High-oxidation-stability mutant of restriction enzyme XhoI and application of high-oxidation-stability mutant

Abstract

The invention discloses a high-oxidation-stability mutant of a restriction enzyme XhoI and application of the high-oxidation-stability mutant of the restriction enzyme XhoI, and the amino acid sequence of the high-oxidation-stability mutant of the restriction enzyme XhoI is as shown in SEQ ID NO. 1. According to the restriction enzyme XhoI mutant, under the simulated oxidation condition that the molar ratio of protein to hydrogen peroxide is 1: 2000, the activity of the restriction enzyme XhoI mutant can still be kept to be 45% of that of a positive control, and the activity of a wild type is only 20%. In other words, the oxidation stability of the restriction enzyme XhoI mutant is more than two times that of a wild type, meanwhile, the mutant still keeps excellent specific activity and has good commercial application value, and the problem that existing restriction enzyme XhoI is oxidized and gradually inactivated due to the fact that the restriction enzyme XhoI is repeatedly exposed in air during use is effectively solved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a high-oxidation-stability mutant of a restriction enzyme XhoI. Background Art

[0002] Restriction endonucleases (REs) are classic molecular cloning enzymes that can recognize and cleave specific DNA sequences. They are important raw materials for synthetic biology, large-molecule nucleic acid drugs, and biomedical research. Restriction enzymes, as active proteins, are easily inactivated when stored at room temperature. They are generally stored at -20°C and have a shelf life of up to two years. However, some REs, such as the commonly used RE XhoI, are still susceptible to interference from external factors during storage and use.

[0003] Currently available commercial XhoI products are all wild-type proteins, encoded by the gene derived from Xanthomonas holcicola, and typically produced through recombinant expression in Escherichia coli. In actual use, commercially available XhoI has been shown to have a shelf life of up to two years if stored continuously at -20°C. However, if repeatedly opened and used for a period of time, its activity may gradually decrease or even be lost within its theoretical shelf life.

[0004] During molecular biology experiments, restriction enzyme products are typically not fully consumed during a single opening. Instead, only a portion of the enzyme solution is withdrawn and used, with the remaining enzyme returned to a low-temperature storage location for future use. This frequent opening and withdrawal results in repeated exposure of XhoI to air, potentially leading to oxidation and gradual inactivation. Therefore, modifying the restriction enzyme XhoI to produce an XhoI mutant with improved oxidative stability is of great significance, as it can effectively extend the lifespan and activity of XhoI mutants. Summary of the Invention

[0005] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides a highly oxidatively stable mutant of the restriction enzyme XhoI and its application, which solves the problem that the existing XhoI is repeatedly exposed to air during use and then gradually inactivated by oxidation.

[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 highly oxidatively stable mutant of the restriction enzyme XhoI, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] Compared with the wild-type XcmI, the high oxidation stability mutant of the restriction enzyme XhoI has cysteine at position 157 replaced by serine (C157S).

[0009] The invention provides a gene encoding a highly oxidatively stable mutant of the restriction enzyme XhoI, wherein the nucleotide sequence of the gene encoding the restriction enzyme XcmI mutant is shown in SEQ ID NO.2.

[0010] The invention relates to the use of the high oxidation stability mutant of the restriction enzyme XhoI in recognizing and cutting DNA sequences.

[0011] Wherein, the DNA sequence is C / TCGAG

[0012] The highly oxidatively stable mutant of the restriction enzyme XhoI is used to recognize and cut DNA sequences under oxidative conditions.

[0013] The kit of the present invention contains the high oxidation stability mutant of the restriction enzyme XhoI with improved activity and stability.

[0014] The kit includes a high oxidation stability mutant enzyme solution of the restriction enzyme XhoI, a buffer, nuclease-free water and necessary components required for the kit.

[0015] Application of the kit of the present invention in identifying and cutting DNA sequences

[0016] Those skilled in the art can obtain the encoding gene of the restriction enzyme XhoI mutant of the present invention through various conventional biological techniques such as molecular cloning and gene synthesis, and obtain the restriction enzyme XhoI mutant protein of the present invention through conventional recombinant expression and protein purification techniques.

[0017] The present invention designs and obtains a restriction enzyme XhoI mutant with improved oxidative stability through molecular simulation and site-directed mutagenesis. The oxidative stability is improved by more than 2 times compared with the wild type.

[0018] The high oxidation stability mutant enzyme of the restriction enzyme XhoI provided by the present invention can be used to develop more efficient kit products.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0020] The restriction enzyme XhoI mutant of the present invention retains 45% of its activity compared to the positive control under simulated oxidative conditions at a protein:hydrogen peroxide molar ratio of 1:2000, while the wild-type retains only 20%. This indicates that the XhoI mutant of the present invention is more than twice as oxidatively stable as the wild-type, while maintaining excellent specific activity, demonstrating its potential for commercial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of the specific activity determination of the XhoI mutant of the present invention and the wild type;

[0022] Figure 2 The oxidative stability of the XhoI mutant of the present invention was compared with that of the wild type. DETAILED DESCRIPTION

[0023] 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.

