Catechol dioxygenase mutant, recombinant vector, preparation method and application thereof

Abstract

The present invention discloses a catechol dioxygenase mutant, a recombinant vector, a preparation method and an application thereof. The mutant catechol dioxygenase is as follows (a1) or (a2): (a1) a polypeptide having the same function as the polypeptide consisting of the amino acid sequence shown in SEQ ID No. 2 obtained by deleting, replacing or adding one or more amino acids from the amino acid sequence shown in SEQ ID No. 2; (a2) a polypeptide having at least 90% homology with the polypeptide consisting of the amino acid sequence shown in SEQ ID No. 2. The mutant catechol dioxygenase of the present invention includes a single point mutant and a combined mutant. Compared with the wild-type catechol dioxygenase, the single point mutant and the combined mutant have a longer half-life at 40° C.; in particular, the combined mutant has a half-life of about 3 times that of the wild-type, showing a superposition effect of the thermal stability of the single point mutant.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a catechol dioxygenase mutant, a recombinant vector, and a preparation method and application thereof. Background Art

[0002] In recent years, environmental problems caused by industrial wastewater pollution have attracted much attention. Aromatic compounds account for a high proportion of these wastewaters, and aromatic compounds are a class of organic substances that are difficult to degrade and have toxic effects on the human body, such as carcinogenicity and neurotoxicity. In nature, there are many bacteria that have the ability to degrade aromatic compounds, such as Acinectobactercalcoaceticu, Pseudomonas sp., Acine tobacter lwoffii, Burkholderia sp., etc., which can use aromatic compounds as the only growth carbon source and completely degrade them into H2O and CO2. Catechol is an important intermediate in the degradation process of aromatic compounds, and catechol dioxygenase is a key enzyme that catalyzes the degradation process. It mainly cleaves catechol through the action of ortho- or meta-dioxygenase to generate muconic acid semialdehyde or muconic acid, thereby breaking the benzene ring. With the development of molecular biology technology, although there have been many studies on the gene expression of catechol dioxygenase, some catechol dioxygenases show low thermal stability because their degradation targets are all stable aromatic compounds, which limits their industrial application. Therefore, it is necessary to carry out enzyme directed evolution modification of existing catechol 1,2-dioxygenase to improve its thermal stability and thus improve its efficiency in degrading aromatic compounds. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a catechol dioxygenase mutant, a recombinant vector and a preparation method and application thereof in view of the deficiencies in the above-mentioned prior art.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is: to provide a mutant catechol dioxygenase (CatA), wherein the mutant catechol dioxygenase is as follows (a1) or (a2):

[0005] (a1) a polypeptide having the same function as the polypeptide consisting of the amino acid sequence shown in SEQ ID No. 2 obtained by deleting, substituting or adding one or more amino acids of the amino acid sequence shown in SEQ ID No. 2;

[0006] (a2) A polypeptide having at least 90% homology with the polypeptide consisting of the amino acid sequence shown in SEQ ID No. 2.

[0007] The study found that the mutant catechol dioxygenase of the above two types (a1) and (a2) have high enzyme stability. The half-life of the mutant catechol dioxygenase can be increased by up to 3 times through experiments.

[0008] Preferably, the mutant catechol dioxygenase is obtained by mutating the amino acid sequence shown in SEQ ID No. 2, and the mutation site is selected from one or a combination of more than one of A95V, L148S, R193Q, and L248D.

[0009] Preferably, the mutation site is A95V, L148S, R193Q, L248D, A95V-L148S, L148S-R193Q, A95V-L248D, R193Q-L248D, A95V-R193Q, L148S-L248D, A95V-L148S-R193Q, A95V-L148S-L248D, A95V-R193Q-L248D, L148S-R193Q-L248D or A95V-L148S-R193Q-L248D.

[0010] Preferably, the amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V single-site mutation is shown in SEQ ID No. 3;

[0011] The amino acid sequence of the mutant catechol dioxygenase corresponding to the L148S single-site mutation is shown in SEQ ID No. 4;

[0012] The amino acid sequence of the mutant catechol dioxygenase corresponding to the R193Q single-site mutation is shown in SEQ ID No. 5;

[0013] The amino acid sequence of the mutant catechol dioxygenase corresponding to the L248D single-site mutation is shown in SEQ ID No. 6;

[0014] The amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V-L148S combined site mutation is shown in SEQ ID No. 7;

[0015] The amino acid sequence of the mutant catechol dioxygenase corresponding to the L148S-R193Q combined site mutation is shown in SEQ ID No. 8;

[0016] The amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V-L248D combined site mutation is shown in SEQ ID No. 9;

[0017] The amino acid sequence of the mutant catechol dioxygenase corresponding to the R193Q-L248D combined site mutation is shown in SEQ ID No. 10;

[0018] The amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V-R193Q combined site mutation is shown in SEQ ID No. 11;

[0019] The amino acid sequence of the mutant catechol dioxygenase corresponding to the L148S-L248D combined site mutation is shown in SEQ ID No. 12;

[0020] The amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V-L148S-R193Q combined site mutation is shown in SEQ ID No. 13;

[0021] The amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V-L148S-L248D combined site mutation is shown in SEQ ID No. 14;

[0022] The amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V-R193Q-L248D combined site mutation is shown in SEQ ID No. 15;

[0023] The amino acid sequence of the mutant catechol dioxygenase corresponding to the L148S-R193Q-L248D combined site mutation is shown in SEQ ID No. 16;

[0024] The amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V-L148S-R193Q-L248D combined site mutation is shown in SEQ ID No.17.

