino acid sequences of the 4 DNA polymerase BstX single-point mutants are SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

SEQ ID NO.3

SEQ ID NO.4

SEQ ID NO.5

SEQ ID NO.6

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: M34L/E169K, M L/N463P, M34L/C556K, E K/N463P, E K/C556K, N463P/C556K, and the amino acid sequences are SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 respectively.

SEQ ID NO.7

SEQ ID NO.8

SEQ ID NO.9

SEQ ID NO.1 0

SEQ ID NO.11

SEQ ID NO.12

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: M34L/E169K/N463P, M L/E169K/C556K, M L/N463P/C556K, E K/N463P/C556K, the amino acid sequences of which are SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, respectively.

SEQ ID NO.13

SEQ ID NO.14

SEQ ID NO.15

SEQ ID NO.16

Or 4 mutation sites are selected from the 4 mutation sites to be combined to obtain 1 DNA polymerase BstX mutant with improved heat stability, wherein the combined mutation sites are as follows: M34L/E169K/N463P/C556K, its amino acid sequence is SEQ ID NO.17.

SEQ ID NO.17

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 20sec, at 72℃for 90sec for 30 cycles, and finally at 72℃for 10min.

After the reaction was completed, the PCR amplification product was detected by 1.5% agarose gel electrophoresis to obtain a 1.0kb 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 50 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 100 mug/mL Kan according to the volume ratio of 1%, and culturing for 12h at 37 ℃ and 220 rpm; 4mL of the bacterial liquid is transferred to a 1L LB culture medium shake flask containing 50 mug/mL Kan, and is cultured for 2.5 hours at 37 ℃ and 220rpm, so that the OD600 reaches about 0.9, and 0.1mM IPTG inducer is added, and the culture is induced for 14 hours at 25 ℃ and 200 rpm. 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 BstXDNA polymerase in a SEQUENCESEARCH 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 amino acid sequences (SEQ ID NO. 2) of BstXDNA polymerase protein being more than 30% 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 files are the sequence files for constructing the evolution tree files, and the aln and fasta files are in different forms;

uploading the fasta files to a Consensu Maker v2.0.0 (http:// www.hiv.lanl.gov/content/sequence/CONSENSUS/content. Html) server, and after modifying the setting parameters 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, 10 different sites are remained, 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 above 10 mutant forms were analyzed in detail one by one according to the crystal structure of BstX DNA polymerase protein, and mutants which could improve the thermal stability of BstX DNA polymerase protein were selected.

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: M34L, E169K, N463P, C556K;

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: M34L, E169K, N463P, C556K, and the corresponding single-point mutants have the amino acid sequences of 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 BstXDNA 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.20 and the downstream amplification primer SEQ ID NO.21 of the mutation site M34L are as follows, respectively:

SEQ ID NO.20：

SEQ ID NO.21：

(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 E169K are as follows:

SEQ ID NO.22：

SEQ ID NO.23：

(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 N463P are respectively as follows:

SEQ ID NO.24：

SEQ ID NO.25：

(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 C556K are as follows:

SEQ ID NO.26：

SEQ ID NO.27：

5′-AACCAGTTCTTTAAGACGTTC-3′；

the amplification conditions were: amplifying for 2min at 95 ℃, then amplifying for 20sec at 56 ℃ and 90sec at 72 ℃ for 30 cycles, and finally amplifying for 10min 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 50 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: M34L/E169K, M34L/N463P, M34L/C556K, E169K/N463P, E169K/C556K, N463P/C556K, the amino acid sequences of the 6 DNA polymerase BstX combined mutants with improved thermostability are SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 respectively;

(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: M34L/E169K/N463P, M34L/E169K/C556K, E169K/N463P/C556K, M34L/N463P/C556K, and 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;

(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 genes encoding DNA polymerase mutants as described in example 1:

(1) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of M34L is SEQ ID NO.28;

SEQ ID NO.28

(2) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of E169K is SEQ ID NO.29;

SEQ ID NO.29

(3) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of N463P is SEQ ID NO.30;

SEQ ID NO.30

(4) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of C556K is SEQ ID NO.31;

SEQ ID NO.31

(5) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of M34L/E169K is SEQ ID NO.32;

SEQ ID NO.32

(6) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of M34L/N463P is SEQ ID NO.33;

SEQ ID NO.33

(7) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of M34L/C556K is SEQ ID NO.34;

CTCGAGSEQ ID NO.34

(8) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of E169K/N463P is SEQ ID NO.35;

SEQ ID NO.35

(9) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of E169K/C556K is SEQ ID NO.36;

SEQ ID NO.36

(10) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of N463P/C556K is SEQ ID NO.37;

SEQ ID NO.37

(11) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of M34L/E169K/N463P is SEQ ID NO.38;

SEQ ID NO.38

(12) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of M34L/E169K/C556K is SEQ ID NO.39;

SEQ ID NO.39

(13) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of E169K/N463P/C556K is SEQ ID NO.40;

SEQ ID NO.40

(14) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of E169K/N463P/C556K is SEQ ID NO.41;

SEQ ID NO.41

(15) The nucleic acid sequence of the BstX mutant of the DNA polymerase with the mutation site of M34L/E169K/N463P/C556K is shown in SEQ ID NO.42.

SEQ ID NO.42

Example 4:

this example investigated the characterization of the enzymatic properties 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 70 ℃; 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

1. A DNA polymerase mutant characterized in that: the DNA polymerase 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 M34L, E169K, N463P, C556K.

2. The DNA polymerase mutant of claim 1, wherein: the mutation site is M34L, E169K, N463P, C556K, M L/E169K, M L/N463P, M L/C556K, E K/N463P, E K/C556K, N463P/C556K, M L/E169K/N463P, M L/E169K/C556K, E K/N463P/C556K, M L/N463P/C556K or M34L/E169K/N463P/C556.

3. The DNA polymerase mutant of claim 1, wherein:

the amino acid sequence of the single-point mutant corresponding to M34L is SEQ ID NO.3;

the amino acid sequence of the combined mutant corresponding to E169K/N463P is SEQ ID NO.17;

4. A method of constructing a DNA polymerase mutant 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;

5. The method for constructing a DNA polymerase mutant according to claim 4, wherein:

the amplification primer sequences of the mutation site M34L are SEQ ID NO.27 and SEQ ID NO.21;

6. A gene encoding the DNA polymerase mutant of 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 DNA polymerase mutant of any one of claims 1-3.

9. Use of a DNA polymerase mutant according to any one of claims 1-3 for catalyzing DNA synthesis.