Bsm DNA polymerase mutant with enhanced tolerance and application thereof

Abstract

The invention discloses a Bsm DNA polymerase mutant with enhanced tolerance and application of the Bsm DNA polymerase mutant, and belongs to the technical field of biology. On the basis of completely retaining 5 '-> 3' polymerase activity and 3 '-> 5' exonuclease proofreading activity, the mutant obviously improves the tolerance to inhibitors, wide pH range, high salt concentration and repeated freeze thawing. The mutant can directly perform high-efficiency and high-fidelity amplification on target nucleic acid in complex samples such as whole blood, soil and excrement containing various inhibitors, a nucleic acid purification step in advance is not needed, and the operation process is greatly simplified. The invention provides a core enzymology tool for pathogen rapid molecular diagnosis, on-site instant detection and difficult template cloning in a high inhibitor environment, and has important application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a Bsm DNA polymerase mutant with enhanced tolerance and application thereof. BACKGROUND

[0002] Bsm DNA polymerase has become a key tool enzyme in the fields of gene cloning, mutation detection and isothermal amplification due to its inherent high fidelity (with both 5'→3' polymerase activity and 3'→5' exonuclease proofreading activity) and strong strand displacement ability. However, its performance has significant limitations when facing complex samples commonly encountered in practical applications. The existing wild-type or conventional Bsm polymerase is sensitive to the reaction environment, and its activity is easily inhibited by common inhibitors in the sample (such as heparin in blood, humic acid in soil, detergent SDS and metal ion chelator EDTA); at the same time, its effective working pH range and salt ion concentration range are relatively narrow. These defects lead to the fact that in the application of direct detection of pathogens, analysis of environmental microorganisms and the like, it is often necessary to perform a time-consuming and possibly target-loss pre-treatment purification step, which is difficult to meet the requirements of process simplification and speed in the application scene of point-of-care testing (POCT) and the like. In addition, the stability of the enzyme preparation itself to repeated freeze-thawing is insufficient, which also brings inconvenience to its storage, transportation and daily use. Therefore, developing a Bsm DNA polymerase mutant with stronger tolerance to complex inhibitors, wide pH and high salt environment, and better stability under the premise of retaining the core functions of high fidelity and strand displacement, has important value for expanding its application in the fields of instant detection, direct analysis of difficult samples and the like. SUMMARY

[0003] The main purpose of the present application is to provide a Bsm DNA polymerase mutant with enhanced tolerance and application thereof, which can effectively solve the problems mentioned in the background art.

[0004] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: A Bsm DNA polymerase mutant with enhanced tolerance is obtained by modifying the amino acid sequence of the wild-type Bsm DNA polymerase, the mutant retains 5'→3' polymerase activity and 3'→5' exonuclease proofreading activity, and the tolerance to inhibitors, wide pH range or high salt concentration environment is significantly enhanced compared with the wild type. The modification is mainly aimed at the key domains affecting the binding of the enzyme to the template DNA and the stability of the microenvironment, which aims to systematically improve its ability to resist adverse physical and chemical conditions without impairing the core catalytic function.

[0005] Preferably, the enhanced tolerance is manifested by an amplification efficiency of no less than 70% of that of the wild-type enzyme under the same conditions in the presence of 1% (w / v) SDS or 50 mM EDTA in the reaction system. The performance bottom line of the mutant in the presence of typical strong inhibitors (ionic detergents and metal chelators) is defined, ensuring its practical reliability in complex sample processing.

[0006] Preferably, it can maintain effective catalytic activity in a buffer system with a pH ranging from 6.0 to 9.5. The risk of reaction failure due to pH fluctuations is reduced.

[0007] Preferably, it can perform nucleic acid amplification in a reaction buffer containing NaCl at a concentration of no more than 200 mM. The improved salt tolerance allows it to work in a reaction system with higher ionic strength, which is beneficial for reducing the dilution or desalting treatment steps of certain samples.