[0024] 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.

[0025] Example 1

[0026] Determination of specific activity of XhoI mutants

[0027] A gene synthesis company was commissioned to synthesize the gene sequence encoding the XhoI mutant (hereinafter referred to as C157S) shown in SEQ ID NO. 2. Following the strategy described in US Patent No. 6,403,354 B1, the mutant C157S protein was obtained through recombinant expression in E. coli and purification. 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 XhoI (i.e., the original unmutated protein, produced by Jiangsu Best Biotechnology Co., Ltd., EG15583) was used as a control.

[0028] Before measuring enzyme activity, prepare the reaction substrate λDNA (HindIII digestion). Take 1 μg λDNA (ThermoFisher, SD0011) for each substrate and add 5 μL Buffer (Bristol-Myers Squibb, packaged with restriction enzyme) and 0.25 μL HindIII (Bristol-Myers Squibb, EG15539) was added to 49 μL with nuclease-free water, reacted at 37°C for 2 hours, and then inactivated at 80°C for 20 minutes.

[0029] Mutant C157S and wild-type XhoI proteins were diluted to 0.01 mg / mL as working solutions. These solutions were then serially diluted in ratios of 1 / 2, 1 / 4, 1 / 8, 1 / 16, and 1 / 32. 1 μL of each of the serially diluted proteins was added to 49 μL of the aforementioned λ DNA (HindIII digested) reaction solution. The reaction was incubated at 37°C for 1 hour and then inactivated at 80°C for 20 minutes. 5 μL of the reaction product was added to 1 μL of 6× DNA Loading Buffer (Bristol-Myers Squibb, EG21915), mixed, and subjected to agarose gel electrophoresis.

[0030] The results are as follows Figure 1 As shown, C157S, like wild-type XhoI, can completely digest λDNA (HindIII digestion) at a dilution of 0.00125 mg / mL. According to the definition of XhoI enzyme activity, one unit (U) is the amount of enzyme required to completely digest 1 μg of λDNA (HindIII digestion) in a 50 μL digestion reaction at 37°C for 1 hour. Calculated specific activities of both C157S and wild-type XhoI are 8×10 5 U / mg, proving that the XhoI mutant of the present invention can still maintain high enzyme activity consistent with the wild type.

[0031] Example 2

[0032] Evaluation of the oxidative stability of XhoI mutants

[0033] Mutant C157S and wild-type XhoI proteins were each prepared into 20 μM (approximately 430 U / μL) solutions. Equal volumes of 40 mM H₂O₂ solution (Aladdin, H112517) were then added to each solution, resulting in a final protein concentration of 10 μM and H₂O₂ concentration of 20 mM, i.e., a molar ratio of protein:H₂O₂ of 1:2000. The solutions were incubated at 25°C for 10, 20, 40, and 60 minutes to simulate oxidation. The reactions were then terminated by the addition of an excess of catalase (Aladdin, C100456).

[0034] The protein solution after oxidation treatment was diluted in a gradient manner according to the method described in Example 1, and the residual enzyme activity was determined. The unoxidized enzyme solution was used as a control, and the relative activity was set as 100%. The results are shown in Figure 1. Figure 2 As shown, after 10 minutes of H₂O₂ oxidation, the relative activity of wild-type XhoI dropped below 50%, while mutant C157S maintained 100% relative activity. After 60 minutes of oxidation, the relative activity of wild-type XhoI had dropped to 20%, while mutant C157S still maintained 45% relative activity, 2.25 times that of the wild-type. This indicates that mutant C157S is significantly more oxidatively stable than the wild-type.

[0035] Comparative Example 1

[0036] Comparative Example 1: Comparative XhoI mutant proteins were prepared using the method of Example 1. Compared to wild-type XhoI, only cysteine at position 70 (C70S) was substituted with serine, or cysteine at position 190 (C190A) was substituted with alanine. The two comparative mutant proteins were oxidized according to the method of Example 2 and then measured for residual relative activity. The residual activity was below 30% at each time point, further demonstrating the advantages of the specific mutations of the mutants of the present invention.

Claims

1. A highly oxidatively stable mutant of the restriction enzyme XhoI, 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 high oxidative stability mutant of the restriction enzyme XhoI according to claim 1, characterized in that Compared with the wild-type XcmI, the high oxidation stability mutant of the restriction enzyme XhoI has cysteine at position 157 replaced by serine (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 XcmI mutant is shown in SEQ ID NO.

2.

4. Use of the high oxidation stability mutant of the restriction enzyme XhoI according to claim 1 in recognizing and cleaving DNA sequences.

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

6. The use according to claim 4, characterized in that The high oxidation stability mutant of the restriction enzyme XhoI is used in recognizing and cutting DNA sequences under oxidation conditions.

7. A kit comprising the highly oxidatively stable mutant of the restriction enzyme XhoI with improved activity and stability according to claim 1.

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

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

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