High oxidative stability mutants of restriction enzyme xhol and use thereof

Abstract

The application discloses a high-oxidation-stability mutant of restriction enzyme Xhol and application thereof, and the amino acid sequence of the high-oxidation-stability mutant of the restriction enzyme Xhol is shown as SEQ ID NO. 1. The mutant of the restriction enzyme Xhol in the application can still retain 45% of the activity of the positive control under the simulated oxidation condition of a protein: hydrogen peroxide molar ratio of 1:2000, while the wild type only has 20%. That is, the oxidation stability of the mutant of the restriction enzyme Xhol is more than twice that of the wild type, and meanwhile, the mutant still maintains excellent specific activity, has good commercial application value, and effectively solves the problem that the existing restriction enzyme Xhol is gradually inactivated due to repeated exposure to air during use and then oxidation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a mutant of restriction enzyme XhoI with high oxidative stability. Background Technology

[0002] Restriction endonucleases (REIs), classic molecular cloning tools, can recognize and cleave specific DNA sequences, making them essential raw materials for synthetic biology, macromolecular nucleic acid drugs, and biomedical research. As a class of active proteins, restriction enzymes are easily inactivated at room temperature and are generally stored at -20°C, where they can remain effective for up to two years. However, some restriction enzymes, such as the commonly used restriction enzyme XhoI, are still susceptible to interference from external factors during storage and use.

[0003] Currently, all commercially available XhoI products are wild-type proteins, and their encoding genes are derived from Xanthomonas oryzae (…). Xanthomonas holcicola XhoI is generally produced through recombinant expression in E. coli. In actual use, it has been found that commercially available XhoI can have a shelf life of up to two years if stored continuously at -20°C; however, if it is repeatedly opened and used for a period of time, its activity may gradually decrease or even be lost within the theoretical shelf life.

[0004] In molecular biology experiments, restriction enzyme products are typically not used entirely upon opening; only a portion of the enzyme solution is used, and the remaining solution is returned to low temperature for later use. This frequent opening and aspiration exposes XhoI to air repeatedly, potentially leading to oxidation and gradual inactivation. Therefore, modifying the restriction enzyme XhoI to obtain a mutant with better oxidative stability is of great significance, as it can effectively extend the shelf life and maintain the activity of the XhoI mutant. Summary of the Invention

[0005] Purpose of the invention: To address the problems existing in the prior art, the present invention provides a mutant of restriction enzyme XhoI with high oxidative stability and its application, which solves the problem that existing XhoI is gradually inactivated due to oxidation caused by repeated exposure to air during use.

[0006] Technical solution: In order to achieve the above objective, the present invention provides a high oxidative stability mutant of restriction enzyme XhoI, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention relates to the coding gene of a high oxidative stability mutant of the restriction enzyme XhoI, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] The high oxidative stability mutant enzyme of restriction enzyme XhoI provided by this invention can be used to develop more efficient reagent kit products.

[0009] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The restriction enzyme XhoI mutant of this invention retains 45% of the activity of the positive control under simulated oxidation conditions of protein:hydrogen peroxide molar ratio of 1:2000, while the wild type retains only 20%. That is, the oxidative stability of the restriction enzyme XhoI mutant of this invention is more than twice that of the wild type, while the mutant still maintains excellent specific activity, and has good commercial application value. Attached Figure Description

[0010] Figure 1 The specific activity determination results of the XhoI mutant and wild type described in this invention; Figure 2 Comparison of oxidative stability between the XhoI mutant and wild type described in this invention. Detailed Implementation

[0011] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional biological experimental methods. Unless otherwise specified, the experimental materials used can be purchased from conventional biochemical reagent manufacturers.

[0012] The mutants in this invention can be obtained using conventional gene synthesis and protein expression purification methods without affecting their function and activity.

[0013] Example 1

[0014] Before measuring enzyme activity, prepare the reaction substrate λDNA (digested with HindIII). Take 1 µg of λDNA (ThermoFisher, SD0011) for each substrate sample and add 5 µL of 10× CutOne. ® Buffer (Blisterine, packaged with restriction enzyme) and 0.25µL LightNing ® HindIII (Bristol-Myers Squibb, EG15539) was added to a nuclease-free water volume of 49 μL, reacted at 37°C for 2 hours, and then inactivated at 80°C for 20 minutes.