[0008] Preferably, the mutation is located in the junction region between the DNA binding domain and the catalytic domain of the enzyme, and includes the introduction of a positively charged amino acid residue. This region is a key site for regulating the interaction between the enzyme and the template DNA and the local charge environment, and the introduction of a positive charge helps to offset the negative interference caused by the inhibitor and stabilize the binding.

[0009] Preferably, the positively charged amino acid residue is lysine (K) or arginine (R). Lysine and arginine are common positively charged amino acids in nature, and their side chain properties are conducive to forming stable charge interactions in protein structures.

[0010] Preferably, the mutant retains no less than 90% of the initial activity after 10 freeze-thaw cycles at -20°C. The loss caused by repeated freeze-thawing is reduced The application of a Bsm DNA polymerase mutant with enhanced tolerance in directly amplifying target nucleic acids in complex biological or environmental samples without the need for prior nucleic acid purification of the samples. Simplifies the workflow, shortens the detection time, and is particularly suitable for on-site detection or high-throughput screening scenarios that require rapid results.

[0011] Preferably, the complex sample includes whole blood, soil extract, fecal suspension, or plant tissue crude extract. These sample types cover typical difficult samples commonly encountered in clinical diagnosis, environmental monitoring, and agricultural research, which are rich in various known PCR inhibitors.

[0012] Compared with the prior art, the present application has the following beneficial effects: The application constructs a mutant with significant advantages in tolerance by site-directed modification of the junction region between the DNA binding domain and the catalytic domain of the wild-type Bsm DNA polymerase, and by introducing modification means such as positively charged amino acid residues. The mutant significantly improves the tolerance to inhibitors such as SDS and EDTA, a wide pH range (6.0-9.5) and high salt concentration (≤200 mM NaCl), and has good freeze-thaw stability on the basis of retaining the original 5'→3' polymerase activity and 3'→5' proofreading function. Based on the above performance, the mutant can be directly used for target nucleic acid amplification of complex samples such as whole blood and soil without prior purification, and provides an effective tool for molecular diagnosis and long fragment cloning under complex conditions. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 It is a mutant domain modification schematic diagram of the application; Figure 2 It is a direct amplification application flowchart of the application; Figure 3 It is an advantage integration effect diagram of the application. DETAILED DESCRIPTION

[0014] In order to make the technical means, creative features, purposes and effects achieved by the application easy to understand, the application will be further described below in combination with specific embodiments.

[0015] I. Experimental materials and reagents Core materials: wild-type Bsm DNA polymerase gene, prokaryotic expression vector pET-28a(+), E. coli BL21(DE3) competent cells, human whole blood samples, farmland soil samples, mouse feces samples, and Arabidopsis thaliana leaf tissues.

[0016] Reagents: high-fidelity DNA polymerase, restriction endonuclease (Nde I / Hind III), T4 DNA ligase, plasmid extraction kit, protein purification kit (Ni-NTA affinity chromatography column), SDS (sodium dodecyl sulfate), EDTA (ethylene diamine tetraacetic acid), NaCl, Tris-HCl buffer, dNTPs mixture, primers (sequences are shown in Table 1), agarose, DNA Marker, and fluorescent quantitative PCR reagent.

[0017] Instruments: PCR instrument, fluorescent quantitative PCR instrument, constant temperature shaker, high-speed refrigerated centrifuge, ultrasonic cell disruptor, electrophoresis instrument, gel imaging system, and pH meter.

[0018] Table 1 Mutant construction primer sequences

[0019] II. Construction of Bsm DNA polymerase mutant (I) Experimental steps 1. Site-directed mutagenesis PCR amplification: using wild-type Bsm DNA polymerase gene as a template, using primer pairs containing K mutations and R mutations in Table 1 for PCR amplification. Reaction system (50 μL): template DNA 1 μL, upstream and downstream primers (10 μM) 2 μL each, dNTPs (10 mM) 1 μL, high-fidelity DNA polymerase 0.5 μL, 10× PCR buffer 5 μL, ddH2O 38.5 μL. Reaction conditions: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 3 min, 30 cycles; 72°C final extension for 10 min.