[0025] The present invention also provides a gene encoding the mutant catechol dioxygenase described above, which is as follows (b1) or (b2):

[0026] (b1) a polynucleotide obtained by deleting, substituting or adding one or more bases from the nucleotide sequence shown in SEQ ID No. 1;

[0027] (b2) A polynucleotide having at least 90% homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 1.

[0028] Preferably, the nucleotide sequence encoding the mutant catechol dioxygenase with mutation site A95V is shown as SEQ ID No. 28;

[0029] The nucleotide sequence encoding the mutant catechol dioxygenase with the mutation site L148S is shown in SEQ ID No. 29;

[0030] The nucleotide sequence encoding the mutant catechol dioxygenase with mutation site R193Q is shown in SE Q ID No. 30;

[0031] The nucleotide sequence encoding the mutant catechol dioxygenase with mutation site L248D is shown in SEQ ID No.31;

[0032] The nucleotide sequence encoding the mutant catechol dioxygenase with mutation site A95V-L148S is shown in SEQ ID No. 32;

[0033] The nucleotide sequence encoding the mutant catechol dioxygenase with mutation site A95V-R193Q is shown in SEQ ID No. 33;

[0034] The nucleotide sequence encoding the mutant catechol dioxygenase with mutation site A95V-L248D is shown in SEQ ID No. 34;

[0035] The nucleotide sequence encoding the mutant catechol dioxygenase with the mutation site L148S-R193Q is shown in SEQ ID No. 35;

[0036] The nucleotide sequence encoding the mutant catechol dioxygenase with mutation sites L148S-L248D is shown in SEQ ID No. 36;

[0037] The nucleotide sequence encoding the mutant catechol dioxygenase with mutation site R193Q-L248D is shown in SEQ ID No. 37;

[0038] The nucleotide sequence encoding the mutant catechol dioxygenase with mutation sites A95V-L148S-R193Q is shown in SEQ ID No. 38;

[0039] The nucleotide sequence encoding the mutant catechol dioxygenase with mutation sites A95V-L148S-L248D is shown in SEQ ID No. 39;

[0040] The nucleotide sequence encoding the mutant catechol dioxygenase with mutation sites A95V-R193Q-L248D is shown in SEQ ID No. 40;

[0041] The nucleotide sequence encoding the mutant catechol dioxygenase with mutation sites L148S-R193Q-L248D is shown in SEQ ID No.41;

[0042] The nucleotide sequence encoding the mutant catechol dioxygenase with mutation sites A95V-L148S-R193Q-L248D is shown in SEQ ID No.42.

[0043] The mutant catechol dioxygenase has excellent thermal stability and can be used for the degradation of catechol compounds. The mutant catechol dioxygenase includes single point mutants and combined mutants. Compared with the wild-type catechol dioxygenase, the thermal stability of the single point mutants and combined mutants is improved, with a maximum difference of 3 times.

[0044] The present invention also provides a recombinant vector comprising the gene as described above. The recombinant vector is a cloning vector or an expression vector. More preferably, the recombinant vector is a pET28a plasmid containing the coding sequence of the mutant catechol dioxygenase.

[0045] Preferably, the method for preparing the recombinant vector comprises the following steps: using a mutation amplification primer pair to perform PCR amplification on a first vector to obtain a recombinant vector, wherein the mutation amplification primer pair contains a nucleotide sequence corresponding to the mutation site of the mutant catechol dioxygenase, and the first vector contains a gene encoding the amino acid sequence shown in SEQ ID No.2.

[0046] Preferably, the mutation amplification primer pair includes at least one of the amplification primer pair for amplifying the mutation site A95V as shown in SEQ ID No. 20 and SEQ ID No. 21, the amplification primer pair for amplifying the mutation site L148S as shown in SEQ ID No. 22 and SEQ ID No. 23, the amplification primer pair for amplifying the mutation site R193Q as shown in SEQ ID No. 24 and SEQ ID No. 25, and the amplification primer pair for amplifying the mutation site L248D as shown in SEQ ID No. 26 and SEQ ID No. 27. In the construction of a single-point mutant of the mutant catechol dioxygenase, one of the above mutation amplification primer pairs is used for PCR amplification. In the construction of multiple-point mutants of the mutant catechol dioxygenase, multiple pairs of the above mutation amplification primer pairs are used for PCR amplification.

[0047] More preferably, before the step of using a mutation amplification primer pair to perform PCR amplification on the first vector, a step of constructing the first vector is also included. The step of constructing the first vector includes: using amplification primer pairs such as sequences shown in SEQ ID No.18 to SEQ ID No.19 to amplify the target gene, the target gene is a coding sequence corresponding to the amino acid sequence shown in SEQ ID No.2; connecting the target gene to an empty vector, transforming, extracting positive plasmids, and obtaining the first vector. Specifically, the sequence shown in SEQ ID No.18 is: 5'-ACTGCTCATATGAGCGTCAAGGTATTC-3'. The sequence shown in SEQ ID No.19 is: 5'-TCAGCTCTCGAGTTACGCGTCGGCGGACGC-3'.

[0048] More preferably, before the step of using the mutation amplification primer pair to perform PCR amplification on the first vector, the method further includes the following step: screening for mutation sites of mutant catechol dioxygenase.

