A restriction endonuclease XcmI mutant with improved activity and stability and its application

Abstract

The present invention discloses a restriction endonuclease XcmI mutant with improved activity and stability and its application, belonging to the field of biotechnology. The restriction endonuclease XcmI mutant of the present invention has an amino acid sequence as shown in SEQ ID NO.1. The XcmI mutant of the present invention has a specific activity that is more than three times that of the wild type. After storage at 37°C for 48 hours, the mutant still retains 100% activity, while the wild type only retains 10% activity. After treatment with 20 mM H2O2 for 30 minutes, the mutant still retains approximately 30% activity, while the wild type only retains 10% activity. The restriction endonuclease XcmI mutant of the present invention has significantly improved activity, thermal stability, and oxidative stability compared to the wild type, and has good application value.

Description

Technical Field

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

[0002] Restriction endonucleases (REs) can recognize and cleave specific DNA sequences. Derived from the bacterial restriction-modification (RM) system, they are important tools and raw materials in fields such as synthetic biology, nucleic acid drugs, in vitro diagnostics, and biomedical research. Based on the composition of the RM system, the characteristics of the DNA recognition sequence and cleavage site, and the need for auxiliary factors, REs can be divided into four major categories: Type I, II, III, and IV. Type II is the primary biomedical tool.

[0003] XcmI is a commonly used Typp II restriction enzyme with a recognition and cleavage sequence of CCANNNNN / NNNNTGG. Its encoding gene is derived from Xanthomonas campestris ( Xanthomonas campestris ), which is generally produced by recombinant expression in E. coli. The specific activity of wild-type XcmI is low, only about 1×10 4 U / mg, resulting in high production costs. On the other hand, wild-type XcmI has poor thermal stability, losing most of its activity after one day of storage at 37°C, and its storage time at 4°C is also extremely limited, affecting the storage and transportation of the enzyme. Furthermore, in actual use, it was found that if a tube of XcmI is repeatedly opened and used for aspiration, even if strict precautions are taken to use and store at low temperatures, XcmI will gradually lose its activity within its shelf life. Analysis suggests that this is due to frequent opening of the lid, which causes XcmI to be frequently exposed to air, leading to oxidation and gradual inactivation.

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

[0005] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides a restriction endonuclease XcmI mutant with improved activity and stability, which solves the problems of low specific activity and poor stability of the existing restriction endonuclease XcmI.

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

[0007] Technical solution: In order to achieve the above-mentioned purpose, 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 shown in SEQ ID NO.1.

[0008] Compared with the wild-type XcmI (NCBI accession number AEN19707.1), the restriction endonuclease XcmI mutant has cysteine ​​at position 114 replaced by alanine (C114A) and cysteine ​​at position 166 replaced by alanine (C166A).

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

[0010] The invention discloses an application of the restriction endonuclease XcmI mutant with improved activity and stability in recognizing and cutting DNA sequences.

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

[0012] The restriction endonuclease XcmI mutant is used to recognize and cut DNA sequences after being incubated at 25-37°C for 24-48 hours.

[0013] The restriction endonuclease XcmI mutant is used in recognizing and cutting DNA sequences after oxidation.

[0014] Furthermore, the restriction endonuclease XcmI mutant is used to recognize and cut DNA sequences after incubation with 5-20 mM H2O2.

[0015] The kit of the present invention contains a restriction endonuclease XcmI mutant with improved activity and stability.

[0016] The kit includes restriction endonuclease XcmI mutant enzyme solution, buffer, nuclease-free water and the necessary components required for the endonuclease kit.

[0017] The kit of the present invention is used in identifying and cutting DNA sequences.

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

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

[0020] The restriction endonuclease XcmI mutant of the present invention has significantly improved thermal stability compared to the wild-type protein. It can still maintain 100% activity after storage at 37°C for 48 hours, while the wild-type protein only has 10% activity after storage at 37°C for 48 hours.

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

[0022] The restriction endonuclease XcmI mutant 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:

[0024] The XcmI mutant described in this invention exhibits over three times the specific activity of wild-type XcmI. After storage at 37°C for 48 hours, the mutant retains 100% activity, while the wild-type retains only 10% activity. After treatment with 20 mM H₂O₂ for 30 minutes, the mutant still retains approximately 30% activity, while the wild-type retains only 10% activity. Compared to the wild-type, the XcmI mutant described in this invention exhibits significantly improved activity, thermal stability, and oxidative stability, which can help reduce production costs, improve stability during storage and use, and extend the product's shelf life. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The results of the specific activity determination of XcmI mutants and wild type are shown, among which C114A / C166A is a mutant;

[0026] Figure 2 Comparison of thermal stability between XcmI mutants and wild type, where C114A / C166A is a mutant;

[0027] Figure 3 Comparison of the oxidative stability of XcmI mutants and wild type, where C114A / C166A is a mutant. DETAILED DESCRIPTION

[0028] The present invention will be further described below with reference to the accompanying drawings and examples. The experimental methods in the following examples, unless otherwise specified, are conventional biological experimental methods. The experimental materials used, unless otherwise specified, can be purchased from conventional biochemical reagent manufacturers.

[0029] The mutants of the present invention can be obtained by conventional gene synthesis and protein expression and purification methods without affecting their functions and activities.