[0020] 2. Vector ligation and transformation: ligate the PCR amplification product (mutant gene) with the Nde I / Hind III double-digested pET-28a(+) vector using T4 DNA ligase (16°C overnight), construct recombinant expression plasmids pET-28a-Bsm-Mut (K mutant) and pET-28a-Bsm-Mut (R mutant). Transform the ligation product into E. coli BL21(DE3) competent cells, and plate on LB plates containing kanamycin, and incubate at 37°C for 12-16 h.

[0021] 3. Positive clone screening and verification: pick single colonies from the plate, inoculate into LB liquid medium containing kanamycin, and incubate at 37°C for 8 h with shaking, then extract the plasmid, verify the fragment size by double digestion, and confirm the correctness of the mutation site by sequencing (ensure that only the target site is mutated, and no other random mutations occur).

[0022] (II) Principle explanation The structural modification core of the present application is the junction region of the DNA binding domain and the catalytic domain of the wild-type Bsm DNA polymerase (as shown in the "mutant domain modification schematic diagram"), which is a key site for regulating the interaction between the enzyme and the template DNA and the local charge environment. By introducing positively charged amino acid residues (lysine K or arginine R) through site-directed mutagenesis, two major functions can be achieved: one is to offset the negative interference of inhibitors (such as SDS with negative charge, heparin with negative charge) in complex samples, maintaining the specific binding of enzyme and DNA; the second is to stabilize the spatial conformation and microenvironment of the enzyme, reducing the damage to the catalytic center under unfavorable conditions (such as extreme pH, high salt), while not affecting the core functions of the 5'→3' polymerase active center and the 3'→5' exonuclease proofreading center.

[0023] III. Expression and purification of Bsm DNA polymerase mutants (I) Experimental steps Induced expression: inoculate the correctly sequenced positive clone into 500 mL of LB liquid medium containing kanamycin, and incubate at 37°C with shaking until the OD600 When the concentration is 0.6-0.8, add IPTG (final concentration 0.5mM) and induce expression at 25℃ for 16h.

[0024] Protein extraction: The induced bacterial culture was centrifuged at 8000 rpm for 10 min to collect the bacterial cells, resuspended in PBS buffer, and then disrupted by an ultrasonic cell disruptor. The supernatant (containing soluble mutant protein) was collected by centrifugation at 12000 rpm for 30 min.

[0025] Affinity purification: The supernatant was purified using a Ni-NTA affinity chromatography column. Impurities were washed with buffer containing 20 mM imidazole, and the target protein was eluted with buffer containing 250 mM imidazole. The elution peak was collected, and protein purity was verified by SDS-PAGE electrophoresis (purity ≥ 90%). The protein was then dialyzed into Tris-HCl buffer (pH 7.5) and stored at -20°C for later use.

[0026] (II) Explanation of Principles High-efficiency expression of mutant proteins was achieved using a prokaryotic expression system (E. coli BL21(DE3)). Ni-NTA affinity chromatography was based on the histidine tag (inherent in the vector) and Ni 2+ The specific binding enables rapid purification of the target protein, ultimately yielding a high-purity mutant enzyme preparation, providing stable experimental materials for subsequent performance verification.

[0027] IV. Performance Verification of BsmDNA Polymerase Mutants (a) Inhibitor Tolerance Verification 1. Experimental Procedure Construct a standard amplification system (50 μL): 2 μL template (human genomic DNA, 10 ng / μL), 1 μL each of forward and reverse primers (10 μM), 1 μL dNTPs (10 mM), 5 μL 10× buffer (pH 7.5), 1 μL mutant enzyme (1 U / μL), and ddH2O to bring the total to 50 μL.

[0028] Experimental groups were set up: SDS and EDTA (final concentration 50mM) were added to the system respectively; the control groups were wild-type BsmDNA polymerase system without inhibitor and wild-type BsmDNA polymerase system with inhibitor.