[0049] Protein engineering is based on the relationship between the structural laws of protein molecules and their biological functions. It uses chemical, physical and molecular biological methods to perform gene modification or gene synthesis to transform existing proteins or create new proteins to meet human needs for production and life. Rational design is the most commonly used method in protein engineering. Computer-aided molecular models are combined with site-directed mutagenesis to achieve protein function optimization, such as improving catalytic activity, thermal stability, acid and alkali resistance, etc. In order to effectively optimize the thermal stability of proteins, the present invention performs homology modeling structural analysis on catechol dioxygenase, and predicts the key amino acid residues that affect the thermal stability of the enzyme, including A95V, L148S, R193Q, and L248D, based on the reported structure-function information.

[0050] The recombinant vector can be used to produce mutant catechol dioxygenase for application in the degradation of aromatic compounds.

[0051] The present invention also provides a recombinant engineering bacterium containing the recombinant vector as described above.

[0052] The present invention also provides the use of the mutant catechol dioxygenase, recombinant vector, and recombinant engineering bacteria as described above in the production and preparation of catechol dioxygenase.

[0053] The beneficial effects of the present invention are:

[0054] The mutant catechol dioxygenase of the present invention includes single point mutants and combined mutants. Compared with the wild-type catechol dioxygenase, the single point mutants and combined mutants have longer half-lives at 40°C; in particular, the combined mutant has a half-life of about 3 times that of the wild-type, showing an additive effect of the thermal stability of the single point mutants.

[0055] The mutant catechol dioxygenase of the invention has high thermal stability, can be used for the degradation of aromatic compounds, and has great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is a schematic diagram of the structure of the mutant catechol dioxygenase protein simulation crystal provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0057] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation of the present invention is described in detail below in conjunction with specific embodiments and drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0058] Unless otherwise specified, the reagents and instruments used in the examples are all conventionally selected in the art. The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions, such as conditions in literature, books, or methods recommended by the kit manufacturer. The reagents used in the examples are all commercially available.

[0059] Example 1

[0060] This embodiment provides a mutant catechol dioxygenase, and the mutant catechol dioxygenase is as follows (a1) or (a2):

[0061] (a1) a polypeptide having the same function as the polypeptide consisting of the amino acid sequence shown in SEQ ID No. 2 obtained by deleting, substituting or adding one or more amino acids of the amino acid sequence shown in SEQ ID No. 2;

[0062] (a2) A polypeptide having at least 90% homology with the polypeptide consisting of the amino acid sequence shown in SEQ ID No. 2.

[0063] Specifically, in a preferred embodiment, the mutant catechol dioxygenase is obtained by mutating the amino acid sequence shown in SEQ ID No. 2, and the mutation site is selected from one or a combination of more than one of A95V, L148S, R193Q, and L248D.

[0064] In a further preferred embodiment, the mutation site is A95V, L148S, R193Q, L248D, A95V-L148S, L148S-R193Q, A95V-L248D, R193Q-L248D, A95V-R193Q, L148S-L248D, A95V-L148S-R193Q, A95V-L148S-L248D, A95V-R193Q-L248D, L148S-R193Q-L248D or A95V-L148S-R193Q-L248D.

[0065] Among them, the amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V single-site mutation is shown in SEQ ID No.3; the amino acid sequence of the mutant catechol dioxygenase corresponding to the L148S single-site mutation is shown in SEQ ID No.4; the amino acid sequence of the mutant catechol dioxygenase corresponding to the R193Q single-site mutation is shown in SEQ ID No.5; the amino acid sequence of the mutant catechol dioxygenase corresponding to the L248D single-site mutation is shown in SEQ ID No.6; the amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V-L148S combined site mutation is shown in SEQ ID No.7; the amino acid sequence of the mutant catechol dioxygenase corresponding to the L148S-R193Q combined site mutation is shown in SEQ ID No.8; the amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V-L248D combined site mutation is shown in SEQ ID The amino acid sequence of the mutant catechol dioxygenase corresponding to the R193Q-L248D combination site mutation is shown in SEQ ID No. 9; the amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V-R193Q combination site mutation is shown in SEQ ID No. 10; the amino acid sequence of the mutant catechol dioxygenase corresponding to the L148S-L248D combination site mutation is shown in SEQ ID No. 11; the amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V-L148S-R193Q combination site mutation is shown in SEQ ID No. 12; the amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V-L148S-L248D combination site mutation is shown in SEQ ID No. 13; the amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V-L148S-L248D combination site mutation is shown in SEQ ID No. 14; the amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V-R193Q-L248D combination site mutation is shown in SEQ ID No.15; the amino acid sequence of the mutant catechol dioxygenase corresponding to the L148S-R193Q-L248D combination site mutation is shown in SEQ ID No.16; the amino acid sequence of the mutant catechol dioxygenase corresponding to the A95V-L148S-R193Q-L248D combination site mutation is shown in SEQ ID No.17.

[0066] Example 2

[0067] This example provides a gene encoding the mutant catechol dioxygenase in Example 1, and the gene is as follows (b1) or (b2):

[0068] (b1) a polynucleotide obtained by deleting, substituting or adding one or more bases from the nucleotide sequence shown in SEQ ID No. 1;

[0069] (b2) A polynucleotide having at least 90% homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 1.

