A mutant and its construction method and application

Abstract

The present invention provides a mutant, whose amino acid sequence has mutations at the following sites on SEQ ID NO.1: S38R; S38R and G34D; S38R and Y128R; S38R and Q332K; S38R, G34D and Y128R; S38R, Y128R and Q332K; S38R, G34D and Q332K; S38R, G34D, Y128R and Q332K. Compared with the wild-type arginine oxidase, the arginine oxidase mutant provided by the present invention has better thermal stability. The arginine oxidase mutant obtained by the construction method provided by the present invention has better thermal stability and still exhibits excellent catalytic activity when oxidizing L-arginine at a higher temperature, and has high application potential in the fields of L-arginine biosensor detection and biochemistry and chemical engineering, etc.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of December 30, 2020, an application number of 202011624067.4, and an invention title of "A Mutant and Its Construction Method and Application". Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular, to a mutant and its construction method and application. Background Art

[0003] L - amino acid oxidase is an important redox enzyme involved in amino acid oxidative metabolism in vivo. It can catalyze the oxidative deamination of L - amino acids with molecular oxygen as the electron acceptor to generate the corresponding keto acids, ammonia (NH 3 ) and hydrogen peroxide (H 2 O 2 ). Some L - amino acid oxidases can specifically recognize specific amino acids without being interfered by other types of amino acids, and thus play an important role in the fields of chiral amine compound resolution, α - keto acid biosynthesis, clinical samples, food, and amino acid content detection in the amino acid fermentation process. Among them, L - arginine oxidase can specifically recognize L - arginine, is an important catalyst for life activities, and has mild catalytic reaction conditions, single products, low energy consumption, and easy product separation, and has been widely used in the fields of food, chemical industry, environmental protection, energy, and medicine. However, due to the fact that the stability and catalytic activity of natural L - arginine oxidase will be greatly reduced under harsh conditions such as high temperature, extreme acidity and alkalinity, organic solvents, unnatural substrates, and product inhibition, its application in industrial production is limited.

[0004] Protein engineering is based on the relationship between the structural rules of protein molecules and their biological functions. Through chemical, physical, and molecular biological means, gene modification or gene synthesis is carried out to modify existing proteins or create a new protein to meet the needs of human production and life. Rational design is the most commonly used method in protein engineering transformation. By using computer-aided molecular models combined with site-directed mutagenesis, protein function optimization can be achieved, such as improving catalytic activity, thermal stability, acid and alkali resistance, etc. In order to effectively optimize the thermal stability of proteins, Markus Wyss et al. proposed the Consensus Concept theory in 2001. Different from the conventional protein rational design method based on the precise structure-function relationship of proteins, the Consensus Concept theory is based on the amino acid sequence information of homologous proteins, and analyzes the information that can improve the thermal stability of enzymes from an evolutionary perspective. Guided by the Consensus Concept theory, this invention conducts an integrated analysis of the L-amino acid oxidase family sequences, and combines bioinformatics assistance to obtain a novel L-arginine oxidase ARod mutant with high stability. No relevant research has been reported currently. Summary of the Invention

[0005] Therefore, the object of this invention is to provide a mutant with thermal stability, the gene sequence of the mutant, and soluble proteins, immobilized enzymes, recombinant engineering cells, and recombinant vectors containing the mutant for catalyzing the oxidation of L-arginine, aiming at the problem of insufficient thermal stability of existing arginine oxidase mutants.

[0006] According to the first aspect disclosed in this application, a mutant is provided, and the amino acid sequence thereof has mutations at the following sites on SEQ ID NO.1:

[0007] S38R;

[0008] S38R and G34D;

[0009] S38R and Y128R;

[0010] S38R and Q332 K;

[0011] S38R, G34D and Y128R;

[0012] S38R, Y128R and Q332K;

[0013] S38R, G34D and Q332K;

[0014] S38R, G34D, Y128R and Q332K.

[0015] In some possible implementation manners, the amino acid sequences corresponding to the mutants are SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.16 respectively.