[0030] Example 1

[0031] Determination of enzyme activity of XcmI mutant:

[0032] We commissioned a gene synthesis company to synthesize the gene sequence encoding the XcmI mutant shown in SEQ ID NO. 2. Following the strategy outlined in US Patent No. 6403354 B1, we used recombinant expression in E. coli and purification to obtain a pure XcmI mutant. The nucleotide sequence of the mutant gene is shown in SEQ ID NO. 2, and the amino acid sequence of the mutant protein is shown in SEQ ID NO. 1. Commercially available, unmodified wild-type XcmI (i.e., the original unmutated protein, obtained from Jiangsu Baishimei Biotechnology Co., Ltd., EG15582) was used as a control.

[0033] The XcmI mutant and wild-type XcmI proteins were diluted to 0.5 mg / mL as working solutions, and then the working solutions were serially diluted in 1.5-fold ratios. 1 μL of protein solution from each serial dilution was added with 1 μg of λ DNA (ThermoFisher, SD0011), 5 μL of 10× CutOne ® Buffer (Bristol-Myers Squibb), make up to 50 μL with nuclease-free water, react at 37°C for 1 hour, and then inactivate at 80°C for 20 minutes. Take 5 μL of the reaction product, add 1 μL of 6× DNA Loading Buffer (Bristol-Myers Squibb, EG21915), mix well, and perform agarose gel electrophoresis.

[0034] The results are as follows Figure 1 As shown, the XcmI mutant of the present invention can completely cleave the substrate λDNA at a dilution of at least 1 / 17.09, or 0.0293 mg / mL; whereas the wild-type can only completely cleave the substrate at a dilution of 1 / 5.06, or 0.0988 mg / mL. According to the definition of XcmI activity, one unit (U) is the amount of enzyme required to completely cleave 1 µg of λDNA in a 50 µL digestion reaction at 37°C for 1 hour. Calculated, 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 was only 1.01×10 4 U / mg, the enzyme activity of mutant XcmI is more than 3 times that of the wild-type protein.

[0035] Example 2

[0036] Comparison of thermal stability between XcmI mutant and wild type:

[0037] Working solutions of the XcmI mutant and wild-type XcmI proteins of Example 1 were incubated at 37°C for 24 h and 48 h, respectively, and then enzyme activity was determined according to the method of Example 1. The specific activity values ​​of the two proteins without 37°C treatment were used as respective controls, and the relative activity was set to 100%.

[0038] The results are as follows Figure 2 As shown in the figure, the XcmI mutant retained 100% activity after incubation at 37°C for 48 hours, while the activity of the wild-type decreased significantly after incubation at 37°C, with the relative activity remaining at only about 10% after 48 hours. This indicates that the XcmI mutant is significantly more thermostable than the wild-type.

[0039] Example 3

[0040] Comparison of oxidative stability between XcmI mutants and wild type:

[0041] To the working solutions of the XcmI mutant and wild-type XcmI proteins described in Example 1, H2O2 (Aladdin, H112517) was added at final concentrations of 5, 10, and 20 mM, respectively. After incubation at 25°C for 30 minutes (i.e., rapid oxidation), the reaction was terminated by adding an excess of catalase (Aladdin, C100456). Enzyme activity was then measured according to the method described in Example 1. The specific activity values ​​of the two proteins without oxidation served as controls, with the relative activity set as 100%.

[0042] The results are as follows Figure 3 As shown in the figure, under the simulated oxidative environment of H2O2 treatment, the enzyme activity of the wild-type XcmI decreases. However, the relative activity of the mutant after treatment with different H2O2 concentrations for 30 minutes is significantly higher than that of the wild-type. After treatment with 20 mM H2O2 for 30 minutes, the mutant still retains approximately 30% of its activity, while the wild-type activity only retains 10%, indicating that the oxidative stability of the XcmI mutant is significantly better than that of the wild-type.

[0043] Comparative Example 1

[0044] Comparative Example 1: Using the method of Example 1, comparative restriction enzyme XcmI mutant proteins were prepared. Compared to wild-type XcmI, only cysteine ​​at position 114 (C114A) or only cysteine ​​at position 166 (C166A) was substituted with alanine. The two mutant proteins were tested according to the method of Example 2. After incubation at 37°C for 48 hours, they retained less than 50% of their activity, demonstrating the importance of the combined mutation of two amino acid positions in the present invention.

Claims

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

1.

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

2.

3. Use of the restriction endonuclease XcmI mutant with improved activity and stability according to claim 1 in recognizing and cleaving a DNA sequence, wherein the restriction endonuclease XcmI mutant recognizes and cleaves a DNA sequence after incubation at 25-37°C for 24-48 hours; or recognizes and cleaves a DNA sequence after incubation in 5-20 mM H2O2.

4. The use according to claim 3, characterized in that The DNA sequence is CCANNNNN / NNNNTGG.

5. A kit comprising the restriction endonuclease XcmI mutant with improved activity and stability according to claim 1.

6. The kit according to claim 5, characterized in that The kit comprises restriction endonuclease XcmI mutant enzyme solution, buffer, nuclease-free water and necessary components required for the endonuclease kit.

7. Use of the kit according to claim 5 in identifying and cleaving DNA sequences.

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