[0070] Specifically, in a preferred embodiment, the nucleotide sequence encoding the mutant catechol dioxygenase with a mutation site of A95V is shown in SEQ ID No.28; the nucleotide sequence encoding the mutant catechol dioxygenase with a mutation site of L148S is shown in SEQ ID No.29; the nucleotide sequence encoding the mutant catechol dioxygenase with a mutation site of R193Q is shown in SEQ ID No.30; the nucleotide sequence encoding the mutant catechol dioxygenase with a mutation site of L248D is shown in SEQ ID No.31; the nucleotide sequence encoding the mutant catechol dioxygenase with a mutation site of A95V-L148S is shown in SEQ ID No.32; the nucleotide sequence encoding the mutant catechol dioxygenase with a mutation site of A95V-R193Q is shown in SEQ ID No.33; the nucleotide sequence encoding the mutant catechol dioxygenase with a mutation site of A95V-L248D is shown in SEQ ID The nucleotide sequence encoding the mutant catechol dioxygenase with a mutation site of L148S-R193Q is shown in SEQ ID No.34; the nucleotide sequence encoding the mutant catechol dioxygenase with a mutation site of L148S-R193Q is shown in SEQ ID No.35; the nucleotide sequence encoding the mutant catechol dioxygenase with a mutation site of L148S-L248D is shown in SEQ ID No.36; the nucleotide sequence encoding the mutant catechol dioxygenase with a mutation site of R193Q-L248D is shown in SEQ ID No.37; the nucleotide sequence encoding the mutant catechol dioxygenase with a mutation site of A95V-L148S-R193Q is shown in SEQ ID No.38; the nucleotide sequence encoding the mutant catechol dioxygenase with a mutation site of A95V-L148S-L248D is shown in SEQ ID No.39; the nucleotide sequence encoding the mutant catechol dioxygenase with a mutation site of A95V-R193Q-L248D is shown in SEQ ID The nucleotide sequence encoding the mutant catechol dioxygenase with mutation sites L148S-R193Q-L248D is shown in SEQ ID No.41; the nucleotide sequence encoding the mutant catechol dioxygenase with mutation sites A95V-L148S-R193Q-L248D is shown in SEQ ID No.42.

[0071] Example 3

[0072] This embodiment provides a recombinant vector comprising the gene of embodiment 1.

[0073] The preparation method of the recombinant vector comprises the following steps: using a mutation amplification primer pair to perform PCR amplification on a first vector to obtain a recombinant vector, wherein the mutation amplification primer pair contains a nucleotide sequence corresponding to the mutation site of the mutant catechol dioxygenase, and the first vector contains a gene encoding the amino acid sequence shown in SEQ ID No.2.

[0074] Among them, the mutation amplification primer pair includes: at least one pair of amplification primer pairs for amplifying the mutation site A95V as shown in SEQ ID No.20 and SEQ ID No.21, amplification primer pairs for amplifying the mutation site L148S as shown in SEQ ID No.22 and SEQ ID No.23, amplification primer pairs for amplifying the mutation site R193Q as shown in SEQ ID No.24 and SEQ ID No.25, and amplification primer pairs for amplifying the mutation site L248D as shown in SEQ ID No.26 and SEQ ID No.27.

[0075] Furthermore, this embodiment provides a detailed method for constructing a mutant catechol dioxygenase, comprising the following steps:

[0076] 1. Cloning of wild-type catechol dioxygenase gene

[0077] The wild-type catechol dioxygenase gene was codon optimized using Escherichia coli as a host cell to obtain an optimized catechol dioxygenase gene (i.e., the target gene), whose nucleic acid sequence is SEQ ID No.1 and the expressed amino acid sequence is SEQ ID No.2. SEQ ID No.1 was used as the target gene, and the upstream amplification primer SEQ ID No.18 and the downstream amplification primer SEQ ID No.19 were used to amplify the target gene. Among them, the sequence shown in SEQ ID No.18 is: 5'-ACTGCT adjacent Hydroquinone dioxygenase TG ATGAGCGTCAAGGTATTC-3' (where the underline is the restriction endonuclease NdeI recognition site); the sequence shown in SEQ ID No.19 is: 5'-TCAGCT CTCGAG TTACGCGTCGGCGGACGC-3' (where the underline is the restriction endonuclease XhoI recognition site). The amplification conditions are: amplification at 95°C for 2 min, then amplification at 56°C for 20 sec, amplification at 72°C for 90 sec, for a total of 30 cycles, and finally amplification at 72°C for 10 min.

[0078] After the reaction is completed, the PCR amplification product is detected by agarose gel electrophoresis with a mass percentage of 1.5%, and a 0.8 kb band is obtained, the length of which is consistent with the expected result. According to the standard operation of the kit, the target fragment is recovered and purified, the target fragment and the pET28a plasmid are double-digested with restriction endonucleases XhoI and NdeI, and then connected with T4 DNA ligase, and the obtained connection product is transformed into Escherichia coli BL21 (DE3) competent cells, and the transformed cells are coated on LB plates containing 50 μg / ml kanamycin, and the positive clone plasmid is extracted and sequenced. The results show that the cloned catechol dioxygenase gene sequence is correct and has been correctly connected to the pET28a plasmid to obtain the recombinant plasmid pET28a-catechol dioxygenase. Among them, the wild-type catechol dioxygenase is derived from Escherichia coli and named Ec catechol dioxygenase protein; the catechol dioxygenase gene is provided by Suzhou Jinweizhi Biotechnology Co., Ltd.; the PCR amplification enzyme is KOD high-fidelity polymerase provided by Toyobo.

[0079] 2. Expression and purification of catechol dioxygenase protein

[0080] The engineered bacteria 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 50 μg / mL Kan, and cultured at 37°C and 220 rpm for 2.5 h until the OD 600 When the pH value reaches about 0.9, 0.1 mM IPTG inducer is added and induced for 14 hours at 25°C and 200 rpm. The E. coli cell suspension harvested after fermentation is ultrasonically disrupted and then subjected to a step of Ni-NTA affinity chromatography to obtain catechol dioxygenase protein with a purity greater than 95%, and its amino acid sequence is SEQ ID No. 2.