[0029] Isothermal amplification conditions: reaction at 65℃ for 60 min. After the reaction, 1% agarose gel electrophoresis was performed. The brightness of the amplified bands was analyzed by a gel imaging system, and the amplification efficiency was calculated (the amplification efficiency of the wild-type system without inhibitors was 100%).

[0030] 2. Principle Explanation SDS is a typical ionic detergent that can disrupt the hydrophobic interactions of proteins, leading to enzyme denaturation, and its negative charge interferes with the electrostatic binding of enzymes to DNA; EDTA is a metal ion chelating agent that can chelate the Mg2+ required for BsmDNA polymerase catalysis. 2+ This directly inhibits enzyme activity (as shown in the "Advantage Integration Effect Diagram" which illustrates the defects of the wild-type enzyme). This validation, by simulating an inhibitory environment in a complex sample, verifies the mutant's anti-interference ability—the introduced positively charged amino acids neutralize the negative charge of SDS, stabilize the enzyme's spatial conformation, reduce the chelating effect of EDTA on metal ions, and ensure that the amplification efficiency is no less than 70% of the wild-type.

[0031] 3. Results The amplification efficiencies of the K mutant and R mutant in the presence of 1% SDS were 78% and 82% of those of the wild type, respectively, and the amplification efficiencies in the presence of 50 mM EDTA were 75% and 79%, respectively; while the wild-type BsmDNA polymerase showed almost no amplification bands in the presence of the above inhibitors.

[0032] (ii) pH adaptability verification 1. Experimental Procedure Prepare reaction buffer solutions with different pH values: pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5.

[0033] Using plasmid DNA as a template, amplification systems were constructed using the above-mentioned buffers. The mutant enzyme and wild-type enzyme served as the experimental group and control group, respectively. After isothermal amplification, the amplification Ct value was detected by real-time PCR, and the relative activity was calculated.

[0034] 2. Principle Explanation Wild-type BsmDNA polymerase has a narrow effective working pH range, while in practical applications, the pH differences between different sample extraction systems (such as acidic soil extracts and alkaline whole blood lysates) can easily lead to enzyme activity loss (Note: pH adaptability broadens its compatibility with different sample extraction systems). This validation uses gradient pH buffers to simulate the pH environment of complex samples, verifying the catalytic stability of the mutant over a wide pH range—the modified mutant achieves effective activity within the pH range of 6.0-9.5 by stabilizing the enzyme's active site conformation and local charge environment, thus reducing the impact of pH fluctuations on the catalytic reaction.

[0035] 3. Results The relative activity of the mutant was ≥80% in the pH range of 6.0-9.5, with the highest activity (≥95%) in the pH range of 7.0-8.5; while the relative activity of the wild-type enzyme was <50% in the pH range of <6.5 or >9.0, which could not meet the pH adaptation requirements of complex samples.

[0036] (III) Verification of high salt tolerance 1. Experimental Procedure An amplification system was constructed with NaCl concentration gradients of 0, 50, 100, 150, and 200 mM. Using Arabidopsis thaliana genomic DNA as a template, mutant and wild-type enzymes were amplified isothermally.

[0037] The amplified products were quantitatively analyzed by agarose gel electrophoresis to calculate the enzyme activity retention rate at different salt concentrations.

[0038] 2. Principle Explanation High-salt environments (high ionic strength) disrupt the electrostatic interaction between enzymes and DNA, preventing the enzyme from effectively binding to the template and thus inhibiting the amplification reaction (Note: Enhanced salt tolerance allows it to function in reaction systems with higher ionic strength). The mutant has been shown to amplify in buffers with ≤200 mM NaCl. This validation simulates the high-salt environment of complex samples (such as soil and plant tissue) to verify the mutant's salt tolerance—the introduction of positively charged amino acids enhances the electrostatic binding stability of the enzyme and DNA, counteracting the interference of high-salt ions, eliminating the need for additional dilution or desalting, and simplifying the sample preparation process.

