Restriction endonuclease XcmI mutant capable of improving activity and stability and application of restriction endonuclease XcmI mutant

Abstract

The invention discloses a restriction enzyme XcmI mutant capable of improving activity and stability and application of the restriction enzyme XcmI mutant, and belongs to the technical field of biology, and the amino acid sequence of the restriction enzyme XcmI mutant is shown as SEQ ID NO.1. The specific activity of the XcmI mutant is more than three times that of a wild type, the mutant can still remain 100% of activity after being stored at 37 DEG C for 48 hours, while only 10% of activity of the wild type remains, and after being treated by 20mM H2O2 for 30 minutes, the mutant can still remain about 30% of activity while only 10% of activity of the wild type remains. Compared with a wild type, the restriction enzyme XcmI mutant disclosed by the invention has the advantages that the activity, the thermal stability and the oxidation stability are remarkably improved, and the restriction enzyme XcmI mutant has a relatively good application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a mutant of restriction endonuclease XcmI with improved activity and stability and its application. Background Art

[0002] Restriction endonucleases (referred to as "restriction enzymes" for short) can recognize and cleave specific DNA sequences. They are derived from the bacterial "restriction-modification" (R-M) system and are important tools and raw materials in the fields of synthetic biology, nucleic acid drugs, in vitro diagnosis, biomedical research, etc. According to the composition mode of the R-M system, the characteristics of DNA recognition sequences and cleavage sites, and the requirements for cofactors, restriction enzymes can be classified into 4 major categories, namely Type I, II, III, and IV. Among them, the main type used as a biomedical tool is Type II.

[0003] XcmI is a relatively commonly used Type II restriction enzyme that recognizes and cleaves the sequence CCANNNNN / NNNNTGG, and its coding gene is derived from Xanthomonas campestris pv. campestris ( Xanthomonas campestris ), and it is generally produced by recombinant expression in Escherichia coli. The specific activity of wild-type XcmI is relatively low, only about 1×10 4 U / mg, resulting in a relatively high production cost. On the other hand, the thermal stability of wild-type XcmI is not good. Most of its activity is lost after being stored at 37°C for 1 day, and its storage time at 4°C is also extremely limited, which affects the storage and transportation of this enzyme. In addition, it has also been found in actual use that if a tube of XcmI is repeatedly opened and aspirated for use, even if strict attention is paid to using and storing it at low temperature, XcmI will gradually lose its activity within the validity period. After analysis, it is considered that repeated opening of the tube causes XcmI to be frequently exposed to air, and thus it is oxidized and gradually inactivated.

[0004] Therefore, it is of great significance to modify restriction endonuclease XcmI to obtain mutants with higher specific activity and stability. Summary of the Invention

[0005] Object of the Invention: Aiming at the problems existing in the prior art, the present invention provides a mutant of restriction endonuclease XcmI with improved activity and stability, and solves the reasons for the relatively low specific activity and poor stability of the existing restriction endonuclease XcmI.

[0006] The present invention also provides the application of the mutant of restriction endonuclease XcmI.

[0007] Technical Solution: In order to achieve the above object, the present invention provides a mutant of restriction endonuclease XcmI with improved activity and stability. The amino acid sequence of the restriction endonuclease XcmI is as shown in SEQ ID NO.1.

[0008] Among them, compared with the wild-type XcmI (NCBI accession number AEN19707.1), the restriction endonuclease XcmI mutant replaces the cysteine at position 114 with alanine (C114A) and replaces the cysteine at position 166 with alanine (C166A).

[0009] The restriction endonuclease XcmI mutant with improved activity and stability according to the present invention, the restriction endonuclease XcmI, the nucleotide sequence of its coding gene is shown in SEQ ID NO.2.

[0010] Application of the restriction endonuclease XcmI mutant with improved activity and stability according to the present invention in recognizing and cleaving a DNA sequence.

[0011] Among them, the DNA sequence is CCANNNNN / NNNNTGG.

[0012] Among them, the application of the restriction endonuclease XcmI mutant in recognizing and cleaving a DNA sequence after incubation at 25-37 °C for 24-48 h.

[0013] Among them, the application of the restriction endonuclease XcmI mutant after oxidation in recognizing and cleaving a DNA sequence.

[0014] Furthermore, the application of the restriction endonuclease XcmI mutant in recognizing and cleaving a DNA sequence after incubation with 5-20 mM H2O2.

[0015] The kit containing the restriction endonuclease XcmI mutant with improved activity and stability according to the present invention.