[0081] 3. Multiple sequence alignment and consensus analysis of catechol dioxygenase homologous proteins

[0082] 3.1. Go to the Pfam database homepage (http: / / pfam.xfam.org / ), enter the amino acid sequence of catechol dioxygenase in the SEQUENCE SEA RCH tool for search, and the server will directly feedback the comparison results of the amino acid sequence of the entire protein family, and display the abundance of various amino acids at each mutation site in the form of a bar graph. The website can also automatically generate a consensus sequence for the protein family.

[0083] 3.2. Input the amino acid sequence shown in SEQ ID No.2 into the NCBI protein database and the Pfam database, use the Blast tool to find all protein sequences with a consistency greater than 30% with the amino acid sequence of catechol dioxygenase protein (as shown in SEQ ID No.2), delete the repeated identical sequences, organize the remaining amino acid sequences into fasta. format, input Clustalx1.83 software for multiple sequence alignment, and output the alignment results in aln., dnd. and fasta. formats, where the dnd. file is a file for constructing an evolutionary tree, and the aln. and fasta. files are sequence files in different formats. Upload the above fasta. file to the Consensus Maker v2.0.0 (http: / / www.hiv.lanl.gov / content / sequence / CONSENSUS / consensus.html) server, and after modifying the setting parameters as needed, the online software will generate a consensus sequence that can be edited later.

[0084] 4. Simulation of the three-dimensional structure of catechol dioxygenase protein and selection of mutation hotspots

[0085] 4.1. The three-dimensional structure prediction of catechol dioxygenase protein (amino acid sequence SEQID No. 2) was obtained using the Swissmodel online tool.

[0086] 4.2. The crystal structure of catechol dioxygenase protein (amino acid sequence as shown in SEQ ID No. 2) was observed by PyMOL. The candidate mutation sites and mutation forms were reviewed based on the structural information to screen out the mutant sites that are most likely to improve the thermal stability of catechol dioxygenase protein. The screening conditions are as follows:

[0087] (1) The criteria for determining a site as a candidate site are:

[0088] ① Most proteins in this family have a high overall abundance of amino acids at this site;

[0089] ②The amino acid at this position is conservative;

[0090] ③ The amino acids with higher frequency of occurrence at this site have significant differences in physical and chemical properties with the amino acids at this site in the catechol dioxygenase protein, such as charge difference, polarity, steric hindrance, etc.

[0091] (2) Remove the area near the active center, i.e., the area away from the catalytic residue ( The amino acid residues within the range are removed, except for those in buried or semi-buried states.

[0092] After the above two steps of screening, there are 8 differential sites remaining, most of which are located on the surface of catechol dioxygenase protein molecules, such as Figure 1 shown.

[0093] (3) Based on the crystal structure of catechol dioxygenase protein, the above eight mutation forms were analyzed in detail one by one, and mutants that may improve the thermal stability of catechol dioxygenase protein were screened out.

[0094] The main judgment criteria are: ① Mutations should eliminate the original forms of forces that are not conducive to thermal stability, such as electrostatic repulsion, charge aggregation, etc.; ② Mutations should not destroy existing forms of forces that are conducive to thermal stability and stable protein structures; ③ Mutations should introduce new forms of forces that are conducive to thermal stability, such as hydrogen bonds, salt bridges, hydrophobic interactions, etc.

[0095] A total of 4 single-point mutants were designed, and their mutation sites were A95V, L148S, R193Q, and L248D, respectively. The thermal stability of the 4 catechol dioxygenase single-point mutants was determined, and 4 mutant catechol dioxygenases with improved thermal stability were screened out, and their mutation sites were A95V, L148S, R193Q, and L248D. The amino acid sequences of the corresponding single-point mutants were SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, and SEQ ID No.6, respectively.

[0096] 5. Construction, expression and purification of mutants

[0097] 5.1. Construction of single-point mutants of catechol dioxygenase protein

[0098] Using the recombinant plasmid pET28a-catechol dioxygenase in step 1 as a template, a pair of complementary oligonucleotides with mutation sites as amplification primers, and KOD high-fidelity enzyme to perform whole-plasmid PCR amplification to obtain a recombinant plasmid with a specific mutation site;

[0099] The amplification primer pairs used were:

[0100] (1) In the amplification primer pair for amplifying the mutation site A95V, the upstream amplification primer is shown in SEQ ID No. 20, and the downstream amplification primer is shown in SEQ ID No. 21. Specifically, the sequence shown in SEQ ID No. 20 is: 5'-GACGCGGCCGACCGCGCGGTGGGCCTCGACGGCGGCACGC-3'. The sequence shown in SEQ ID No. 21 is: 5'-GCGTGCCGCCGTCGAGGCCCACCGCGCGGTCGGCCGCGTC-3'.

[0101] (2) In the amplification primer pair for amplifying the mutation site L148S, the upstream amplification primer is shown in SEQ ID No. 22, and the downstream amplification primer is shown in SEQ ID No. 23. Specifically, the sequence shown in SEQ ID No. 22 is: 5'-GGCGCCGACGGCAAGCCGTCGGCCGGTGCGCTCGTCG-3'. The sequence shown in SEQ ID No. 23 is: 5'-CGACGAGCGCACCGGCCGACGGCTTGCCGTCGGCGCC-3'.