[0016] According to the second aspect disclosed in the present application, the gene sequence is configured as any one of SEQ ID NO.18, SEQ ID NO.21, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, and SEQ ID NO.31, where:

[0017] The nucleic acid sequence encoding the mutant with the mutation site S38R is SEQ ID NO.18;

[0018] The nucleic acid sequence encoding the mutant with the mutation sites G34D and S38R is SEQ ID NO.21;

[0019] The nucleic acid sequence encoding the mutant with the mutation sites S38R and Y128R is SEQ ID NO.24;

[0020] The nucleic acid sequence encoding the mutant with the mutation sites S38R and Q332K is SEQ ID NO.25;

[0021] The nucleic acid sequence encoding the arginine oxidase mutant with the mutation sites G34D, S38R, and Y128R is SEQ ID NO.27;

[0022] The nucleic acid sequence encoding the arginine oxidase mutant with the mutation sites G34D, S38R, and Q332K is SEQ ID NO.28;

[0023] The nucleic acid sequence encoding the mutant with the mutation sites S38R, Y128R, and Q332K is SEQ ID NO.29;

[0024] The nucleic acid sequence encoding the mutant with the mutation sites G34D, S38R, Y128R, and Q332K is SEQ ID NO.31.

[0025] Furthermore, the nucleic acid sequences of the amplification primer pair for the mutation site G34D are SEQ ID NO.32 and SEQ ID NO.33.

[0026] Further, the nucleic acid sequences of the amplification primer pair for the mutation site S38R are SEQ ID NO.34 and SEQ ID NO.35.

[0027] Further, the nucleic acid sequences of the amplification primer pair for the mutation site Y128R are SEQ ID NO.36 and SEQ ID NO.37.

[0028] Further, the nucleic acid sequences of the amplification primer pair for the mutation site Q332K are SEQ ID NO.38 and SEQ ID NO.39.

[0029] According to the third aspect disclosed in the present application, a soluble protein is provided, which is configured to include the aforementioned mutant.

[0030] According to the fourth aspect disclosed in the present application, an immobilized enzyme is provided, which is configured to include the aforementioned mutant.

[0031] According to the fifth aspect disclosed in the present application, a recombinant engineering cell is provided, which is configured to include the coding sequence of the aforementioned mutant.

[0032] According to the sixth aspect disclosed in the present application, a recombinant vector is provided, which is configured to include the coding sequence of the aforementioned mutant.

[0033] According to the seventh aspect disclosed in the present application, the application of the aforementioned mutant, soluble protein, immobilized enzyme, recombinant engineering cell or recombinant vector in the catalytic oxidation of L-arginine is provided.

[0034] The mutants provided by the disclosure of the present application include single-point mutants and combined mutants. Compared with the wild-type arginine oxidase, the half-life of its single-point mutants and combined mutants is longer at 42°C; especially for the combined mutants, they show the superimposed effect of the thermal stability of the single-point mutants, and their half-life is about 6 times that of the wild-type arginine oxidase. Therefore, the mutants provided by the disclosure of the present application have better thermal stability and are suitable for catalytically oxidizing L-arginine at a higher temperature.

[0035] The construction method of the mutants provided by the disclosure of the present application is different from the rational design based on the precise structure-function relationship of proteins. The present invention takes the Consensus Concept theory as the guiding ideology, analyzes the information that can improve the thermal stability of enzymes from an evolutionary perspective, integrates and analyzes the sequences of the amino acid oxidase family, and combines bioinformatics and crystallography methods to assist, so as to obtain novel arginine oxidase mutants with high stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the accompanying drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0037] Figure 1 Shows the molecular structure of a natural arginine oxidase of an embodiment disclosed in the present application. The arrow in the figure indicates the mutation site. Detailed embodiments

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Therefore, the detailed description of the embodiments of the present invention provided in the following drawings is not intended to limit the scope of the claimed invention, but merely represents the selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0039] In the mutants of arginine oxidase provided by the disclosure of the present application, the natural arginine oxidase is a wild-type arginine oxidase derived from Oceanobacter kriegii, and its amino acid sequence is SEQ ID NO.1.