[0015] Both the mutant C157S and wild-type XhoI proteins were diluted to 0.01 mg / mL as working solutions, and then serially diluted at ratios of 1 / 2, 1 / 4, 1 / 8, 1 / 16, and 1 / 32. 1 μL of each serially diluted protein solution was added to 49 μL of the prepared λDNA (HindIII digestion) reaction solution, and incubated at 37°C for 1 hour, followed by inactivation at 80°C for 20 minutes. 5 μL of the reaction product was then added to 1 μL of 6× DNA Loading Buffer (Bristol-Myers Squibb, EG21915), mixed thoroughly, and subjected to agarose gel electrophoresis.

[0016] The results are as follows Figure 1 As shown, C157S, like wild-type XhoI, can still completely digest λDNA (HindIII digestion) even when diluted to 0.00125 mg / mL. According to the definition of XhoI enzyme activity, one enzyme activity unit (U) is the amount of enzyme required to completely digest 1 µg of λDNA (HindIII digestion) fragment in a 50 µL digestion reaction system at 37°C for 1 hour. Calculations show that the specific activity of both C157S and wild-type XhoI is 8 × 10⁻⁶. 5 U / mg, demonstrating that the XhoI mutant of the present invention can still maintain the same high enzyme activity as the wild type.

[0017] Example 2

[0018] Evaluation of the oxidative stability of the XhoI mutant The mutant C157S and wild-type XhoI proteins were each prepared into 20 µM (approximately 430 U / µL) solutions. Then, an equal volume of 40 mM H2O2 solution (Aladdin, H112517) was added to each solution, bringing the final protein concentration to 10 µM and the final H2O2 concentration to 20 mM, i.e., a molar ratio of protein to H2O2 of 1:2000. The solutions were incubated at 25°C for 10 min, 20 min, 40 min, and 60 min, respectively, to simulate the oxidation process. The reaction was then terminated by adding excess catalase (Aladdin, C100456).

[0019] The oxidized protein solution was serially diluted according to the method described in Example 1, and the remaining enzyme activity was measured. The unoxidized enzyme solution was used as a control, and the relative activity was set to 100%. The results are as follows: Figure 2As shown, after 10 min of H2O2 oxidation treatment, the relative activity of wild-type XhoI decreased to below 50%, while the mutant C157S maintained 100% relative activity. After 60 min of oxidation treatment, the relative activity of wild-type XhoI had decreased to 20%, while the mutant C157S still maintained 45% relative activity, which is 2.25 times that of the wild type. This indicates that the oxidative stability of the mutant C157S is significantly higher than that of the wild type.

[0020] Comparative Example 1 Comparative Example 1 used the method of Example 1 to prepare a comparative XhoI mutant protein, which, compared to wild-type XhoI, only had serine replacing cysteine ​​at position 70 (C70S) or alanine replacing cysteine ​​at position 190 (C190A). The two comparative mutant proteins were then subjected to oxidation treatment according to the method of Example 2, and their remaining relative activity was measured. All time points showed activity below 30%, further demonstrating the advantages of the specific mutations in the mutants of this invention.

Claims

1. A mutant of restriction enzyme XhoI with high oxidative stability, characterized in that, The amino acid sequence of the highly oxidatively stable mutant of the restriction enzyme XhoI is shown in SEQ ID NO.

1.

2. The highly oxidatively stable mutant of restriction enzyme XhoI according to claim 1, characterized in that, The high oxidative stability mutant of the restriction enzyme XhoI, compared with wild-type XhoI, has serine replacing cysteine ​​at position 157 (C157S).

3. A gene encoding a highly oxidatively stable mutant of the restriction enzyme XhoI, characterized in that, The nucleotide sequence of the gene encoding the restriction enzyme XhoI mutant is shown in SEQ ID NO.

2.

4. The use of a highly oxidatively stable mutant of the restriction enzyme XhoI as described in claim 1 in recognizing and cutting DNA sequences.

5. The application according to claim 4, characterized in that, The DNA sequence is C / TCGAG.

6. The application according to claim 4, characterized in that, Application of the highly oxidatively stable mutant of the restriction enzyme XhoI in recognizing and cleaving DNA sequences under oxidative conditions.

7. A kit containing a high oxidative stability mutant of the restriction enzyme XhoI, which enhances activity and stability as described in claim 1.

8. The reagent kit according to claim 7, characterized in that, The kit includes a highly oxidatively stable mutant enzyme solution of restriction enzyme XhoI, buffer, nuclease-free water, and other necessary components required for the kit.

9. The use of the kit of claim 7 in identifying and cutting DNA sequences.

Images