[0102] (3) In the amplification primer pair for amplifying the mutation site R193Q, the upstream amplification primer is shown in SEQ ID No. 24, and the downstream amplification primer is shown in SEQ ID No. 25. Specifically, the sequence shown in SEQ ID No. 24 is: 5'-GACGGCAAGTACGAATTCCAGACGCTGATGCCGGTCGGC-3'. The sequence shown in SEQ ID No. 25 is: 5'-GCCGACCGGCATCAGCGTCTGGAATTCGTACTTGCCGTC-3'.

[0103] (4) In the amplification primer pair for amplifying the mutation site L248D, the upstream amplification primer is shown as SEQ ID No. 26, and the downstream amplification primer is shown as SEQ ID No. 27. Specifically,

[0104] The sequence shown in SEQ ID No. 26 is: 5'-CAACATCGAAGGCGACCCGGACATCTGGGACGACTTCGC-3'. The sequence shown in SEQ ID No. 27 is: 5'-GCGAAGTCGTCCCAGATGTCCGGGTCGCCTTCGATGTTG-3'.

[0105] The amplification conditions are: amplification at 95°C for 2 minutes, then amplification at 56°C for 20 seconds, amplification at 72°C for 90 seconds, a total of 30 cycles, and finally amplification at 72°C for 10 minutes; gel recovery of PCR amplification products, digestion of gel recovery products with DpnI enzyme at 37°C for 2 hours, degradation of the initial template; transformation of digestion products into Escherichia coli BL21 (DE3) competent cells, coating on LB agar plates containing 50μg / mL kanamycin, culturing at 37°C overnight, screening positive clones, sequencing verification, and obtaining recombinant bacteria containing single-point mutants of catechol dioxygenase. Among them, the above-mentioned KOD high-fidelity enzyme was provided by Taka Ra Company; the above-mentioned DpnI enzyme was provided by Fermentas Company.

[0106] 5.2. Construction of combined mutants of catechol dioxygenase protein

[0107] Using a construction method similar to that of single-point mutants, single-point mutants with improved stability are cumulatively combined, and multiple mutation sites are selected in the amino acid sequence shown in SEQ ID No. 2 for combination, such as selecting 2 to 4 mutation sites from the above 4 mutation sites for combination, to obtain different catechol dioxygenase combination mutants:

[0108] (1) By selecting two mutation sites for combination, six mutant catechol dioxygenases can be constructed, and the combined mutation sites are: A95V / L148S, A95V / R193Q, A95V / L248D, L148S / R193Q, L148S / L248D, R193Q / L248D. The amino acid sequences of the six mutant catechol dioxygenases are SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, and SEQ ID No.12, respectively.

[0109] (2) Three mutation sites are selected for combination to construct four mutant catechol dioxygenases, and the combined mutation sites are: A95V / L148S / R193Q, A95V / L148S / L248D, A95V / R193Q / L248D, L148S / R193Q / L248D. The amino acid sequences of the four mutant catechol dioxygenases are SEQ ID No.13, SEQ ID No.14, SEQ ID No.15, and SEQ ID No.16, respectively.

[0110] (3) Four mutation sites are selected for combination to construct a mutant catechol dioxygenase, wherein the combined mutation sites are: A95V / L148S / R193Q / L248D. The amino acid sequence of the mutant catechol dioxygenase is SEQ ID No. 17.

[0111] Among them, the coding sequence encoding the mutant catechol dioxygenase with a mutation site of A95V is shown in SEQ ID No. 28. The coding sequence encoding the mutant catechol dioxygenase with a mutation site of L148S is shown in SEQ ID No. 29. The coding sequence encoding the mutant catechol dioxygenase with a mutation site of R193Q is shown in SEQ ID No. 30. The coding sequence encoding the mutant catechol dioxygenase with a mutation site of L248D is shown in SEQ ID No. 31. The coding sequence encoding the mutant catechol dioxygenase with a mutation site of A95V / L148S is shown in SEQ ID No. 32. The coding sequence encoding the mutant catechol dioxygenase with a mutation site of A95V / R193Q is shown in SEQ ID No. 33. The coding sequence encoding the mutant catechol dioxygenase with a mutation site of A95V / L248D is shown in SEQ ID No. 34. The coding sequence encoding the mutant catechol dioxygenase with mutation sites L148S / R193Q is shown in SEQ ID No. 35. The coding sequence encoding the mutant catechol dioxygenase with mutation sites L148S / L248D is shown in SEQ ID No. 36. The coding sequence encoding the mutant catechol dioxygenase with mutation sites R193Q / L248D is shown in SEQ ID No. 37. The coding sequence encoding the mutant catechol dioxygenase with mutation sites A95V / L148S / R193Q is shown in SEQ ID No. 38. The coding sequence encoding the mutant catechol dioxygenase with mutation sites A95V / L148S / L248D is shown in SEQ ID No. 39. The coding sequence encoding the mutant catechol dioxygenase with mutation sites A95V / R193Q / L248D is shown in SEQ ID No. 40. The nucleic acid sequence encoding the mutant catechol dioxygenase with mutation sites L148S / R193Q / L248D is shown in SEQ ID No. 41. The nucleic acid sequence encoding the mutant catechol dioxygenase with mutation sites A95V / L148S / R193Q / L248D is shown in SEQ ID No. 42.