[0040] In some embodiments, the amino acid sequence of the mutant is as follows (a) or (b):

[0041] (a) Substitute, delete, or add at least one amino acid of the amino acid sequence SEQ ID NO.1 of the natural arginine oxidase, and the amino acid sequence of the mutant has more than 90% homology with SEQ ID NO.1; or

[0042] (b) Substitute, delete, or add at least one amino acid of the amino acid sequence SEQ ID NO.1 of the natural arginine oxidase, and the amino acid sequence of the mutant has the same function as SEQ ID NO.1.

[0043] In some embodiments, the amino acid sequence of the mutant is any one of SEQ ID NOs. 2 to 16. Specifically, a single-site mutation is performed at a certain site in the amino acid sequence shown in SEQ ID NO. 1 to obtain 5 single-site mutants of arginine oxidase, and the mutation sites are: G34D, S38R, Y128R, S195R, Q332K. The properties of these 5 single-site mutants of arginine oxidase are measured, and 4 arginine oxidase mutants with improved thermal stability are screened out. The mutation sites are: G34D, S38R, Y128R, Q332K, and their amino acid sequences are SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 respectively. And,

[0044] Multiple mutation sites are selected for combination in the amino acid sequence shown in SEQ ID NO. 1:

[0045] 1) If 2 mutation sites are selected from the above 4 mutation sites for combination, 6 arginine oxidase mutants with improved thermal stability are obtained respectively. The combined mutation sites are: G34D / S38R, G34D / Y128R, G34D / Q332K, S38R / Y128R, S38R / Q332K, Y128R / Q332K, and their amino acid sequences are SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 respectively.

[0046] 2) If 3 mutation sites are selected from the above 4 mutation sites for combination, 4 arginine oxidase mutants with improved thermal stability are obtained respectively. The combined mutation sites are:

[0047] G34D / S38R / Y128R,

[0048] G34D / S38R / Q332K,

[0049] S38R / Y128R / Q332K,

[0050] G34D / Y128R / Q332K, and their amino acid sequences are SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15 respectively.

[0051] 3) If 4 mutation sites are selected from the above 4 mutation sites for combination, 1 arginine oxidase mutant with improved thermal stability is obtained. The combined mutation site is: G34D / S38R / Y128R / Q332K, and its amino acid sequence is SEQ ID NO. 16.

[0052] The embodiments disclosed in the present application provide gene sequences encoding the aforementioned mutants, and the corresponding relationships between the gene sequences and amino acid sequences with different mutation sites are as follows:

[0053] The nucleic acid sequence encoding the mutant with the mutation site G34D is SEQ ID NO.17;

[0054] The nucleic acid sequence encoding the mutant with the mutation site S38R is SEQ ID NO.18;

[0055] The nucleic acid sequence encoding the mutant with the mutation site Y128R is SEQ ID NO.19;

[0056] The nucleic acid sequence encoding the mutant with the mutation site Q332K is SEQ ID NO.20;

[0057] The nucleic acid sequence encoding the mutant with the mutation sites G34D and S38R is SEQ ID NO.21;

[0058] The nucleic acid sequence encoding the mutant with the mutation sites G34D and Y128R is SEQ ID NO.22;

[0059] The nucleic acid sequence encoding the mutant with the mutation sites G34D and Q332K is SEQ ID NO.23;

[0060] The nucleic acid sequence encoding the mutant with the mutation sites S38R and Y128R is SEQ ID NO.24;

[0061] The nucleic acid sequence encoding the mutant with the mutation sites S38R and Q332K is SEQ ID NO.25;

[0062] The nucleic acid sequence encoding the mutant with the mutation sites Y128R and Q332K is SEQ ID NO.26;