[0039] 3. Results The mutant retained ≥85% of its activity when the NaCl concentration was ≤200mM; while the wild-type enzyme retained <40% of its activity when the NaCl concentration was ≥150mM, and was completely inactive at 200mM NaCl.

[0040] (iv) Freeze-thaw stability verification 1. Experimental Procedure The purified mutant enzyme and wild-type enzyme were aliquoted into 10 μL tubes and placed in a -20°C freezer for freeze-thaw cycles (freeze-thaw cycle: freeze at -20°C for 2 h → thaw at room temperature for 30 min, which is 1 cycle), and 10, 15 and 20 cycles were performed respectively.

[0041] After each cycle, 1 μL of enzyme was taken for amplification reaction (standard system, no inhibitors), and enzyme activity was detected by real-time PCR. The activity retention rate was calculated (with the enzyme activity before freezing and thawing as 100%).

[0042] 2. Principle Explanation The freeze-thaw stability of enzyme preparations directly affects their storage, transportation, and daily use—repeated freeze-thaw cycles can easily lead to protein conformational denaturation and loss of activity (Note: This stability indicator ensures the consistency of enzyme preparation performance during routine storage, transportation, and frequent handling). The mutant is required to retain ≥90% of its activity after 10-20 freeze-thaw cycles at -20°C. This validation simulates real-world freeze-thaw scenarios to verify the structural stability of the mutant—the modified enzyme maintains its core catalytic activity by optimizing the interactions of local structural domains, reducing conformational changes during freeze-thaw cycles.

[0043] 3. Results The mutant enzyme retained 98%, 95%, and 92% of its activity after 10, 15, and 20 freeze-thaw cycles, respectively; while the wild-type enzyme retained only 72% of its activity after 10 freeze-thaw cycles and decreased to 55% after 20 freeze-thaw cycles.

[0044] V. Validation of Direct Amplification Application for Complex Samples (I) Experimental Procedure Sample processing: Whole blood sample: Take 1 μL of human whole blood (unpurified) and add it to the amplification system; Soil extract: Take 1g of farmland soil, add 10mL of sterile water, shake for 10min and take 1μL of supernatant (unpurified). Fecal suspension: Take 0.1g of mouse feces, add 1mL of sterile water, vortex to mix, and take 1μL of the supernatant (unpurified). Crude extract of plant tissue: Take 0.1g of Arabidopsis thaliana leaves, add 100μL of sterile water, grind and centrifuge to obtain 1μL of supernatant (unpurified).

[0045] Amplification reaction: A direct amplification system (50 μL, pH 7.5, NaCl concentration 100 mM) was constructed using mutant enzymes. The target genes (human β-actin gene, soil nitrogen-fixing bacteria nifH gene, mouse GAPDH gene, Arabidopsis thaliana ACT2 gene) were used as amplification targets. The amplification was carried out isothermally at 65℃ for 30 min.

[0046] Control group setup: Traditional workflow: After the above samples are purified using a nucleic acid extraction kit, an equal amount of template is amplified using a wild-type enzyme; Negative control group: template-free amplification system.

[0047] Results detection: Agarose gel electrophoresis was used to analyze the amplified bands, and quantitative real-time PCR was used to calculate the target recovery rate.

[0048] (II) Explanation of Principles Complex samples (whole blood, soil, feces, etc.) are rich in various PCR inhibitors (such as heparin in whole blood, humic acid in soil, and bile salts in feces), and their pH and salt concentration fluctuate greatly (as shown in the "Direct Amplification Application Flowchart"). Traditional processes require 1-2 hours for nucleic acid purification (which can easily lead to target loss). However, the mutant of this invention, through the synergistic advantages of "inhibitor tolerance + wide pH + high salt tolerance", can directly cope with the adverse environment of complex samples without prior purification, achieving rapid amplification. The core value lies in simplifying the process and shortening the time (completed within 30 minutes), meeting the needs of point-of-care testing (POCT) and high-throughput screening.