[0112] Test Example 1

[0113] Characterization of the enzymatic properties of mutant catechol dioxygenase

[0114] The wild-type catechol dioxygenase and the mutant catechol dioxygenase provided in Example 1 were subjected to a thermal stability test according to a conventional catechol dioxygenase stability determination method, specifically:

[0115] The enzyme solution was incubated at a certain temperature, and samples were taken at different treatment times to determine the residual activity percentage of catechol dioxygenase or mutant catechol dioxygenase. The ln value of the residual activity percentage was plotted against time t (min). The slope of the straight line was the inactivation constant kinact, which was expressed by t 1 / 2 = ln2 / kinact to obtain the half-life of the wild-type catechol dioxygenase or mutant catechol dioxygenase at this temperature. The test results are shown in Table 1.

[0116] Table 1. Enzymatic properties of wild-type catechol dioxygenase and mutant catechol dioxygenase

[0117]

[0118] As can be seen from Table 1, the 15 mutant catechol dioxygenases include single point mutants and combined mutants. Compared with the wild-type catechol dioxygenase, the single point mutants and combined mutants have longer half-lives at 40°C; especially the combined mutant, whose half-life is about 3 times that of the wild-type, showing the superposition effect of the thermal stability of the single point mutants. In summary, the mutant catechol dioxygenase of the present invention has high thermal stability, can be used for the degradation of aromatic compounds, and has great application potential.

[0119] Example 4

[0120] This embodiment provides a recombinant engineered bacterium, which contains the recombinant vector of Example 3.

[0121] Example 5

[0122] This embodiment provides the use of the mutant catechol dioxygenase, recombinant vector, and recombinant engineered bacteria in the production and preparation of catechol dioxygenase.

[0123] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details. Applicant: Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences Invention Name: Catechol dioxygenase mutant, recombinant vector, preparation method and application thereof SEQ ID No.1 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGCCGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGCTCGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCGCACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGCTGATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA。 SEQ ID No.2 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAAGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPLAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFRTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPLIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.3 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAVGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPLAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFRTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPLIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.4 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAAGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPSAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFRTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPLIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.5 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAAGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPLAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFQTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPLIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.6 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAAGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPLAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFRTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPDIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.7 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAVGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPSAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFRTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPLIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.8 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAVGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPLAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFQTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPLIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.9 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAVGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPLAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFRTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPDIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.10 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAAGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPSAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFQTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPLIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.11 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAAGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPSAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFRTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPDIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.12 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAAGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPLAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFQTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPDIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.13 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAVGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPSAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFQTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPLIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.14 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAVGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPSAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFRTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPDIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.15 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAVGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPLAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFQTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPDIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.16 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAVGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPLAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFQTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPDIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.17 MSVKVFDTKEVQDLLKAAANLNGDAGNARFRQIVHRLLSDLFKAIDDLDITPDEVWAGVNYLNKLGQDGEAALLAAGIGLEKYLDIRMDAADRAVGLDGGTPRTIEGPLYVAGAPVRDGVAKIDLDDDADAGPLVIRGTVTGADGKPSAGALVECWHANSKGFYSHFDPTGAQTAFNLRGAVRTDADGKYEFQTLMPVGYGCPPQGATQQLLNGLGRHGNRPAHVHFFVSGDGHRKLTTQFNIEGDPDIWDDFAYATREELIPHVVDKTGGAALGMKSDAYKEIEFDIVLTPLLDGRDNQVVHRPRASADA。 SEQ ID No.18 ACTGCTCATATG ATGAGCGTCAAGGTATTC SEQ ID No.19 TCAGCTCTCGAG TTACGCGTCGGCGGACGC SEQ ID No.20 GACGCGGCCGACCGCGCGGTGGGCCTCGACGGCGGCACGC SEQ ID No.21 GCGTGCCGCCGTCGAGGCCCACCGCGCGGTCGGCCGCGTC SEQ ID No.22 GGCGCCGACGGCAAGCCGTCGGCCGGTGCGCTCGTCG SEQ ID No.23 CGACGAGCGCACCGGCCGACGGCTTGCCGTCGGCGCC SEQ ID No.24 GACGGCAAGTACGAATTCCAGACGCTGATGCCGGTCGGC SEQ ID No.25 GCCGACCGGCATCAGCGTCTGGAATTCGTACTTGCCGTC SEQ ID No.26 GACGGCAAGTACGAATTCCAGACGCTGATGCCGGTCGGC SEQ ID No.27 GCCGACCGGCATCAGCGTCTGGAATTCGTACTTGCCGTC SEQ ID No.28 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGTGGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGCTCGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCGCACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGCTGATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA。 SEQ ID No.29 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGCCGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGTCGGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCGCACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGCTGATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA。 SEQ ID No.30 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGCCGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGCTCGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCAAACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGCTGATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA。 SEQ ID No.31 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGCCGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGCTCGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCGCACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGGACATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA。 SEQ ID No.32 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGTGGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGTCGGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCGCACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGCTGATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA。 SEQ ID No.33 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGTGGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGCTCGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCAAACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGCTGATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA。 SEQ ID No.34 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGTGGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGCTCGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCGCACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGGACATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA。 SEQ ID No.35 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGCCGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGTCGGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCAAACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGCTGATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA。 SEQ ID No.36 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGCCGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGTCGGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCGCACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGGACATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA。 SEQ ID No.37 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGCCGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGCTCGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCAAACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGGACATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA。 SEQ ID No.38 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGTGGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGTCGGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCAGACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGCTGATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA。 SEQ ID No.39 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGTGGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGTCGGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCGCACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGGACATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA。 SEQ ID No.40 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGTGGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGCTCGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCAAACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGGACATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA。 SEQ ID No.41 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGCCGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGTCGGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCAAACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGGACATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA。 SEQ ID No.42 ATGAGCGTCAAGGTATTCGATACGAAGGAAGTGCAGGATCTGCTGAAGGCCGCCGCGAACCTGAACGGCGACGCCGGCAATGCGCGCTTTCGCCAGATCGTGCATCGTCTGCTGAGCGACCTGTTCAAGGCGATCGACGATCTCGACATCACGCCCGACGAAGTCTGGGCCGGCGTCAACTATCTGAACAAGCTCGGCCAGGATGGTGAAGCGGCGCTGCTCGCGGCCGGCATCGGGCTCGAGAAGTATCTGGACATCCGGATGGACGCGGCCGACCGCGCGGTGGGCCTCGACGGCGGCACGCCGCGCACGATCGAAGGGCCGCTGTACGTGGCAGGCGCGCCCGTGCGCGATGGCGTCGCGAAGATCGATCTCGACGACGATGCCGACGCGGGCCCGCTCGTGATCCGCGGCACCGTGACGGGCGCCGACGGCAAGCCGTCGGCCGGTGCGCTCGTCGAATGCTGGCACGCGAACTCGAAGGGCTTCTACTCGCACTTCGATCCGACCGGCGCGCAGACCGCGTTCAACCTGCGCGGCGCGGTGCGCACCGATGCCGACGGCAAGTACGAATTCCAGACGCTGATGCCGGTCGGCTACGGCTGCCCGCCGCAAGGCGCGACGCAGCAGCTGCTGAACGGCCTCGGCCGCCACGGCAACCGCCCGGCGCATGTGCACTTCTTCGTGTCCGGCGACGGCCATCGCAAGCTGACGACGCAGTTCAACATCGAAGGCGACCCGGACATCTGGGACGACTTCGCGTACGCGACGCGCGAGGAACTGATTCCGCACGTGGTCGACAAGACCGGCGGCGCCGCGCTCGGCATGAAGAGCGATGCGTACAAGGAAATCGAGTTCGACATCGTGCTGACGCCGCTGCTCGACGGCCGCGACAACCAGGTCGTCCATCGTCCGCGCGCGTCCGCCGACGCGTAA