[0063] The nucleic acid sequence of the arginine oxidase mutant encoding the mutant with the mutation sites G34D, S38R and Y128R is SEQ ID NO.27;

[0064] The nucleic acid sequence of the arginine oxidase mutant encoding the mutant with the mutation sites G34D, S38R and Q332K is SEQ ID NO.28;

[0065] The nucleic acid sequence encoding the mutant with the mutation sites S38R, Y128R and Q332K is SEQ ID NO.29;

[0066] The nucleic acid sequence of the mutant with coding mutation sites G34D, Y128R, and Q332K is SEQ ID NO.30;

[0067] The nucleic acid sequence of the mutant with coding mutation sites G34D, S38R, Y128R, and Q332K is SEQ ID NO.31.

[0068] Furthermore, the nucleic acid sequences of the amplification primer pair for the mutation site G34D are SEQ ID NO.32 and SEQ ID NO.33.

[0069] Furthermore, the nucleic acid sequences of the amplification primer pair for the mutation site S38R are SEQ ID NO.34 and SEQ ID NO.35.

[0070] Furthermore, the nucleic acid sequences of the amplification primer pair for the mutation site Y128R are SEQ ID NO.36 and SEQ ID NO.37.

[0071] Furthermore, the nucleic acid sequences of the amplification primer pair for the mutation site Q332K are SEQ ID NO.38 and SEQ ID NO.39.

[0072] The embodiments disclosed in this application also provide a construction method for the aforementioned mutant, and the method includes:

[0073] S1: Cloning of the arginine oxidase gene

[0074] The natural wild-type arginine oxidase gene is codon-optimized with Escherichia coli as the host cell to obtain an optimized gene sequence, whose nucleic acid sequence is SEQ ID NO.25, and the amino acid sequence expressed by this nucleic acid sequence is SEQ ID NO.1.

[0075] Using SEQ ID NO.1 as the target gene, the following amplification primer pair is used to amplify the target gene:

[0076] F: 5’-ACTGCT CATATG ATGGATTCTAATAACCAATCCCCTCAG-3’ (where the underlined part is the recognition site of the restriction enzyme NdeI);

[0077] R: 5’-TCAGCT CTCGAG CTGTTGCGCGTGGTTTATGCCTTCG-3’ (where the underlined part is the recognition site of the restriction enzyme XhoI).

[0078] The amplification conditions are: amplify at 95°C for 2 min, then amplify at 56°C for 20 sec and at 72°C for 90 sec, for a total of 30 cycles, and finally amplify at 72°C for 10 min.

[0079] After the reaction ended, the PCR amplification product was detected by 1.5% agarose gel electrophoresis, and a 1.0 kb band was obtained, the length of which was consistent with the expected result. According to the standard operation of the kit, the target fragment was recovered and purified. The target fragment and the pET28a plasmid were double digested with the restriction endonucleases XhoI and NdeI, and then ligated with T4 DNA ligase. The obtained ligation product was transformed into Escherichia coli BL21(DE3) competent cells. The transformed cells were spread on an LB plate containing 100 μg / ml kanamycin, and the plasmid of the positive clone was extracted and sequenced. The results showed that the cloned arginine oxidase gene sequence was correct and had been correctly inserted into the pET28a plasmid, and the recombinant plasmid pET28a containing the arginine oxidase gene sequence was obtained.

[0080] Among them, the PCR amplification enzyme was KOD high-fidelity polymerase (Toyobo).

[0081] S2: Expression and purification of arginine oxidase

[0082] The engineered bacteria (recombinant plasmid pET28a containing the arginine oxidase gene sequence) in the glycerol tube were inoculated into a 4 mL LB medium test tube containing 100 μg / mL Kan at a volume ratio of 1%, and cultured at 37 °C and 220 rpm for 12 h; 4 mL of the bacterial solution was transferred to a 1 L LB medium shake flask containing 100 μg / mL Kan and cultured at 37 °C and 220 rpm for 2.5 h to make the OD 600 reach 0.6 - 0.8, 1 mM IPTG inducer was added, and induced culture was carried out at 25 °C and 200 rpm for 14 h. The Escherichia coli cell suspension harvested after fermentation was ultrasonically disrupted and then treated by one-step Ni-NTA affinity chromatography to obtain arginine oxidase with a purity > 95%, and its amino acid sequence was SEQ ID NO.1.

