A mutant, its construction method and application

Abstract

The application provides a mutant, and the amino acid sequence of the mutant is mutated at the following positions in SEQ ID NO. 1: Y128R; Y128R, G34D; Y128R, S38R; Y128R, Q332K; Y128R, G34D, S38R; Y128R, G34D, Q332K; Y128R, S38R, Q332K; Y128R, G34D, S38R, Q332K. The arginine oxidase mutant provided by the application has better thermal stability compared with wild-type arginine oxidase. The arginine oxidase mutant obtained by the construction method provided by the application has better thermal stability, and when oxidizing L-arginine at a higher temperature, the arginine oxidase mutant still has excellent catalytic activity, and has high application potential in the fields of L-arginine biosensor detection and biological chemical industry.

Description

[0001] This application is a divisional application of Chinese patent application filed on December 30, 2020, with application number 202011624067.4 and invention title "A mutant and its construction method and application". Technical Field

[0002] This invention relates to the field of biotechnology, and more specifically, to a mutant, its construction method, and its application. Background Technology

[0003] L-amino acid oxidases are important oxidoreductases involved in amino acid oxidative metabolism in organisms. They catalyze the deamination of L-amino acids using oxygen molecules as electron acceptors, producing the corresponding keto acids, ammonia (NH3), and hydrogen peroxide (H2O2). Some L-amino acid oxidases can specifically recognize specific amino acids without interference from other types of amino acids, thus playing an important role in the resolution of chiral amine compounds, α-keto acid biosynthesis, clinical samples, and the detection of amino acid content in food and amino acid fermentation processes. Among them, L-arginine oxidase can specifically recognize L-arginine and is an important catalyst for life activities. Its catalytic reaction is mild, produces a single product, consumes little energy, and the product is easily separated, making it widely used in the food, chemical, environmental, energy, and pharmaceutical fields. However, the stability and catalytic activity of natural L-arginine oxidase are greatly reduced under harsh conditions such as high temperature, extreme pH, organic solvents, non-natural substrates, and product inhibition, limiting its application in industrial production.

[0004] Protein engineering is based on the structural regularities of protein molecules and their relationship with biological functions. It utilizes chemical, physical, and molecular biological methods to modify or synthesize existing proteins or create new ones to meet human needs for production and daily life. Rational design is the most commonly used method in protein engineering, employing computer-aided molecular models combined with site-directed mutagenesis to optimize protein function, such as improving catalytic activity, thermal stability, and acid / alkali resistance. To effectively optimize protein thermal stability, Markus Wyss et al. proposed the Consensus Concept theory in 2001. Unlike conventional rational protein design methods based on precise structure-function relationships, the Consensus Concept theory is based on the amino acid sequence information of homologous proteins, analyzing information from an evolutionary perspective that can improve enzyme thermal stability. This invention, guided by the Consensus Concept theory, integrates and analyzes the sequences of the L-amino acid oxidase family, and with the assistance of bioinformatics, obtains a novel highly stable L-arginine oxidase ARod mutant. No related research has been reported to date. Summary of the Invention

[0005] Therefore, the purpose of this invention is to address the problem of insufficient thermal stability of existing arginine oxidase mutants by providing a thermally stable mutant, the gene sequence of the mutant, and a soluble protein containing the mutant, an immobilized enzyme, recombinant engineered cells, and a recombinant vector for catalyzing the oxidation of L-arginine.

[0006] According to the first aspect disclosed in this application, a mutant is provided, wherein the amino acid sequence of SEQ ID NO.1 is mutated at the following sites:

[0007] Y128R;

[0008] Y128R, G34D;

[0009] Y128R, S38R;

[0010] Y128R, Q332K;

[0011] Y128R, G34D, S38R;

[0012] Y128R, G34D, Q332K;

[0013] Y128R, S38R, Q332K;

[0014] Y128R, G34D, S38R, Q332K.

[0015] In some possible implementations, the amino acid sequences corresponding to the mutants are SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively.

[0016] According to a second aspect disclosed in this application, the gene sequence is configured as any one of SEQ ID NO.19, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.30, and SEQ ID NO.31, wherein:

[0017] The nucleic acid sequence encoding the mutant with the mutation site Y128R is SEQ ID NO.19;

[0018] The nucleic acid sequence of the mutant encoding the mutation sites G34D and Y128R is SEQ ID NO.22;

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

[0020] The nucleic acid sequence encoding the mutant with mutation sites Y128R and Q332K is SEQ ID NO.26;

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

[0022] The nucleic acid sequence of the mutant encoding the mutation sites S38R, Y128R, and Q332K is SEQ ID NO.29;

[0023] The nucleic acid sequence of the mutant encoding the mutation sites G34D, Y128R, and Q332K is SEQ ID NO.30;

[0024] The nucleic acid sequence of the mutant encoding 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] 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.