Claims

1. A mutant catechol dioxygenase, characterized in that: The mutant catechol dioxygenase is obtained by mutating the amino acid sequence shown in SEQ ID No. 2, and the mutation site is L148S, R193Q, L148S-R193Q, R193Q-L248D, L148S-L248D, or L148S-R193Q-L248D; The amino acid sequence of the mutant catechol dioxygenase corresponding to the L148S single-site mutation is shown in SEQ ID No. 4; The amino acid sequence of the mutant catechol dioxygenase corresponding to the R193Q single-site mutation is shown in SEQ ID No. 5; The amino acid sequence of the mutant catechol dioxygenase corresponding to the L148S-R193Q combined site mutation is shown in SEQ ID No. 10; The amino acid sequence of the mutant catechol dioxygenase corresponding to the R193Q-L248D combined site mutation is shown in SEQ ID No. 12; The amino acid sequence of the mutant catechol dioxygenase corresponding to the L148S-L248D combined site mutation is shown in SEQ ID No. 11; The amino acid sequence of the mutant catechol dioxygenase corresponding to the L148S-R193Q-L248D combined site mutation is shown in SEQ ID No.

16.

2. A gene encoding the mutant catechol dioxygenase as claimed in claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence encoding the mutant catechol dioxygenase with the mutation site L148S is shown in SEQ ID No. 29; The nucleotide sequence encoding the mutant catechol dioxygenase with mutation site R193Q is shown in SEQ ID No. 30; The nucleotide sequence encoding the mutant catechol dioxygenase with the mutation site L148S-R193Q is shown in SEQ ID No. 35; The nucleotide sequence encoding the mutant catechol dioxygenase with mutation sites L148S-L248D is shown in SEQ ID No. 36; The nucleotide sequence encoding the mutant catechol dioxygenase with mutation site R193Q-L248D is shown in SEQ ID No. 37; The nucleotide sequence encoding the mutant catechol dioxygenase with mutation sites L148S-R193Q-L248D is shown in SEQ ID No.

41.

4. A recombinant vector, characterized in that: It comprises the gene according to claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that The preparation method of the recombinant vector comprises the following steps: using a mutation amplification primer pair to perform PCR amplification on a first vector to obtain a recombinant vector, wherein the mutation amplification primer pair contains a nucleotide sequence corresponding to the mutation site of the mutant catechol dioxygenase, and the first vector contains a gene encoding the amino acid sequence shown in SEQ ID No.

2.

6. The recombinant vector according to claim 5, characterized in that The mutation amplification primer pair includes: at least one pair of amplification primer pairs for amplifying the mutation site L148S as shown in SEQ ID No.22 and SEQ ID No.23, amplification primer pairs for amplifying the mutation site R193Q as shown in SEQ ID No.24 and SEQ ID No.25, and amplification primer pairs for amplifying the mutation site L248D as shown in SEQ ID No.26 and SEQ ID No.

27.

7. A recombinant engineered bacterium, characterized in that: Contains the recombinant vector according to claim 4.

8. Use of the mutant catechol dioxygenase according to claim 1, the recombinant vector according to any one of claims 4 to 6, and the recombinant engineered bacteria according to claim 7 in the production and preparation of catechol dioxygenase.

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