[0083] S3: Multiple sequence alignment and Consensus analysis of arginine oxidase homologous proteins

[0084] S301: Enter the Pfam database homepage (http: / / pfam.xfam.org / ), input the amino acid sequence of arginine oxidase in the SEQUENCE SEARCH tool for searching, and the server will directly feedback the alignment results of the amino acid sequences of the entire protein family. The abundances of various amino acids at each mutation site are displayed in the form of a histogram, and this website can also automatically generate the consensus sequence of this protein family;

[0085] S302: Input the amino acid sequence shown in SEQ ID NO.1 into the protein database of NCBI and the Pfam database. Using the Blast tool, find all protein sequences with an amino acid sequence identity greater than 50% to arginine oxidase, delete the repeated identical sequences among them, organize the remaining amino acid sequences into the fasta format, and input them into the Clustalx1.83 software for multiple sequence alignment. The alignment results are output in the aln., dnd., and fasta. formats, where the dnd. file is the file for constructing the phylogenetic tree, and the aln. and fasta. files are sequence files in different forms;

[0086] Upload the above fasta. file to Consensus Maker v2.0.0

[0087] (https: / / www.hiv.lanl.gov / content / sequence / CONSENSUS / consensus.html)

[0088] server. After modifying the setting parameters as needed, this online software will generate a consensus sequence that can be edited later.

[0089] S303: Compare the amino acid sequence of arginine oxidase with the consensus sequence of this family and the abundance map of amino acids at each site.

[0090] S4: Simulation of the three-dimensional structure of arginine oxidase and selection of mutation hotspots

[0091] S401: Predict the three-dimensional structure of arginine oxidase (SEQ ID NO.1) through the swissmodel online tool;

[0092] S402: Observe the crystal structure of arginine oxidase (amino acid sequence SEQ ID NO.1) using PyMOL, review the above-mentioned candidate mutation sites and mutation forms according to the structural information, and screen out the mutation sites that are most likely to improve the thermal stability of arginine oxidase. The screening conditions are as follows:

[0093] (1) The criteria for determining a site as a candidate site are:

[0094] ① The overall amino acid abundance at this site in most proteins of this family is relatively high;

[0095] ② The amino acid at this site is conserved;

[0096] ③The amino acids with a high frequency of occurrence at this site have significant differences in physical and chemical properties from the amino acids of arginine oxidase at this site, such as hydrogen bonds, charge differences, polarity strengths, and steric hindrance sizes.

[0097] (2) Except for the vicinity of the active center, that is, the amino acid residues within the range from the catalytic residue (glutamic acid at position 104) are removed, and the amino acid residues in the buried or semi-buried state are removed.

[0098] After the above two-step screening, a total of 25 differential sites remain at this time, and most of them are located on the surface of the arginine oxidase molecule, as Figure 1 shown, and the arrow indicates the mutation site.

[0099] (3) According to the crystal structure of arginine oxidase, each of the above 25 mutation forms is analyzed in detail to screen out the mutants that may improve the thermal stability of arginine oxidase.

[0100] The main judgment criteria are as follows: ① The mutation should eliminate the original force forms that are not conducive to thermal stability, such as electrostatic repulsion and charge aggregation; ② The mutation should not destroy the existing force forms conducive to thermal stability and the stable protein structure; ③ The mutation should introduce new force forms conducive to thermal stability, such as hydrogen bonds, salt bridges, and hydrophobic interactions.

[0101] A total of 5 single-point mutants are designed, and their mutation sites are respectively:

[0102] G34D, S38R, Y128R, S195R, Q332K;

[0103] The activities of these 5 arginine oxidase single-point mutants are measured, and 4 arginine oxidase mutants with improved thermal stability are screened out. Their mutation sites are: G34D, S38R, Y128R, Q332K, and the amino acid sequences of the corresponding single-point mutants are SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 respectively.