[0027] 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.

[0028] 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.

[0029] According to a third aspect disclosed in this application, a soluble protein is provided, which is configured to include the mutants described above.

[0030] According to the fourth aspect disclosed in this application, an immobilized enzyme is provided, which is configured to include the mutant as described above.

[0031] According to the fifth aspect disclosed in this application, a recombinant engineered cell is provided, which is configured to include the coding sequence of the mutant as described above.

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

[0033] According to the seventh aspect disclosed in this application, the use of the aforementioned mutants, soluble proteins, immobilized enzymes, recombinant engineered cells, or recombinant vectors in the catalytic oxidation of L-arginine is provided.

[0034] The mutants disclosed in this application include single-point mutants and combined mutants. Compared with wild-type arginine oxidase, both the single-point mutants and combined mutants have longer half-lives at 42°C. In particular, the combined mutants exhibit a synergistic effect of the thermal stability of the single-point mutants, with a half-life approximately six times that of wild-type arginine oxidase. Therefore, the mutants disclosed in this application have better thermal stability and are suitable for catalytic oxidation of L-arginine at higher temperatures.

[0035] The mutant construction method disclosed in this application differs from rational design based on the precise structure-function relationship of proteins. This invention is guided by the Consensus Concept theory, analyzes information that can improve the thermal stability of enzymes from an evolutionary perspective, integrates and analyzes the amino acid oxidase family sequences, and combines bioinformatics and crystallography methods to obtain novel arginine oxidase mutants with high stability. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 The figure shows the molecular structure of a natural arginine oxidase according to an embodiment of this application. The arrows in the figure indicate the mutation sites. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0039] In the mutant of arginine oxidase disclosed in this application, the natural arginine oxidase is the 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) At least one amino acid of the amino acid sequence of SEQ ID NO.1 of natural arginine oxidase is substituted, deleted, or added, and the amino acid sequence of the mutant has more than 90% homology with SEQ ID NO.1; or

[0042] (b) At least one amino acid of the amino acid sequence of natural arginine oxidase in SEQ ID NO.1 is replaced, deleted, or added, 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 NO. 2 to 16. Specifically, a single-point mutation is performed at a site selected in the amino acid sequence shown in SEQ ID NO. 1, resulting in five single-point mutants of arginine oxidase. The mutation sites are G34D, S38R, Y128R, S195R, and Q332K. These five single-point mutants of arginine oxidase are then subjected to property testing, and four arginine oxidase mutants with improved thermostability are selected. The mutation sites are G34D, S38R, Y128R, and Q332K, and their amino acid sequences are SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively.

[0044] Multiple mutation sites were selected and combined within the amino acid sequence shown in SEQ ID NO.1:

[0045] 1) If two mutation sites are selected from the above four mutation sites for combination, the following six thermostability-enhanced arginine oxidase mutants are obtained. The combined mutation sites are: G34D / S38R, G34D / Y128R, G34D / Q332K, S38R / Y128R, S38R / Q332K, and 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, and SEQ ID NO.11, respectively.

[0046] 2) If three mutation sites are selected from the above four mutation sites for combination, four arginine oxidase mutants with improved thermostability are obtained. The combined mutation sites are:

[0047] G34D / S38R / Y128R

[0048] G34D / S38R / Q332K,

[0049] S38R / Y128R / Q332K,

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

[0051] 3) If four mutation sites are selected from the above four mutation sites and combined, a mutant of arginine oxidase with improved thermostability can be obtained. The combined mutation sites are: G34D / S38R / Y128R / Q332K, and its amino acid sequence is SEQ ID NO.16.

[0052] The embodiments disclosed in this application provide gene sequences encoding the aforementioned mutants, and the correspondence between these gene sequences and amino acid sequences with different mutation sites is as follows:

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

[0054] The nucleic acid sequence of the mutant encoding the S38R mutation site 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 Q332K mutation site is SEQ ID NO.20;

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

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

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

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

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

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

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

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

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

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

[0067] The nucleic acid sequence of the mutant encoding the 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 method for constructing the aforementioned mutant, the method comprising:

[0073] S1: Cloning of the arginine oxidase gene

[0074] The natural wild-type arginine oxidase gene was codon optimized using Escherichia coli as the host cell to obtain the 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 primer pair was used to amplify the target gene:

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

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

[0078] The amplification conditions were as follows: amplification at 95℃ for 2 min, then at 56℃ for 20 sec, then at 72℃ for 90 sec, for a total of 30 cycles, and finally at 72℃ for 10 min.