[0049] (III) Results The mutant enzyme effectively amplified all unpurified complex samples, and agarose gel electrophoresis showed clear target bands with a target recovery rate of ≥85%. Although the traditional process group (purification + wild-type enzyme) also obtained amplification bands, the process took more than 2 hours and the target recovery rate (78%-82%) was lower than that of the mutant direct amplification group. When wild-type enzymes were directly amplified from unpurified samples, no obvious amplification bands were observed (inhibitors and salt concentrations interfered with enzyme inactivation).

[0050] VI. Results and Conclusions In this embodiment, a mutant with enhanced tolerance was successfully constructed by introducing positively charged amino acid residues (K / R) into the DNA-binding domain-catalytic domain junction region of BsmDNA polymerase using site-directed mutagenesis. This mutant retains the 5'→3' polymerase activity and 3'→5' exonuclease proofreading activity of the wild-type enzyme, while also meeting the following performance requirements: In the presence of 1% SDS or 50mM EDTA, the amplification efficiency is no less than 70% of that of the wild type; It maintains effective catalytic activity within the pH range of 6.0-9.5; It can be amplified in systems with ≤200mM NaCl; Activity retention rate ≥90% after 20 freeze-thaw cycles at -20℃; It can directly amplify complex samples such as whole blood and soil without nucleic acid purification, and the reaction is completed within 30 minutes.

[0051] The mutant of this invention overcomes the shortcomings of wild-type BsmDNA polymerase, such as sensitivity to complex environments and insufficient stability (as shown in the "Advantage Integration Effect Diagram"). It provides an effective tool for rapid molecular diagnosis of pathogens under high inhibitory environments (such as virus detection in whole blood) and long-fragment DNA cloning (such as functional gene cloning of soil microorganisms), and has important practical application value.

[0052] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A mutant of Bsm DNA polymerase with enhanced tolerance, characterized in that: The mutant is obtained by modifying the amino acid sequence of wild-type Bsm DNA polymerase, retains 5'→3' polymerase activity and 3'→5' exonuclease proofreading activity, and has significantly enhanced tolerance to inhibitors, wide pH range or high salt concentration environment compared with the wild type.

2. The Bsm DNA polymerase mutant with enhanced tolerance according to claim 1, wherein: The enhanced tolerance is specifically manifested as: in the presence of 1% (w / v) SDS or 50 mM EDTA in the reaction system, the amplification efficiency is not lower than 70% of the amplification efficiency of the wild-type enzyme under the same conditions.

3. The Bsm DNA polymerase mutant with enhanced tolerance according to claim 1, wherein: It can maintain effective catalytic activity in a buffer system with a pH range of 6.0 to 9.

5.

4. The Bsm DNA polymerase mutant with enhanced tolerance according to claim 1, wherein: It can perform nucleic acid amplification in a reaction buffer containing a concentration of not higher than 200 mM NaCl.

5. The Bsm DNA polymerase mutant with enhanced tolerance according to claim 1, wherein: The mutation is located at the junction region of the DNA binding domain and the catalytic domain of the enzyme, and includes the introduction of a positively charged amino acid residue.

6. The Bsm DNA polymerase mutant with enhanced tolerance according to claim 5, wherein: The positively charged amino acid residue is lysine (K) or arginine (R).

7. The Bsm DNA polymerase mutant with enhanced tolerance according to claim 1, wherein: After 10-20 °C freeze-thaw cycles, the activity retention rate of the mutant is not lower than 90% of the initial activity.

8. Use of a Bsm DNA polymerase mutant according to any one of claims 1 to 7 for direct amplification of target nucleic acids in complex biological or environmental samples, characterized in that: The application does not require pre-nucleic acid purification of the sample.

9. Use according to claim 8, characterized in that: The complex sample includes whole blood, soil extract, fecal suspension or plant tissue crude extract.

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