[0104] S5: Construction, expression, and purification of mutants

[0105] S501: Construction of arginine oxidase single-point mutants

[0106] Using the recombinant plasmid in S1 (recombinant plasmid pET28a containing the arginine oxidase gene sequence) as a template and a pair of complementary oligonucleotides with mutation sites as amplification primers, whole plasmid PCR amplification is carried out with KOD high-fidelity enzyme (TakaRa Company) to obtain a recombinant plasmid with specific mutation sites;

[0107] The amplification primer pairs used are:

[0108] (1) The nucleic acid sequences of the upstream and downstream amplification primers for the mutation site G34D are as follows:

[0109] F (SEQ ID NO.32):

[0110] 5’-TCAGCTGTCGACATGGATTCTAATAACCAATCCCCTCAG-3’;

[0111] R (SEQ ID NO.33):

[0112] 5’-ACTGCTGCTAGCCTGTTGCGCGTGGTTTATGCCTTCGTATG-3’;

[0113] (2) The nucleic acid sequences of the upstream and downstream amplification primers for the mutation site S38R are as follows:

[0114] F (SEQ ID NO.34):

[0115] 5’-TCAGCTGGATCCATGGATTCTAATAACCAATCCCCTCAG-3’;

[0116] R (SEQ ID NO.35):

[0117] 5’-ACTGCTAGATCTCTGTTGCGCGTGGTTTATGCCTTCGTATG-3’;

[0118] (3) The nucleic acid sequences of the upstream and downstream amplification primers for the mutation site Y128R are as follows:

[0119] F (SEQ ID NO.36):

[0120] 5’-TCAGCTGGTACCATGGATTCTAATAACCAATCCCCTCAG-3’;

[0121] R (SEQ ID NO.37):

[0122] 5’-ACTGCTAAGCTTCTGTTGCGCGTGGTTTATGCCTTCGTATG-3’;

[0123] (4) The nucleic acid sequences of the upstream and downstream amplification primers for the mutation site Q332K are as follows:

[0124] F (SEQ ID NO.38):

[0125] 5’-TCAGCTATCGATATGGATTCTAATAACCAATCCCCTCAG-3’;

[0126] R(SEQ ID NO.39):

[0127] 5’-ACTGCTCATATGCTGTTGCGCGTGGTTTATGCCTTCGTATG-3’;

[0128] The amplification conditions were as follows: amplify at 95°C for 2 min, then amplify at 56°C for 20 sec and at 72°C for 90 sec for a total of 30 cycles, and finally amplify at 72°C for 10 min; recover the PCR amplification product by gel extraction, digest the gel-extracted product with DpnI enzyme (from Fermentas) at 37°C for 2 h to degrade the initial template; transform the digested product into competent Escherichia coli BL21(DE3) cells, spread them on an LB agar plate containing 100 μg / mL kanamycin, culture overnight at 37°C, screen for positive clones, verify by sequencing, and obtain a recombinant bacterium containing a single-point mutant of arginine oxidase;

[0129] S502: Construction of the combined mutant of arginine oxidase

[0130] Using a construction method similar to that of the single-point mutant, accumulate and combine the single-point mutants with improved stability, select multiple mutation sites for combination in the amino acid sequence shown in SEQ ID NO.1, such as selecting 2 - 4 mutation sites from the above 4 mutation sites for combination to obtain different combined mutants of arginine oxidase:

[0131] (1) Select 2 mutation sites for combination, and 6 mutants of arginine oxidase with improved thermal stability and combined mutants of arginine oxidase can be constructed. The combined mutation sites are respectively:

[0132] G34D / S38R, G34D / Y128R, G34D / Q332K, S38R / Y128R, S38R / Q332K, Y128R / Q332K,

[0133] The amino acid sequences of these 6 combined mutants of arginine oxidase with improved thermal stability are SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11 respectively;