[0079] After the reaction was complete, the PCR amplification product was detected by 1.5% agarose gel electrophoresis, yielding a 1.0 kb band, the length of which was in line with the expected result. Following the standard procedure of the kit, the target fragment was recovered and purified. The target fragment and the pET28a plasmid were double-digested using restriction endonucleases XhoI and NdeI, and then ligated using T4 DNA ligase. The ligation product was transformed into *E. coli* BL21(DE3) competent cells. The transformed cells were plated on LB plates containing 100 μg / ml kanamycin, and positive clone plasmids were extracted and sequenced. The results showed that the cloned arginine oxidase gene sequence was correct and had been correctly inserted into the pET28a plasmid, resulting in the recombinant plasmid pET28a containing the arginine oxidase gene sequence.

[0080] The PCR amplification enzyme used 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) from the glycerol tube were inoculated at a volume ratio of 1% into a 4 mL LB medium tube containing 100 μg / mL Kan, and cultured at 37℃ and 220 rpm for 12 h. 4 mL of the bacterial culture was then transferred to a 1 L LB medium shake flask containing 100 μg / mL Kan, and cultured at 37℃ and 220 rpm for 2.5 h to allow OD to develop. 600 When the concentration reaches 0.6–0.8, 1 mM IPTG inducer is added, and the mixture is induced and cultured at 25°C and 200 rpm for 14 h. The E. coli cell suspension harvested after fermentation is ultrasonically disrupted, and then subjected to a one-step Ni-NTA affinity chromatography process to obtain arginine oxidase with a purity >95%, the amino acid sequence of which is SEQ ID NO.1.

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

[0084] S301: Go to the Pfam database homepage (http: / / pfam.xfam.org / ), enter the amino acid sequence of arginine oxidase in the SEQUENCE SEARCH tool to search, and the server will directly return the comparison results of the amino acid sequences of the entire protein family, displaying the abundance of various amino acids at each mutation site in the form of a bar chart. The website can also automatically generate the consensus sequence of the protein family.

[0085] S302: Input the amino acid sequence shown in SEQ ID NO.1 into the NCBI protein database and Pfam database. Use the Blast tool to find all protein sequences with an amino acid sequence similarity greater than 50% to arginine oxidase. Delete any duplicate sequences. Organize the remaining amino acid sequences into fasta. format and input them into Clustalx 1.83 software for multiple sequence alignment. Output the alignment results in alan., dnd., and fasta. formats. The dnd. file is the phylogenetic tree file, while the alan. and fasta. files are sequence files in different formats.

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

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

[0088] After modifying the server settings as needed, the 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 the family and the abundance plot of amino acids at each site.

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

[0091] S401: The three-dimensional structure of arginine oxidase (SEQ ID NO.1) was predicted using the Swissmodel online tool;

[0092] S402: The crystal structure of arginine oxidase (amino acid sequence SEQ ID NO.1) was observed using PyMOL. Based on the structural information, the above-mentioned candidate mutation sites and mutation modes were reviewed to screen for the mutant sites most likely to improve the thermostability of arginine oxidase. The specific screening conditions are as follows:

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

[0094] ①Most proteins in this family have a generally high amino acid abundance at this site;

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

[0096] ③ The amino acids that appear more frequently at this site have significant differences in physicochemical properties compared to the amino acids at this site in arginine oxidase, such as differences in hydrogen bonds, charge, polarity, and steric hindrance.

[0097] (2) Remove the area near the active site, i.e., the area away from the catalytic residue (glutamic acid at position 104). The amino acid residues within the range, excluding amino acid residues that are embedded or partially embedded.

[0098] After the above two screening steps, a total of 25 differential sites remain, most of which are located on the surface of arginine oxidase molecules, such as... Figure 1 As shown, the arrow points to the mutation site.

[0099] (3) Based on the crystal structure of arginine oxidase, the above 25 mutation forms were analyzed in detail one by one, and mutants that may improve the thermal stability of arginine oxidase were screened out.

[0100] The main criteria for judgment are: ① Mutations should eliminate existing forces that are detrimental to thermal stability, such as electrostatic repulsion and charge accumulation; ② Mutations should not destroy existing forces that are beneficial to thermal stability and stable protein structures; ③ Mutations should introduce new forces that are beneficial to thermal stability, such as hydrogen bonds, salt bridges, and hydrophobic interactions.