[0016] Among them, the kit includes the restriction endonuclease XcmI mutant enzyme solution, Buffer, nuclease-free water, and the necessary components required for an endonuclease kit.

[0017] Application of the kit according to the present invention in recognizing and cleaving a DNA sequence.

[0018] Those skilled in the art can obtain the coding gene of the restriction enzyme XcmI mutant according to the present invention through various conventional biological techniques such as molecular cloning and gene synthesis, and obtain the restriction enzyme XcmI mutant protein according to the present invention through conventional recombinant expression and protein purification techniques.

[0019] The restriction endonuclease XcmI mutant according to the present invention has a specific activity more than 3 times that of the wild-type protein, at least reaching 3.42×10 4 U / mg.

[0020] The XcmI mutant restriction endonuclease described in the present invention has significantly improved thermal stability compared to the wild-type protein, and can still maintain 100% activity after being stored at 37 °C for 48 h, while the wild-type only retains 10% activity after being stored at 37 °C for 48 h.

[0021] The XcmI mutant restriction endonuclease described in the present invention also has significantly improved oxidative stability compared to the wild-type protein. In a simulated strong oxidation environment treated with 20 mM H2O2 for 30 min, it can still retain more than 30% activity, while the wild-type only retains 10% activity under the same conditions.

[0022] The XcmI mutant restriction endonuclease provided by the present invention can be used to develop more efficient kit products.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The specific activity of the XcmI mutant in the present invention is more than 3 times that of the wild-type XcmI. This mutant can still retain 100% activity after being stored at 37 °C for 48 h, while the wild-type only retains 10% activity. After being treated with 20 mM H2O2 for 30 min, this mutant can still retain about 30% activity, while the wild-type only retains 10% activity. The XcmI mutant described in the present invention has significantly improved activity, thermal stability and oxidative stability compared to the wild-type, which is beneficial to reducing production costs, improving stability during storage and use, and extending the product shelf life. Description of the Drawings

[0024] Figure 1 It is the determination result of the specific activity of the XcmI mutant and the wild-type, where C114A / C166A is the mutant; Figure 2 It is the comparison of the thermal stability of the XcmI mutant and the wild-type, where C114A / C166A is the mutant; Figure 3 It is the comparison of the oxidative stability of the XcmI mutant and the wild-type, where C114A / C166A is the mutant. Detailed Embodiments

[0025] The present invention will be further described below in conjunction with the drawings and embodiments. The experimental methods in the following embodiments are all conventional biological experimental methods unless otherwise specified. The experimental materials used can all be obtained from conventional biochemical reagent manufacturers unless otherwise specified.

[0026] The mutant in the present invention can be obtained by using conventional gene synthesis and protein expression and purification methods, without affecting its function and activity.

[0027] Example 1

[0028] Determination of the enzyme activity of the XcmI mutant: Entrust a gene synthesis company to synthesize the XcmI mutant coding gene sequence shown in SEQ ID NO. 2. Referring to the strategy of Patent US 6403354 B1, Escherichia coli recombinant expression and purification were used to obtain pure XcmI mutant. The nucleotide sequence of the mutant coding gene is as shown in SEQ ID NO.2, and the amino acid sequence of the mutant protein is as shown in SEQ ID NO.1. At the same time, commercially available unmodified wild-type XcmI (i.e., the original unmutated protein, Jiangsu Bristol-Myers Squibb Biotechnology Co., Ltd., EG15582) was used as a control.

[0029] Dilute both the XcmI mutant and wild-type XcmI proteins to 0.5 mg / mL as the working solution, and then perform gradient dilution on the working solution at a 1.5-fold ratio respectively. Take 1 μL of the protein liquid with different gradient dilutions, add 1 µg of λDNA (ThermoFisher, SD0011), 5 µL of 10× CutOne ® Buffer (Bristol-Myers Squibb), and make up to 50 μL with nuclease-free water. After reacting at 37°C for 1 hour, inactivate at 80°C for 20 minutes. Take 5 μL of the reaction product, add 1 μL of 6× DNA LoadingBuffer (Bristol-Myers Squibb, EG21915), mix well, and then perform agarose gel electrophoresis.