[0134] (2) Select 3 mutation sites for combination, and 4 combined mutants of arginine oxidase with improved thermal stability can be constructed. The combined mutation sites are respectively:

[0135] G34D / S38R / Y128R, G34D / S38R / Q332K, S38R / Y128R / Q332K, G34D / Y128R / Q332K,

[0136] The amino acid sequences of these 4 combination mutants of arginine oxidase with improved thermal stability are SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15 respectively;

[0137] (3) By selecting 4 mutation sites for combination, 1 combination mutant of arginine oxidase with improved thermal stability can be constructed, and its combined mutation sites are respectively:

[0138] G34D / S38R / Y128R / Q332K,

[0139] The amino acid sequence of this 1 combination mutant of arginine oxidase with improved thermal stability is SEQ ID NO.16.

[0140] Experiment - Enzymatic Property Characterization of Arginine Oxidase Mutants

[0141] The natural wild-type arginine oxidase and various arginine oxidase mutants provided in the examples were subjected to thermal stability tests. According to the conventional method for determining the activity of arginine oxidase, specifically:

[0142] Incubate the enzyme solution at a certain temperature, take samples at different treatment times, measure the percentage of residual activity of arginine oxidase or arginine oxidase mutants, plot the ln value of the percentage of residual activity against the time t (min), and the slope of the straight line is the inactivation constant kinact. The half-life of this wild-type arginine oxidase or arginine oxidase mutant at this temperature is obtained from t1 / 2 = ln2 / kinact.

[0143] The experimental results show that among the above-mentioned various arginine oxidase mutants, the thermal stabilities of 4 single mutants and 11 combination mutants are significantly improved, as shown in Table 1:

[0144] Table 1. Enzymatic Property Characterization of Wild-Type Arginine Oxidase, Single Mutants and Combination Mutants

[0145]

[0146]

[0147] As can be seen from Table 1, the arginine oxidase mutants provided by the present invention include single-point mutants and combined mutants. Compared with the wild-type arginine oxidase, the half-life of its single-point mutants and combined mutants is longer at 42°C; in particular, the combined mutants show a superimposed effect of the thermal stability of the single-point mutants, and its half-life is about 6 times that of the wild-type arginine oxidase. Based on this, the arginine oxidase mutants provided by the present invention have better thermal stability and are suitable for catalytically oxidizing L-arginine at a higher temperature.

[0148] Although the present invention has been further described in detail by preferred embodiments, the present invention is not limited to the disclosed examples, and those skilled in the art can derive other variations therefrom without departing from the scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A mutant, characterized in that its amino acid sequence has mutations at the following sites on SEQ ID NO.1: S38R.

2. The mutant according to claim 1, characterized in that the amino acid sequence corresponding to the mutant is SEQ ID NO.

3.

3. A gene encoding the mutant according to claim 2, characterized in that the gene sequence is configured as SEQ ID NO.18, wherein: the nucleic acid sequence encoding the mutant with the mutation site S38R is SEQ ID NO.

18.

4. The gene according to claim 3, characterized in that the nucleic acid sequences of the amplification primer pair for the mutation site S38R are SEQ ID NO.34 and SEQ ID NO.

35.

5. A soluble protein, characterized in that it is configured to include the mutant according to any one of claims 1 to 2.

6. An immobilized enzyme, characterized in that it is configured to include the mutant according to any one of claims 1 to 2.

7. A recombinant engineering cell, characterized in that it is configured to include the coding sequence of the mutant according to any one of claims 1 to 2.

8. A recombinant vector, characterized in that it is configured to include the coding sequence of the mutant according to any one of claims 1 to 2.

9. Use of the mutant according to any one of claims 1 to 2, the soluble protein according to claim 5, the immobilized enzyme according to claim 6, the recombinant engineering cell according to claim 7 or the recombinant vector according to claim 8 in the catalytic oxidation of L-arginine.

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