[0101] Five single-point mutants were designed, with the following mutation sites:

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

[0103] The activity of the five single-point mutants of arginine oxidase was determined, and four mutants with improved thermostability were screened out. The mutation sites were G34D, S38R, Y128R, and Q332K, and the amino acid sequences of the corresponding single-point mutants were 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] Construction of S501: Arginine Oxidase Single-Point Mutant

[0106] Using the recombinant plasmid pET28a (containing the arginine oxidase gene sequence) in S1 as a template, and a pair of complementary oligonucleotides with mutation sites as amplification primers, the whole plasmid was amplified by KOD high-fidelity enzyme (TakaRa) 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: amplification at 95℃ for 2 min, followed by amplification at 56℃ for 20 sec, amplification at 72℃ for 90 sec, for a total of 30 cycles, and finally amplification at 72℃ for 10 min. The PCR amplification products were recovered from the gel and digested with DpnI enzyme (Fermentas) at 37℃ for 2 h to degrade the initial template. The digested products were transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing 100 μg / mL kanamycin, and cultured overnight at 37℃. Positive clones were screened and sequenced to verify the results, yielding recombinant bacteria containing a single-point mutant of arginine oxidase.

[0129] Construction of S502: Arginine Oxidase Combinatorial Mutants

[0130] Using a construction method similar to that for single-point mutants, single-point mutants with improved stability were cumulatively combined. Multiple mutation sites were selected from the amino acid sequence shown in SEQ ID NO.1 for combination. For example, 2 to 4 mutation sites were selected from the above 4 mutation sites for combination to obtain different arginine oxidase combined mutants.

[0131] (1) By selecting two mutation sites for combination, six arginine oxidase mutants and combined arginine oxidase mutants with improved thermostability can be constructed. The combined mutation sites are as follows:

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

[0133] The amino acid sequences of the six thermostable arginine oxidase mutant combinations 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) By selecting three mutation sites for combination, four combined mutants of arginine oxidase with improved thermostability can be constructed. The combined mutation sites are as follows:

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

[0136] The amino acid sequences of the four thermostable arginine oxidase mutant combinations are SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15, respectively.

[0137] (3) By selecting four mutation sites for combination, a combined mutant of arginine oxidase with improved thermostability can be constructed. The combined mutation sites are as follows:

[0138] G34D / S38R / Y128R / Q332K,

[0139] The amino acid sequence of this thermostability-enhanced arginine oxidase combination mutant is SEQ ID NO.16.

[0140] Experiment—Characteristics of the enzymatic properties of arginine oxidase mutants

[0141] The thermal stability of natural wild-type arginine oxidase and various arginine oxidase mutants provided in the examples was tested according to the conventional method for determining arginine oxidase activity.

[0142] The enzyme solution was incubated at a certain temperature, and samples were taken at different treatment times to determine the percentage of residual activity of arginine oxidase or arginine oxidase mutant. The ln value of the residual activity percentage was plotted against time t (min), and the slope of the straight line was the inactivation constant kinact. The half-life of the wild-type arginine oxidase or arginine oxidase mutant at that temperature was obtained by t1 / 2 = ln2 / kinact.

[0143] Experimental results show that among the various arginine oxidase mutants, the thermal stability of 4 single-point mutants and 11 combined mutants was significantly improved, as shown in Table 1:

[0144] Table 1. Enzymatic properties of wild-type arginine oxidase, single-point mutants, and combinatorial mutants

[0145]

[0146]

[0147] As shown in Table 1, the arginine oxidase mutants provided by this invention include single-point mutants and combined mutants. Compared with wild-type arginine oxidase, both the single-point mutants and combined mutants have longer half-lives at 42°C. In particular, the combined mutants exhibit a synergistic effect of the thermal stability of the single-point mutants, with a half-life approximately six times that of wild-type arginine oxidase. Based on this, the arginine oxidase mutants provided by this invention have better thermal stability and are suitable for catalytic oxidation of L-arginine at higher temperatures.

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

Claims

1. A mutant, characterized in that, Its amino acid sequence is SEQ ID NO.1, with mutations occurring only at the following sites: Y128R; Y128R, S38R.

2. The mutant according to claim 1, characterized in that, The amino acid sequences corresponding to the mutants are SEQ ID NO.4 and SEQ ID NO.9, respectively.

3. A soluble protein, characterized in that, It is configured to include any of the mutants described in claims 1 to 2.

4. An immobilized enzyme, characterized in that, It is configured to include any of the mutants described in claims 1 to 2.

5. A recombinant engineered cell, characterized in that, It is configured to include the coding sequence of any of the mutants described in claims 1 to 2.

6. A recombinant vector, characterized in that, It is configured to include the coding sequence of any of the mutants described in claims 1 to 2.

7. The use of any of the mutants described in claims 1 to 2, the soluble protein described in claim 3, the immobilized enzyme described in claim 4, the recombinant engineered cell described in claim 5, or the recombinant vector described in claim 6 in the catalytic oxidation of L-arginine.

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