[0030] The results are as Figure 1 shown. For the XcmI mutant of the present invention, it can still completely digest the substrate λDNA when diluted to at least 1 / 17.09 times, that is, 0.0293 mg / mL; while the wild type can only be diluted to 1 / 5.06, that is, 0.0988 mg / mL at the lowest to completely digest the substrate. According to the definition of XcmI enzyme activity, one enzyme activity unit (U) is the amount of enzyme required to completely digest 1 µg of λDNA in a 50 µL enzyme digestion reaction system at 37°C for 1 hour. After calculation, the specific activity of the XcmI mutant of the present invention is at least 3.42×10 4 U / mg, while the specific activity of the wild type is only 1.01×10 4 U / mg. The enzyme activity of the mutant XcmI is more than 3 times that of the wild-type protein.

[0031] Example 2

[0032] Comparison of the thermal stability between the XcmI mutant and the wild type: Incubate the working solutions of the XcmI mutant and wild-type XcmI proteins in Example 1 at 37°C for 24 h and 48 h respectively, and then determine the enzyme activity according to the method in Example 1. Use the specific activity values of the two proteins without 37°C treatment as their respective controls, set as relative activity 100%.

[0033] The results are as follows Figure 2 shown. After incubation at 37 °C for 48 h, the XcmI mutant still retained 100% of its activity; while the wild type showed a significant decrease in activity after incubation at 37 °C, and the relative activity was only about 10% after 48 h. This indicates that the thermal stability of the XcmI mutant is significantly higher than that of the wild type.

[0034] Example 3

[0035] Comparison of the oxidative stability between the XcmI mutant and the wild type: To the working solutions of the XcmI mutant and the wild type XcmI protein in Example 1, H2O2 (Aladdin, H112517) with a final concentration of 5, 10, and 20 mM was added respectively. After incubation at 25 °C for 30 min (i.e., rapid oxidation treatment), an excess amount of catalase (Aladdin, C100456) was added to terminate the reaction. Then, the enzyme activity was measured according to the method of Example 1. The specific activity values of the two proteins without oxidation treatment were used as their respective controls, set as 100% relative activity.

[0036] The results are as follows Figure 3 shown. In the simulated oxidative environment treated with H2O2, the enzyme activity of the wild type XcmI decreased. However, the relative activities of the mutant after treatment with different concentrations of H2O2 for 30 min were significantly higher than those of the wild type. After treatment with 20 mM H2O2 for 30 min, the mutant still retained about 30% of its activity, while the activity of the wild type was only 10%. This indicates that the oxidative stability of the XcmI mutant is significantly better than that of the wild type.

[0037] Comparative Example 1 In Comparative Example 1, the method of Example 1 was used to prepare a comparative restriction enzyme XcmI mutant protein. Compared with the wild type XcmI, only cysteine at position 114 was replaced with alanine (C114A) or only cysteine at position 166 was replaced with alanine (C166A). The two mutant proteins obtained were tested according to the method of Example 2. After incubation at 37 °C for 48 h, they could only retain less than 50% of their activity, indicating the importance of the combined mutation of the two amino acid sites in the present invention.

Claims

1. A mutant of restriction endonuclease XcmI with improved activity and stability, characterized in that, In the XcmI mutant of the restriction endonuclease, compared with wild-type XcmI, cysteine at position 114 is replaced with alanine (C114A), and cysteine at position 166 is replaced with alanine (C166A); the amino acid sequence of the XcmI mutant of the restriction endonuclease is as shown in SEQ ID NO.

1.

2. The XcmI restriction endonuclease mutant with improved activity and stability according to claim 1, characterized in that, For the XcmI mutant of the restriction endonuclease, the nucleotide sequence of its encoding gene is as shown in SEQ ID NO.

2.

3. Application of the XcmI mutant of the restriction endonuclease with improved activity and stability as claimed in claim 1 in recognizing and cleaving a DNA sequence.

4. The application according to claim 3, wherein The DNA sequence is CCANNNNN / NNNNTGG.

5. The application according to claim 3, characterized in that, Application of the XcmI mutant of the restriction endonuclease in recognizing and cleaving a DNA sequence after incubation at 25-37 °C for 24-48 h.

6. The application according to claim 3, wherein Application of the XcmI mutant of the restriction endonuclease after oxidation in recognizing and cleaving a DNA sequence.

7. A kit containing the XcmI mutant of the restriction endonuclease with improved activity and stability as claimed in claim 1.

8. The kit according to claim 7, wherein The kit includes the enzyme solution of the XcmI mutant of the restriction endonuclease, Buffer, nuclease-free water, and the necessary components required for an endonuclease kit.

9. Application of the kit as claimed in claim 7 in recognizing and cleaving a DNA sequence.

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